Trace Elements in Man and Animals 10

Trace Elements in Man and Animals 10

TEMA 10 Chairman Alain E. Favier Joseph Fourier University, Grenoble, France

International Parent Committee M. Anke, Friedrich Schiller University, Jena, Germany J. R. Arthur, Rowett Research Institute, Aberdeen, UK I. Bremner, Rowett Research Institute, Aberdeen, UK N. Costa, Murdoch University, Perth, Australia P. W. F. Fischer, Health Canada, Ottawa, Canada R. Gibson, University of Otago, Dunedin, New Zealand J. King, USDA, ARS, WHNRC, San Francisco, California B. Momcilovic, Institut Research Health, Zagreb, Croatia J. Proshaska, University of Minnesota, Duluth, Minnesota R. Sunde, University of Missouri, Columbia, Missouri

Advisory Scientific Committee A. Favier, Joseph Fourier University, Grenoble, France R. A. Anderson, Beltsville Human Nutrition Research Center, Beltsville, Maryland P. Braetter, Hahn Meitner Institut Berlin Gmbh, Berlin, Germany P. Chappuis, Hospital Lariboisière, Paris, France J. Neve, University of Pharmacy, Bruxelles, Belgium Y. Rayssiguier, INRA, Clermond Ferrand, France

Local Organizing Committee A. M. Roussel, Treasurer, Joseph Fourier University, Grenoble, France A. Alcaraz, Secretary, Hospital, Grenoble, France

Trace Elements in Man and Animals 10 Edited by

A. M. Roussel Joseph Fourier University, Grenoble, France

R. A. Anderson Beltsville Human Nutrition Research Center, Beltsville, Maryland and

A. E. Favrier Joseph Fourier University, Grenoble, France

KLUWER ACADEMIC PUBLISHERS NEW YORK, BOSTON, DORDRECHT, LONDON, MOSCOW

eBook ISBN: Print ISBN:

0-306-47466-2 0-306-46378-4

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ACKNOWLEDGMENTS

The financial support of the following sponsors is gratefully acknowledged. Without their

generous support the meeting would not have been possible

Activation Laboratory Ltd, Ancaster, Canada Albion Laboratories, Inc.—Inobio, Romilly sur Andelle, France AMBI Nutrition Company—Nutrition 21, San Diego, California ADIR—I.R.I.S. Courbevoie, France Astra Calvé, Rueil Malmaison, France Banque Populaire du Dauphiné et des Alpes du Sud Beghin Meiji Industries, Neuilly sur Seine, France Candia Cedillac SA, Lyon, France Compagnie Des Salins du Midi et Des Salines de l’Est, Aigues-Mortes, France Diepal-nsa, Villefranche sur Saône, France Estèe Lauder Companies, Oevel, Belgium Ingen Inter Genetic Techn, Rungis, France INRA, Clermont Ferrand, France Labcatal S.A, Montrouge, France Laboratory Boiron, Ste Foy Les Lyon, France Laboratory C.C.D., Paris, France Laboratory Des Granions, Monaco Laboratory Randox, Mauguio, France Laboratory Richelet, Paris, France Laboratory Robapharm—Pierre Fabre Medicament, Castres, France Laboratory Roche Nicholas S.A., Gaillard, France Mairie de Grenoble, Grenoble, France Micromass UK Ltd, Cheshire, UK Nestle Research Center, Lausanne, Switzerland Nicomed—AS SERO, Billingstad Norway Perkin Elmer S.A., Versailles, France Pharma Nord, Zaventem, Belgium Roche Diagnostic S.A, Meylan, France Selenium Tellurium Development Association, Inc., Grimbergen, Belgium Université Joseph Fourier, Grenoble, France Region Rhône Alpes, Charbonnieres-Les-Bains, France S.A des Eaux Minérales D’Evian, Evian, France U.S. Borax Inc., Newport Coast, California

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PREFACE

The Tenth International Symposium on Trace Elements in Man and Animals (TEMA 10) was held on the south bank of Lake Geneva at the base of the snowcapped Alps in Evian, France, May 2–7, 1999. The TEMA Symposium, which has evolved into one of the premier meetings regarding human and animal trace metal nutrition and metabolism, was held initially in Aberdeen, Scotland in 1969. Subsequent symposia have been held in Madison, Wisconsin, USA, 1973; Freising, Germany, 1977; Perth, Australia, 1981; Aberdeen, Scotland, 1984; Pacific Grove, California, USA, 1987; Dubrovnik, Croatia, 1990; Dresden, Germany, 1993 and Banff, Canada, 1996. TEMA 10 was the largest of all the TEMA meetings attracting 479 participants from 41 countries. The meeting commenced with the Underwood Lecture entitled “Metallothionein and zinc metabolism” presented by Robert Cousins and was followed by 48 invited lectures, 125 oral presentations, three poster sessions, and three workshops. This large attendance demonstrates that TEMA remains the most attended of all meeting focusing on the biology of trace metals, certainly because of the quality of its scientific content. The successive scientific programs of all TEMA meetings facilitate the description of the evolution of the research on trace elements. TEMA 10 consolidated the progress in the knowledge on molecular and cellular biology of metals. Various lecture and communications presented new data on zinc and copper transport mechanism, providing a global view on the molecular metabolism of these metals in the human body. Other particularly dramatic results have been presented on the epidemiology of trace element deficiency and supplementation. The results of large recent trials were presented demonstrating the beneficial effect of selenium for human health. Apart from selenium, which has gained a spectacular growth of interest in the scientific field with the discovery of new selenoproteins and biological role, the year of 1999 was also marked by particular progress of research on boron and chromium. The meeting was designed for maximal scientific and social interaction of the participants and all sessions were extremely well attended even though the beautiful weather and surroundings were tempting distractions. A morning stroll along Lake Geneva prior to the morning program often resulted in meeting many of the TEMA participants and often “mini TEMA sessions” occurred along the lake. The ideas and friendships exchanged at these and the many other impromptu meetings further exemplified the benefits of attending this symposium. The enthusiasm and spirited discussions at the scientific sessions extended to the on-site dining facilities which allowed for excellent interactions of the participants and a chance to sample fine French cuisine and French hospitality. The social program was truly enjoyed by all and gave many visitors a “taste of the French way” and made it difficult to return to the rigors of daily life. The social program vii

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Preface

commenced with a Cocktail Reception to welcome all to TEMA. The following day, participants were welcomed to Evian and the region by the Mayor of Evian. It is the custom of the TEMA meetings to have one afternoon where all the participants and guests go on a joint outing to relax, enjoy the local scenery, and have a chance to interact with all the participants in an informal atmosphere. The afternoon was really one of the lasting memories of the meeting with a musical cruise on Lake Geneva highlighted by a private guided tour of Chillon Feodal Castle, on the Swiss side of the lake. The social program was highlighted by the formal dinner held in the Evian Royal Casino complete with professional night club dancers in “unique French attire.” This meeting would not have been possible without the special financial support of Evian Mineral Water Company, Eridiana Beghin Say, Borax Company, Labcatal, Robapharm, and Nutrition 21. We would like to express our sincere thanks to the members of the scientific Committee, Richard Anderson (USA), Ian Bremner (UK), John Arthur (UK), Petter Braetter (Germany), Phillippe Chappuis (France), Jean Neve (Belgium), and Yves Rayssiguier (France) for their time and their expertise. We thank the parental committee of TEMA that is responsible for the conservation of the spirit of TEMA for trusting in us when asking us to organize the tenth session of this great meeting. We also thank the graduate students and staff of the laboratory LBSO from Grenoble for their efforts in ensuring that the meeting was conducted smoothly with no major problems. A special and truly deserved thank you to Arlette Alcaraz and Isabelle Michel who worked tirelessly for more than three years to make the meeting not only possible but also in reaching such a high standard and also for the completion of the Proceedings. We wish you many pleasant memories of TEMA 10 and look forward to meeting you at TEMA 11 in California in 2002. We are confident that TEMA 11 will build upon TEMA 10 and provide even better scientific and social programs. The Editors, A. E. Favier, A. M. Roussel, R. A. Anderson

CONTENTS

UNDERWOOD MEMORIAL LECTURE 1.

Integrative Aspects of Zinc Metabolism and Function Robert J. Cousins

1

I. CELLULAR AND MOLECULAR BIOLOGY OF TRACE ELEMENTS Trace Elements Cellular Metabolims 2.

The Copper Transporting ATPases in Human Disease Jonathan D. Gitlin

3.

Regulation and Function of the Copper Ion Transport Machinery Jaekwon Lee and Dennis J. Thiele

4.

Control of Gene Expression of Glutathione Peroxidase-1 and Other Selenoproteins in Rats and Cultured Cells Roger A. Sunde and Jacqueline K. Evenson

5.

Cellular and Subcellular Distribution of Selenium and Selenium-Containing Proteins in the Rat Dietrich Behne, Henning Pfeifer, Doris Röthlein, and Antonios Kyriakopoulos

9 15

21

29

35

6.

The Mechanism and Regulation of Zinc Transport in Yeast David J. Eide

7.

Metallothionein Facilitates Zinc Absorption in Zinc Deficient Mice but Limits Absorption in Zinc Replete Mice P. Coyle, J. C. Philcox, and A. M. Rofe

43

Metallothionein and Innate Activation of Primary Human and Mouse Monocytes James Koropatnick, Suzanne Dale, and Rudolfs K. Zalups

47

8.

ix

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Contents

9.

Role of Metallothionein on Zn, Cu, Cd Au, and Ag Accumulation in Hepatic Cytosol of Heavy Metal-Injected Rats S. Saito and K. Yoshida

10.

Molecular Cloning of a Human cDNA Regulated by Heavy Metals M. G. Pagliuca, S. Cigliano, R. Lerose, and A. Leone

11.

Distribution of Copper Transported ATP7B in Embryo and New Born Rat T. Hosokawa, M. Okabe, M. Kurasaki, A. Hata, F. Endo, K. Nagano, I. Matsuda, K. Urakami, and T. Saito

12.

13.

51

55

59

Interaction between Copper and Iron Metabolism in the Human Intestinal Caco-2 Cells I. Gabrielli, Y. Sambuy, S. Ferruzza, and M. L. Scarino

63

Induction of Metallothioneins by Zinc (Zn) Supplementation Quantification in Young Rat Tissues G. S. Henriques and S. M. F. Cozzolino

65

Trace Elements and Oxidative Stress 14.

Iron, Oxidative Stress, the HFE Gene, and Lung Cancer J. M. McCord, S. K. Bose, and B. Gao

67

15.

Selenium and the Protection against Peroxynitrite Helmut Sies, Lars-Oliver Klotz, Stefan M. Schieke, Karlis Briviba, and Gavin E. Arteel

71

16.

Cooperative Effects of Zinc/Selenium and Thiols in the Protection against UV-Induced Genomic DNA Damage Marie-Jeanne Richard, Nathale Emonet-Piccardi, Christine Didier, Eric Jourdan, Marie-Thérèse Leccia, Marie-Odile Parat, Jean Cadet, Jean C. Béani, and Alain Favier

77

17.

Trace Elements and Enzyme Redox Centers Marc Fontecave

83

18.

Importance of Trace Elements in Transcription Factor Activation Jacques Piette

89

Trace Elements: Metabolism and Oxidative Modifications of Lipoproteins Y. Rayssiguier and A. Mazur

97

19.

20.

Co-localization of Cu/Zn-Superoxide Dismutase (SOD-1), Nitric Oxide Synthase (NOS), and Zn/Cu-Metallothionein (MT) in Rat Brain Masashi Okabe, Toshiyuki Hosokawa, Shigeru Saito, Takeshi Saito, Masaaki Kurasaki, and Hidesuke Shimizu

105

Contents

21.

22.

23.

24.

25.

26.

27.

28.

29.

30.

Effect of Dolomite and Thiamine Supplementation on Serum Total Antioxidant Status and Bioelements Concentration in LeadIntoxicated Rats Z. Krejpcio and R. Wojciak

xi

111

Protective Effects of Antioxidants on Iron-Induced Free Radical-Mediated Damaging Processes in Humans and Animals Igor Afanas’ev, Elena Ostrachovich, Irina Deeva, and Ludmila Korkina

115

Complexes of Flavonoids with Iron and Copper as a New Way of Decreasing the Oxidative Damage Induced by Transition Metal Supplementation L. G. Korkina, E. A. Ostrachovich, G. A. Ibragimova, and I. B. Afanas’ev

119

Oncogene Activation and Apoptosis as Possible Mechanism of Antitumour Effect of Ferric-Sorbitol-Citrate Marija Poljak-Blazi, Marijeta Kralj, and Marijana Popovic-Hadzija

123

Changes in Oxidant and Antioxidant Status in Rats Fed Different Amounts of Selenium and Exposed to J. Gromadzinska, W. Wasowicz, K. Rydzynski, and J. Neve

127

Zinc Protects Genomic DNA from Solar Light Injury: A Possible Role for Zinc-Induced Metallothionein? E. Jourdan, N. Emonet-Piccardi, A. Favier, J. C. Beani, and M. J. Richard The Effect of Carbamylation on Glutathione Peroxidase and CopperZinc Superoxide Dismutase Heather E. Roxborough, Cheryl Reid, Jane McEneny, Caroline Mercer, Dorothy McMaster, Maireard O’Hare, and Ian S. Young

129

131

Anthracyclin Resistance of GLC4 Tumoral Cells and Intracellular Selenium Metabolism M. Andriollo, P. Guiraud, M. J. Richard, and A. Favier

133

Antiproliferative Ability of Ferric-Sorbitol-Citrate and Ferrocenes for Malignant Cell Line, Hep2 and F10 M. Poljak-Blazi, A. Ferle Vidovic, V. Rapic, and D. Škare

135

Modulation of SP1 and Binding Activity by Aluminum in HeLa Cells C. Garrel, M. Osman, and A. Favier

137

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31.

Antioxidant Micronutriments Insulin-Sensitivity P. Faure, F. Couzy, D. Barclay, E. Rossini, M. J. Richard, J. Arnaud, A. Favier, and S. Halimi

32.

Exacerbated Immune Stress Response in Early Magnesium Deficiency in the Rat A. Mazur, C. Malpuech-Brugère, W. Nowacki, E. Rock, and Y. Rayssiguier

33.

34.

35.

36.

Zinc Protects Human Endothelial Vascular Cells against the Glucose Induced Cytotoxicity K. Lalanne, S. Bouvard, N. Wiernsperger, P. Faure, A. Favier, and S. Halimi Effects of Elevated Dietary Cu and Cd Concentrations on Oxidative Stress, Cell Proliferation, and Apoptosis in Atlantic Salmon (Salmon Salar L.) Parr Marc H. G. Berntssen, Sjoerd E. Wendelaar Bonga, and Amund Maage Modulation of Copper Deficiency Induced Oxidative Stress by Dietary Polyphenol in the Rat I. Bureau, C. Feillet-Coudray, E. Gueux, E. Rock, A. Mazur, and Y. Rayssiguier Thioredoxin/Thioredoxin Reductase System: Their Roles in DNA Stability and Cell Adaptation to Ultraviolet Radiation C. Didier, M.-J. Richard, J. C. Beani, and A. Favier

138

139

140

141

142

143

37. Toxic Effect of Ciprofloxacin May be the Result of

a Free Radical Pathway A. Gürbay, B. Gonthier, D. Daveloose, F. Hincal, and A. Favier

144

Transport, Speciation and Distribution of Trace Elements 38.

Trace Element Speciation in Human Body Fluids Peter Brätter, Andrea Raab, and Andrea N. Richarz

39.

Gelfiltration and Anion-Exchange Chromatography for the Separation of Vanadium Binding Proteins in Plasma of Rats, Rabbits, and Humans K. De Cremer and K. R. Cornelis

40.

Metabolism of Selenate Administered into Rats: Speciation of Selenium by HPLC-ICP MS Kazuo T. Suzuki and Yamato Shiobara

145

153

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Contents

41. Fractionation of Soluble Selenium Compounds from Fish using SizeExclusion Chromatography with On-Line Detection by Inductively Coupled Plasma Mass Spectrometry: Comparison with other Techniques Gunilla Önning and Ingvar A. Bergdahl 42. Metabolism of Arsenic from Seaweed by Man and Animals: Speciation in Body Fluids using Liquid Chromatography Inductively Coupled Plasma Mass Spectrometry Jörg Feldmann

43. Speciation of Selenium in Food and Animal Feed using Ion Chromatography On-Line Microwave Pretreatment Hydride Generation Atomic Absorption Spectrometry Magnus Johansson, Guy Bordin, and Adela R. Rodriguez 44.

Selenium True Absorption and Tissue Concentration of Rats at Dietary Selenite, Seleno Cysteine, and Seleno Methionine W. Windisch and M. Kirchgeßner

xiii

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165

169

173

45. Effects of Selenium Status on Selenium Incorporation into Plasma Fractions and Excretion in Urine in Men Infused with Selenite Y. Xia, J. Butler, M. Janghorbani, P. Ha, P. Whanger, J. Olesik, and L. Daniels

175

46. Magnesium Excretion and Redistribution in Rat Tissues after Disulfiram Experimental Administration L. Kovatsi, M. Tsougas, H. Tsoukali, and D. Psaroulis

177

Selenium and Arsenic Distribution in Soluble Protein Fractions in Liver of Hens Fed with Added V. Stibilj, I. Falnoga, D. Cestnik, and R. Jacimovic

178

Metal Distribution in Metallothioneins of Cytosols from Human Cirrhotic Livers C. Wolf, P. Brätter, and U. Rösick

180

47.

48.

49.

50.

Effect of Retinol Deficiency on Liver Concentration of Fe, Cr, Zn, Mn, Co, Cd, and Pb in Rat A. C. Anzulovich, L. B. Oliveros, L. D. Martínez, M. Roura, and M. S. Giménez Vitamin A Deficiency Modify the Antioxidant Defenses and the Trace Elements Level in Rat Liver A. C. Anzulovich, L. Oliveros, D. Martinez, M. Baucells, and M. S. Gimenez

181

183

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Contents

51. Transport of Silver and Its Potential Interactions with Copper

Metabolism in the Rat S. Hanson, S. Donley, H. Rim, and M. C. Linder 52.

Dietary Vanadium, P-ATPase-7a Expression and the Influence on Lysyl Oxidase and Cu Accumulation in Rat Skin and Liver Robert B. Rucker, Chang Tai Cui, Eskouhie H. Tchaparian, Alyson E. Mitchell, Michael Clegg, Janet Y. Uriu-Hare, and Carl L. Keen II.

184

186

HUMAN PHYSIOLOGY AND STATUS OF TRACE ELEMENTS

Trace Elements in Food Chain, Diets and Intakes 53.

Recommended Dietary Intakes for Trace Elements: New Trends Janet C. King

54.

Trace Element Addition to Foods: Technological and Nutritional Aspects Denis Barclay

189

195

55.

Trace Elements in Intravenous Nutrition A. Shenkin

201

56.

Trace Element Intake and Balance in Adults in Central Europe M. Anke, M. Glei, W. Dorn, R. Müller, J. Vormann, M. Müller, M. Jahritz, M. Seifert, S. Holzinger, S. Drobner, B. Röhrig, C. Rother, L. Angelow, and G. O. Latunde-Dada

209

57.

Nutrient Risk Assessment: Implications for Food Fortification Policy Mary R. L’Abbé, Kevin A. Cockell, Sheila Dubois, and William H. Ross

215

58.

Vanadium—An Essential Element for Animals and Humans? M. Anke, H. Illing-Günther, H. Gürtler, S. Holzinger, M. Jaritz, S. Anke, and U. Schäfer

221

59.

Effect of Iron Fortification of Infant Weaning Foods on Mineral Absorption C. Martínez, G. López, G. Ros, T. Fox, and S. J. Fairweather-Tait

227

An Approach to Assessing the Safety of Trace Element Supplementation D. H. Shrimpton

228

60.

61.

Strontium Transfer in the Food Chain of Humans M. Anke, M. Seifert, M. Jaritz, E. Lösch, and E. Hartmann

229

Contents

62.

63.

64.

65.

66.

xv

Content of the Mineral Elements in the Light Diets Used for Alimentation of the Pensioner House Residents in Warsaw A. Klos and J. Bertrandt

231

Determination of Mineral Levels in Walnut Cultivars after Microwave Mineralisation F. Lavédrine, A. Ravel, A. Villet, V. Ducros, and J. Alary

232

Average Intake of Trace Elements in Military Food Services in France P. Le Francois and D. Argaud

233

Use of Haem Iron Concentrate in the Fortification of Weaning Foods C. Martínez, G. Ros, M. J. Periago, and G. López

235

Mineral Content in Four Spanish Fish after Bone Addition Isabel Martínez, Mariá Jesús Periago, Marina Santaella y Gaspar Ros

67. Trace Elements in Austrian Food

237

239

A. Sima, M. Wilplinger, S. Zöchling, S. Heumann, U. Schaller, and W. Pfannhauser 68. Trace Element (Cu, Fe, Zn) Intakes in Cuba

240

T. Verdura, J. Arnaud, P. Fleites, M. Chassagne, A. Favier, R. Perez-Cristia, J. Barnouin, and the SECUBA group 69.

70.

Distribution of Trace Elements and Minerals in Pulp and Peel of Apples and of Persimmons Z. Zachwieja, J. Piotrowicz, M. Folta, H. Barton, S. Gorinstein, M. Zemser, and S. Traktenberg Mineral Elements in the Diet Planned for Consumption for Residents of the Pensioner House in Warsaw J. Bertrandt and A. Klos

242

244

245

71.

Zinc is Scarce and Expensive N. Darmon and A. Briend

72.

Estimated Daily Intakes and Concentrations of Essential Trace Elements in Infant Formulas I. Navarro, J. I. Alvarez, and A. Martin

247

Clinical Evaluation of “Designer Eggs” as a Source of Essential Micronutrients for Humans P. Surai and A. MacPherson

248

73.

xvi

74.

75.

Contents

Plasma Levels of Selenium, Selenoprotein P, and Glutathione Peroxidase and Their Correlations to Fish Intake and Serum Levels of Thyropropin and Thyroid Hormones—A Study on Latvian Fish Consumers L. Hagmar, M. Persson-Moschos, B. Åkesson, and A. Schütz Interlaboratory Comparison Studies into Determination of Metals Content in Foods, Organized by the National Institute of Hygiene Krystyna Starska, Maria Wojciechowska-Mazurek, Elzbieta Brulinska-Ostrowska, and Kazimierz Karlowski

250

252

Trace Elements Bioavailability 76.

Bioavailability of Trace Elements in Human Diet Susan J. Fairweather-Tait

77.

The Influence of Various Food Ingredients and Their Combinations on in Vitro Availability of Iron and Zinc in Cereal Based Vegetarian Meals V. V. Agte, K. V. Tarwadi, and S. A. Chiplonkar

78.

Is Increasing Ascorbic Acid Intake from Foods an Effective Strategy for Improving Dietary Iron Bioavailability? An Evaluation Using Dietary Algorithms, Iron Isotopes, and a Food-Based Community Intervention Trial in Rural Mexico Lindsay H. Allen, Olga Garcia Obregon, Margarita Diaz, Steven Abrams, Suzanne Murphy, and Jorge L. Rosado

255

261

267

78a. Iron Intake from Mixed Meals in Cameroon

A. Bell, F. Ndigui, and P. Effa 79.

80.

81.

Bioavailability of Calcium and Zinc from Various Infant Formulae with and without Thickening Agents Douwina Bosscher, Kristien Van Dyck, Harry Robberecht, Micheline Van Caillie-Bertrand, and Hendrik Deelstra Absorption of Selenium from Biosynthetically Labelled Foods in Humans C. Atherton, T. Fox, S. Fairweather-Tait, J. Dainty, J. Lewis, M. Baxter, H. Crews, and N. Langford Tissue Zinc Uptake as a Measure of the Relative Bioavailability of Supplemental Zinc Sources for Domestic Animals C. B. Ammerman, M. Sandoval, P. R. Henry, R. C. Littell, and R. D. Miles

271

275

279

283

Contents

82.

Effects of Whole Wheat Flour and Fermentable Carbohydrates on Intestinal Absorption of Trace Elements in Rats C. Coudray, H. W. Lopez, M. A. Levrat-Verny, J. Bellanger, C. Rémésy, and Y. Rayssiguier

83.

Bioavailability of Different Sources of Protected Zinc Daniel D. Gallaher, Cynthia M. Gallaher, Stephanie Shulman, Andrea McElhome, Kyle A. Brokken, and Gerry Shurson

84.

Cobalt Deficiency Induced Hyperhomocysteinemia and Oxidative Status of Cattle G. I. Stangl, F. J. Schwarz, and M. Kirchgessner

85.

86.

87.

88.

89.

90.

91.

92.

Role of Diet Related Habits and Cooking Practices on Bioavailability of Iron, Copper, and Zinc and Status of Iron in Vegetarians V. V. Agte, M. Indumadhavi, V. Kakade, S. Palkar, S. Girigosavi, K. Tarwadi, and S. A. Chiplonkar

xvii

287

293

299

305

Dialysability of Calcium, Iron, and Zinc in Beans, Chick Peas, and Lentils V. Sebastiá, R. Barberá, R. Farré, and M. J. Lagarda

306

Effect of Zinc Depletion on Iron Transport across Isolated Duodenal Sacs Peter W. F. Fischer and Bartholomeus Belonge

308

Mineral Element Status in Rats Fed Diets with Extruded or not Extruded Evening Primrose (Oenothera Paradoxa) M. A. Gralak, H. Leontowicz, M. Leontowicz, A. Bogucka-Sciezynska, and G. W. Kulasek Combined Effects of Complex Carbohydrates and a Resistant Protein on Intestinal Absorption and Status of Iron and Zinc in the Rat D. Grizard, C. Coudray, M. Tahiri, J. C. Tressol, Y. Van Doesum, Y. Rayssiguier, and C. Barthomeuf Nutrient Risk Assessment: Implications for Food Fortification Policy Mary R. L’Abbé, Kevin A. Cockell, Sheila Dubois, and William H. Ross Design of Product as Source of Variance in “in Vitro” Mineral Availability of Homogenised Weaning Foods A. B. Olivares, C. Martínez, and G. Ros Iron Dialysability in Enteral Diets C. H. Azevedo, A. P. Galhardo, and C. Colli

309

311

313

314

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Trace Element Status 93.

94.

95.

96.

New Indices for Assessment of Trace Element Status and Requirement, with a Special Focus on Selenium Jean Nève Are Young Women with Low Iron Stores at Risk of Zinc as Well as Iron Deficiency? R. S. Gibson, A-L Heath, N. Prosser, W. Parnell, U. M. Donovan, T. Green, K. E. McLaughlin, D. L. O’Connor, W. Bettger, and C. M. Skeaff Assessment of Femurs and Testis as Parameters for Zinc Bioavailability from Different Food Sources G. S. Henriques, L. F. C. Pedrosa, E. L. Dantas, J. M. de Moura, C. L. Araujo, and S. M. F. Cozzolino The Effects of Low Protein Diet and Light Deprivation on Zinc Status and Gonad Function in Adult Male Rats N. Virgona, M. Kamiyama, T. Yano, and T. Esashi

97.

Zinc Nutritional Status in Obese Children and Adolescents D. N. Marreiro, M. Fisberg, and S. M. F. Cozzolino

98.

Reference Values of Selected Trace Elements in the Serum of Term Newborns from the Urban Area of Rome Alessandro Alimonti, Francesco Petrucci, Francesco Laurenti, and Sergio Caroli

99.

Haptoglobin Polymorphism Association with Magnesium and Lipid Profile in Healthy Teenagers M. Bicho, C. P. Monteiro, L. Sardinha, S. Llobet, P. Marques Vidal, M. J. Halpern, and M. J. Laires

317

323

329

330

331

332

333

100. Urinary Lithium: Distribution Shape, Reference Values, and Evaluation

of Exposure Based on Inductively Coupled Plasma Argon Emission Spectrometry K. Usuda, K. Kono, T. Watanabe, T. Dote, H. Nishiura, M. Shimahara, N. Hashiguchi, and H. Takeishi 101.

102.

Assessment of Nonheme Iron Status in the Whole Blood, Plasma, and Serum: Healthy Neonates and Patients with Iron Overload O. M. Mykhaylyk, N. A. Dudchenko, T. A. Orlova, N. M. Pyasetska, and I. P. Lubyanova Lipid Peroxidation and Zinc and Copper Status in Healthy Adults after Ginseng Ingestion A. Sanchez-Mayoral, R. De Miguel Romera, and L. Perez Gallardo

335

336

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Contents

103.

104.

xix

Variability of Multiple Nutritional Elements in Hair of One Man Over Two Decades L. M. Klevay, D. M. Christopherson, and T. R. Shuler

339

Effect of Magnesium Deficiency on Enterocyte Ca, Fe, Cu, and Zn Content E. Planells, N. Sanchez-Morito, P. Aranda, and J. Llopis

340

105. Serum Copper, Zinc, and Selenium Levels with Regard to Psychological Stress in Men A. Pizent, J. Jurasovic, M. Pavlovic, and S. Telišman 106.

Urinary Iodine and Thyroid Status of New Zealand Residents C. D. Thomson, S. Woodruffe, A. Colls, and T. D. Doyle

107.

Serum Silicon Concentration of Healthy Persons in the Antwerp Region (Belgium) K. Van Dyck, H. Robberecht, R. Van Cauwenbergh, and H. Deelstra

341

343

345

Trace Elements in Pregnancy, Lactation, Growth

347

108.

Zinc and Human Pregnancy Michael Hambidge, Nancy Krebs, and Laura Caulfield

109.

Trace Element Nutrition during Pregnancy and Lactation—Effect on Milk Trace Elements Bo Lönnerdal

353

Glutathione Peroxidase is Not a Functional Marker of Selenium Status in the Neonatal Period L. A. Daniels, R. A. Gisbon, and K. Simmer

359

110.

111.

112.

113.

Trace Element Transfer from the Mother to the Newborn— Investigations on Triplets of Colostrum, Maternal, and Umbilical Cord Sera Michael Krachler, Erich Rossipal, and Dusanka Micetic-Turk Relationship between Trace Elements, Activities of Antioxidant Enzymes in Maternal and Umbilical Cord Blood in Poland W. Wasowicz, J. Gromadzinska, K. Szram, K. Rydzynski, P. Wolkanin, Zb Pietrzak, and J. Tomczak Compensatory Changes in Selenoenzymes in Full Term Human Placentae Induced by Environmental Conditions P. Zagrodzki, L. Zamorska, and M. Zadrozna

365

369

373

xx

114.

Contents

Maternal Anemia at Delivery, Outcome of Pregnancy and Infant Birth Weight, in a Group of Low Socioeconomic Level, in Greater Buenos Aires M. L. de Portela, L. López, S. H. Langini, S. Fleischman, A. Weisstaub, M. García, O. Moreno, and C. R. Ortega Soler

375

115.

Trace Element and Vitamin Deficiencies in French Pregnant Women I. Hininger, M. Favier, H. Faure, J. Arnaud, F. Khatir, J. Thoulon, E. Hariveau, A. Favier, and A. M. Roussel

377

116.

Vinegar Drink to Improve Iron Status during Pregnancy U. Heins, C. Koebnick, and C. Leitzmann

379

117.

Evolution of Copper Contents from Colostrum to Transitional Human Milk M. D. Silvestre, M. J. Lagarda, R. Farré, C. Martinez-Costa, J. Brines, and G. Clemente

118.

119.

120.

121.

Ceruloplasmin Oxidase Expression in the Rowett Hooded Rat and Human Placenta during Pregnancy L. Gambling, W. Bruce-Johnson, R. G. Lea, M. J. Bingham, and H. J. McArdle Anemia and Its Therapy with Peroral Antianemics in Pregnancy and Puerperium M. Mara, V. Eretova, J. Zivny, J. Kvasnicka, A. Umlaufova, and E. Marova

381

383

385

Requirements for the Essential Trace Elements Iron and Zinc during Pregnancy Shahla M. Wunderlich

387

Urinary Selenium in Preterm Infants: Effect of Inorganic Versus Organic Selenium Intake L. A. Daniels, R. A. Gibson, and K. Simmer

388

122.

Trace Element Balance Study in Very Low Birthweight Infants Andrea Raab, Andrea Loui, Peter Brätter, and Michael Obladen

123.

Nutritional Status of Iron of Pre-School Children from the City of Juiz de Fora, MG, Brazil E. L. Chicourel, A. F. Pinto, Ae. C. Lassance, M. Fisberg, and S. M. F. Cozzolino

390

391

Trace Elements in Exercise and Extreme Conditions 124.

Exercise Effects on Trace Element Metabolism Richard A. Anderson

393

Contents

xxi

125. Altitude Related Changes in Red Blood Cell Membrane Lipids and

Proteins. Possible Links with Redox Claus Behn, Manuel Ivan Estrada, Eliseo Hibert Dávila, Oscar Araneda, Max González, Alejandro Carrasco, Rudy Soria, Mauricio Araos, Mercedes Villena, Wilma Téllez, Hilde Spielvogel, Enrique Vargas, Jorge Cajigal, Gloria Celedan, and Gustavo Gonzolez 126.

127.

Effect of Supplementation with Brazil Nuts (CP, Bertholletia excelsa H. B. R.), in Capoeira Players on Selenium (Se) Concentration and Glutathione Peroxidase’s Activity (GSH-Px, E.C.1.11.1.9) V. F. Coutinho, V. B. Bittencourt, and S. M. F. Cozzolino Effects of Weight Loss and Weight-Bearing Exercise on Blood and Organ Concentrations of Lead and Some Essential Metals Francis W. Kemp, Shenggao Han, Wenjie Li, David Tiber, Chenzang Wang, David Sepulveda, Justin Holmes, and John D. Bogden

399

405

407

Trace Elements in Aging 128.

Trace Element Deficiencies and Supplementations in the Elderly Anne-Marie Roussel

129.

Serum and Urine Selenium Changes in a Group of Elderly during One Year of Selenium Supplementation J. Kvícala, V. Zamrazil, and J. Jiránek

130.

Blood and Skin Antioxidant Status in Werner Syndrome (4 patients) M. T. Leccia, M. J. Richard, G. Borla Darve, J. C. Béani, A. Favier, and P. Amblard

131.

Effects of Aluminum on Glutamate Metabolism: A Possible Explanation for Its Toxicity in the Aged Rats and in an Experimental Model of Aluminum Overload Cécile Struys-Ponsar, Olivier Guillard, and Phillippe van den Bosch de Aguilar

132.

133.

Zinc-Induced Excessive Glutamate Release May Cause Accelerated Senescence with Defect in Learning and Memory in Senescence Accelerated Mouse Takeshi Saito, Noriko Nakagawa, Kyoko Takahashi, Dan Li, Yuwako Yamamoto, Hiroko Sasaki, Nozomu Nakamura, Kazuhiro Ogawa, Hiroyoshi Fujita, Masaaki Kurasaki, Masaski Okabe, and Toshiyuki Hosokawa Serum Selenium and Glutathione Peroxidase Activity in Octogenarian and Nonagenarian Subjects in Belfast I. M. Rea, D. McMaster, A. Murphy, and C. Mercer

409

417 421

425

429

433

xxii

134.

135.

Contents

Long Term Tropical Antioxidant Treatment Provides Protection against Clinical Signs of Photoaging L. Vaillant, L. Declercq, D. Malvy, J. C. Béani, J. Bazex, D. Maes, and S. Hercberg Lactulose Stimulates Calcium Absorption in Postmenopausal Women Dose-Dependently E. G. H. M. van den Heuvel, Th. Muijs, W. van Dokkum, and G. Schaafsma

136.

Selenium and Cognitive Decline in the Elderly—The EVA Study C. Berr, B. Balansard, J. Arnaud, A. M. Roussel, and A. Alpérovitch

137.

Does Hormonal Replacement Therapy Influence Antioxidant Status and LDL Oxidizability in Post-Menopausal Women? I. Bureau, F. Laporte, V. Ducros, H. Faure, Y. Rayssiguier, A. Favier, and A. M. Roussel

138.

139.

140.

141.

142.

Metallothionein Expression and Its Significance in the Brain Aging of Dog Akinori Shimada, Masahiko Satoh, and Chiharu Tohyama Vitamin and Mineral Intakes and Biochemical Status in a Spanish Aged Women Group. Effects of Body Mass Index P. Valera, P. Garcia-Garcia, R. M. Ortega, A. Lopez-Sobaler, and A. M. Requejo Biomarkers of Mouse Aging: Modifications of Minerals and Antioxidant Enzymes Heng-Kuan Wong, Jacqueline Riondel, and Alain Favier Does Hormonal Replacement Therapy Influence Trace Element Status in Post-Menopausal Women? I. Bureau, J. Arnaud, R. Anderson, M. J. Richard, F. Laporte, A. Favier, and A. M. Roussel Relationship between Serum Selenium and Red Cell and Plasma Glutathion Peroxidase Levels: Effect of Supplementation with Nutritional Doses of Antioxidants in Elderly and Adult Subjects Paul Preziosi, Josiane Arnaud, Marie-Jeanne Richard, Pilar Galan, Denis Malvy, Serge Briancon, Anne-Marie Roussel, Alain Favier, and Serge Hercberg

437

441

442

443

444

446

448

449

450

Epidemiology of Trace Elements 143.

Intervention Studies on Antioxidant Trace Elements. Special Focus on Selenium Serge Hercberg

453

Contents

144.

Selenium Status in Northern Ireland and France: The Prime Study Dorothy McMaster, Pedro Marques-Vidal, Pierre Ducimetière, Phillipe Amouyel, Dominique Arveiler, Alun Evans

145.

Determinants of Blood Selenium Concentrations in France (SU VI MAX Study) J. Arnaud, A. M. Roussel, P. Preziosi, P. Galan, S. Hercberg, A. Favier, and the SU VI MAX group

146.

147.

Iodine Content of Water and Selenium Level in Corn and Wheat Consumed in an Endemic Goiter Area B. Giray and F. Hincal

471

149.

Effect of Copper Supplementation in Middle Aged People on Plasma Anti-Oxidants and Red Blood Cell Oxidizability: Foodcue Study E. Rock, A. Mazur, Y. Rayssiguier, C. Kehoe, J. M. O’Connor, M. P. Bonham, and J. J. Strain

152.

153.

154.

467

469

Selenium Deficiency in South-West Bohemia J. Kvícala, V. Zamrazil, and V. Jiránek

151.

463

Selenium Status and Antioxidant Enzyme Activities in High School Children from an Endemic Goiter Area B. Giray, T. Teziç, Y. Gedik, A. Öktem, and F. Hincal

148.

150.

xxiii

Plasma and Urine Selenium of Cows from Various Regions of the Czech Republic and Its Comparison with Corresponding Human Population Selenium Indexes J. Kvicala and V. Kroupová A Comparative Study of Blood Antioxidant Parameters in Two Portuguese Urban Populations A. M. Viegas-Crespo, M. C. Santos, M. L. Pavão, P. A. Lopes, and J. Nève

473

475

477

479

Comparison of Trace Elements and Macronutrients in Breast Milk of Women from Eight Different Geographical Locations G. M. Radzanowski, J. Jackson, K. Pramuk, and S. M. Kaup

481

Selenium Concentration in Human Milk and the Daily Se Intake by Breast-Fed Infants in the Western Part of Poland B. A. Zachara and A. Pilecki

482

Effect on Biochemical Markers of Two-Years Supplementation with Antioxidants in the SU.VI.MAX Study: Interest of Nutritional Doses Serge Hercberg, Henri Faure, Paul Preziosi, Josiane Arnaud, Marie-Jeanne Richard, Pilar Galan, Denis Malvy, Serge Briancon, Anne-Marie Roussel, and Alain Favier

484

Contents

xxiv

155.

156.

Iodine Nutrition of French Adults Issued from the SU.VI.MAX Cohort Pierre Valeix, Marjorie Zarebska, Paul Preziosi, Bruno Pelletier, and Pilar Galan Dietary Iron Intake and Iron Status of French Adults Participating in the SU.VI.MAX Cohort Pilar Galan, Paul Preziosi, Bernadette Fieux, Marjorie Zarebska, Serge Briancon, Denis Malvy, Anne-Marie Roussel, Alain Favier and Serge Hercberg

486

488

III. Trace Elements in Human Diseases Diabetes 157.

Insulin Enhancing Effects of Vanadium John H. McNeill

491

158.

Is Zinc Essential to Modulate Insulin Sensitivity? Patrice Faure, Serge Halimi, Sophie Bouvard, Olivier Ramon, Karine Lalane, Anne Marie Roussel, and Alain Emile Favier

497

159.

Role of Chromium in Glucose Intolerance, Diabetes, Total Parenteral Nutrition, and Body Composition Richard A. Anderson, William Cefalu, Khursheed N. Jeejeebhoy, and Gilbert R. Kaats

160.

Evaluation of Zinc in Children with Type 1 Diabetes Mellitus L. P. C. Pedrosa, A. Spínola-Castro, M. Matsumoto, J. Len, F. Schwartzman, L. P. Camargo, and S. M. F. Cozzolino

161.

Zinc Sulphate Induced Metallothionein in Pancreatic Islets of Mice and Protected from Streptozotocin-Induced Diabetes Patricia Ohly, Claudia Dohle, Josef Abel, and Helga Gleichmann

162.

163.

164.

Water Content of Minerals Associated with Type 2 Diabetes Mellitus of Samples Collected on the Navajo Reservation Judith Hallfrisch, Claude Veillon, Kristine Patterson, A. David Hill, Irene Benn, Bessie Holiday, Ruby Ross, Sylvia Zhonnie, Frances Price, and Ann Sorenson

503

511

515

519

Dietary Copper Deficiency Causes Elevation of Early and Advanced Glycation End-Products J. T. Saari and G. M. Dahlen

523

The Use of Stable Isotope Tracers to Explorate Zinc and Selenium Metabolism in Insulin Dependent Patients M. Bertouze, P. Faure, V. Ducros, S. Halimi, and A. Favier

527

Contents

165.

166.

167.

168.

169.

170.

171.

172.

xxv

Zinc Modulates the Glucose Induced Rat Aortic Smooth Cell Proliferation S. Bouvard, P. Faure, A. Favier, M. Leconte, and S. Halimi Metabolic Effects of Dietary Chromium-L-Methionine in Horses and Beef Cattle M. T. Socha, S. L. Ralston, R. Raub, E. B. Kegley, C. K. Swenson, A. B. Johnson, and T. M. Fakler Zinc Protects HeLa Cells against the Glucose Induced Cytotoxic Effect P. Faure, S. Bouvard, A. Favier, and S. Halimi Oxidative Stress and Antioxidant Trace Element Status in Patients with Non Insulin-Dependent Diabetes A. Kerkeni, A. M. Roussel, A. Othmane, S. Majhoub, N. Zouari, F. Najjar, J. Arnaud, A. Favier, and R. A. Anderson Zinc Deficiency Increases the Deleterious Effect of High Fructose Diet on Fetal Development and Free Radical Activity in Rats B. Lachil, P. Faure, C. Ribuot, J. Arnaud, M. J. Richard, M. Sève, and A. Favier Zinc 100 ppm Does Not Improve Insulin Sensitivity of High Fructose Fed Rats Leading to Insulin Resistance E. Rossini, S. Bouvard, M. J. Richard, S. Halimi, and A. Favier Design of New Oxovanadium (IV) Complexes for Treatment of Diabetes. Bioavailability, Speciation, and Tissue Targeting Considerations K. H. Thompson, J. H. McNeill, and C. Orvig The Metallic Component of the Glucose Tolerance Factor, G. T. F.: Cobalt against Chromium F. Silió, A. Santos, and B. Ribas

528

530

532

534

536

538

539

540

Cardiovascular Diseases and Oxidative Stress 173.

The Interaction between Dietary Copper and Excess Iron—Increases the Risk of Heart Disease Meira Fields and Charles G. Lewis

174.

Selenium and Chronic Heart Failure Michel de Lorgeril, Patricia Salen, and Michèle Accominotti

175.

Antioxidant Status of Patients with Unstable Angina Pectoris and Acute Myocardial Infarction Momcilo B. Mihailovic, Zorana Vasiljevic, Sladjana Sobajic, Ivan B. Jovanovic, Olivera Pesut, and Gordana Matic

543 547

551

xxvi

176.

177.

178.

Contents

The Effect of Zinc Deficiency on Parameters of Lipoprotein Metabolism and Lipolysis in Rats Fed Different Fats K. Eder, S. Wild, and M. Kirchgessner

555

Plasma Level of Antioxidant Minerals (Cu, Zn, Mn, and Se) and Fe: A Comparison of Patients with Cerebro-vascular Disease and Healthy Adults in Korea I. S. Kwun, H. S. Jang, and C. S. Kwon

559

Trace Elements as Determined by ICP-MS Analysis in Patients with Coronary Artery Disease Ellen Burgess, Robert Audette, Merril Knudtson, and George Wyse

563

179. The Effect of Zinc Deficiency on Some Key Enzymes of Lipid Metabolism in Rats Fed Olive Oil or Linseed Oil K. Eder, K. Waldhauser, and M. Kirchgessner 180.

181.

182.

183.

Lipid Concentrations of Low-Density Lipoproteins and Their Oxidative Susceptibility in Zinc-Deficient Rats K. Eder and M. Kirchgessner Comparative Evaluation of Trace Elements Contents in Aortic Wall and in Hair Samples of Young Males A. V. Skalny, A. A. Zhavoronkov, A. L. Chernyaev, and A. V. Koudrine Heart Disease Risk in the UK as Influenced by Environmental Factors and Defined by Hair Calcium Concentration A. MacPherson and J. Bacsó Regulation of Hepatic Cholesterol and Lipoprotein Metabolism in Copper-Deficient Rats A. Mazur, C. Sérougne, C. Moundras, B. Bayle, F. Millat, and Y. Rayssiguier

184.

Copper Body Status and Cardiovascular Diseases G. Mielcarz

185.

Increased Plasma Glutathione Peroxidase Activity in Patients with Acute Myocardial Infarction B. A. Zachara, M. Ukleja-Adamowicz, J. Lecka, and E. Nartowicz

565

566

568

569

571

573

574

Other Human Diseases 186.

Selenium in Cancer Prevention M. P. Rayman and L. C. Clark

575

187.

Trace Elements in Hypertension Gianfranco Vivoli, Margherita Bergomi, Paola Borella, and Sergio Rovesti

581

Contents

188.

189.

190.

191.

192.

193.

194.

195.

196.

197.

Persistence of Goiter Despite Oral Iodine Supplementation in Goitrous Children with Iron-Deficiency Anemia in the Côte d’Ivoire Michael Zimmermann, Pierre Adou, Toni Torresani, Christophe Zeder, and Richard Hurrell Trace Elements in Patients Treated by On-Line Hemodiafiltration. The Blood Chromium Behaviour A. Baj, G. Bregante, R. D’Angelo, G. Bonforte, F. Toffoletto, and M. Surian Relationships between Trace Element Nutriture and Progression of Human Immunodeficiency Virus Infection John D. Bogden, Joan H. Skurnick, Francis W. Kemp, Shenggao Han, Joan Lloyd, Herman Baker, and Donald B. Louria Iron and Manganese Homeostasis in Chronic Liver Disease: Relationship to Pallidal T1-Weighted Magnetic Resonance Signal Hyperintensity Elise A. Malecki, Attila G. Devenyi, Todd F. Barron, Timothy J. Mosher, Paul Eslinger, Claire V. Flaherty-Craig, and Lorenzo Rossaro Effect of Depression and of Antidepressant Therapy on Serum Zinc Levels—A Preliminary Clinical Study Malgorzata Schlegel-Zawadzka, Andrzej Zieba, Dominika Dudek, Miroslaw Krosniak, Maria Szymaczek, and Gabriel Nowak Abnormalities of Iron Homeostasis in the Pregnancy Syndrome Pre-Eclampsia M. P. Rayman, J. Barlis, S. Sokari, R. W. Evans, C. W. G. Redman, and L. J. King

xxvii

587

591

597

601

607

611

Lipid Peroxidation in Patients with Diseases Known to Affect Trace Element Status Mariana Vlad, P. J. Porr, G. Uza, and Maria Zirbo

617

Selenium Status of Breast and Gastroinstestinal Cancer Patients in Turkey B. Avsar and I. G. Gökmen

623

Zinc and Selenium Intake in Nondialysed Patients with Chronic Renal Failure D. Mafra, D. I. T. Favaro, and S. M. F. Cozzolino

625

Zinc Nutritional Status in Patients with Chronic Renal Failure, Residents in São Paulo, Brazil D. Mafra, L. Cuppari, and S. M. F. Cozzolino

626

xxviii

198.

199.

200.

Contents

Iron, Zinc, Copper Levels of Thalassemia Patients of Northern Cyprus R. Oktekin and G. Gökmen Blood Markers of Oxidative Stress in Patients with Amyotrophic Lateral Sclerosis D. Bonnefont-Rousselot, M. C. Jaudon, L. Lacomblez, B. Bourely, V. Doppler, C. Bizard, F. Salachas, G. Bensimon, J. Delattre, and V. Meininger Glutathione Peroxidase Activity Is Reduced in Haemodialysis Patients H. E. Roxborough, C. M. Loughrey, C. Mercer, D. McMaster, and I. S. Young

627

629

631

633

201.

Copper and Zinc Levels in Patients with Colorectal Polyps K. Linke, G. Mielcarz, and K. Zietek

202.

Total Superoxide Dismutase Activity and Glutathione Peroxidase Activity in Plasma of Phenylketonuric Subjects Supplemented with Selenium M. Calomme, P. Cos, V. Ramaekers, B. François, M. Van Caillie-Bertrand, and D. Vanden Berghe

634

The Content of Selected Bioelements in Hair of Children with Infantile Cerebral Palsy (Paralysis Cerebralis Infantilis, Diplegia) R. W. Wójciak and Z. Krejpcio

636

203.

638

204.

Serum Copper in Juvenile Rheumatoid Arthritis O. M. S. Amancio, D. M. A. Chaud, and M. O. E. Hilário

205.

Serum Copper in Juvenile Rheumatoid Arthritis: Influence of Dietary Copper D. M. A. Chaud, O. M. S. Amancio, and Hilário M. O. E.

639

Gallium Nitrate and Zinc Content in Peripheral Blood Lymphocytes of Patients with Lung Cancer A. V. Koudrine and A. V. Skalny

640

206.

207.

208.

Antioxidant Systems in Normal and Preeclamptic Algerian Pregnant Women Brahim Lachili, Josiane Arnaud, Henri Faure, Marie Jeanne Richard, Anwar Bouabsa, Anne Marie Roussel, and Alain Favier Effect of Erythropoietin Therapy and Selenium Supplementation on Some Antioxidant Parameters in Blood of Uremic Patients on Long-Term Hemodialysis B. A. Zachara, A. Adamowicz, U. Trafikowska, A. Pilecki, A. Trafikowska, E. Nartowicz, and J. Manitius

641

643

Contents

xxix

Trace Element Toxicity and Environmental Exposure 209.

210.

211.

Arsenic Groundwater Contamination and Sufferings of People in West Bengal-India and Bangladesh U. K. Chowdhury, B. K. Biswas, T. Roy Chowdhury, B. K. Mandal, G. Samanta, G. K. Basu, C. R. Chanda, D. Lodh, K. C. Saha, D. Chakraborti, S. C. Mukherjee, S. Roy, S. Kabir, and Q. Quamruzzaman Heavy Metals and Persistent Organic Pollutants in Newborn Correlated with Maternal Smoking Bente Deutch and Jens C. Hansen

651

Copper and Early Childhood Cirrhosis (ECC)—A Retrospective Study in Germany H. H. Dieter, W. Schimmelpfennig, E. Meyer, and M. Tabert

655

212.

Difference in Penetration of Metals into the Brain Takeshi Minami, Yuko Sakita, Yuko Okazaki, and Ryouhei Amano

213.

Exposure to Platinum-Group Metals Released by Automotive Catalytic Converters: The Case of Urban Youngsters S. Caroli, F. Petrucci, B. Bocca, M. Krachler, F. Forastiere, and A. Alimonti

214.

645

Urinary Copper and Mortality among Inhabitants Living in a Cadmium Polluted Area in Japan M. Nishijo, H. Nakagawa, Y. Morikawa, M. Tabata, K. Miura, K. Higashiguchi, T. Seto, T. Kido, K. Nogawa, K. Mizukoshi, and M. Nishi

663

667

671

675

215.

Serum Selenium in Relation to Biomarkers of Lead in Men J. Jurasovic, A. Pizent, and S. Telišman

216.

Hepatotoxic Effects Associated to Short and Long Term Exposure to Excess Dietary Copper in Rats E. Aburto, A. Cribb, and C. Fuentealba

679

The Biological and Medical Importance of the Interactions between Nickel and Zinc, Magnesium and Manganese in Vivo M. Anke, A. Trüpschuch, and G. Gunstheimer

685

Effect of Aluminum on Manganese Superoxide Dismutase (MnSOD) Expression C. Garrel, C. Carron, and A. Favier

687

217.

218.

219.

Indicators for Sensitisation to Ni, Co, and Cr, a Eu-Project Olof Vesterberg

688

xxx

220.

221.

222.

223.

224.

225.

226.

227.

Contents

Fish Consumption and Mercury Speciation in Hair of Indigenous Population of the Amazon Antonio C. Barbosa, José G. Dórea, Jurandir R. Souza, and Glauce L. Oliveira Metabolism of Phospholipids in Pituitary Gland: Effect of Ion Cadmium Ana María Calderoni and María Sofía Giménez Investigations on the Elements Status Markers in Humans. Lead Significant Correlation between Pb Content in Milk Teeth and Blood of 6-Year-Old Children H. Barton, Z. Zachwieja, and M. Folta Morphologic and Biochemical Assessment of the Role of Lipid Peroxidation in the Pathogenesis of Copper Toxicity in CopperLoaded Rats E. Aburto, A. Cribb, and I. C. Fuentealba Effect of Dolomite and Thiamine Supplementation on Tissue Trace Elements Concentrations in Lead-Intoxicated Rats Z. Krejpcio, D. Olejnik, R. W. Wojciak, and J. Gawêcki Acute Human Molybdenum Toxicity from a Dietary Molybdenum Supplement—A New Member of the “Lucor Metallicum” Family Berislav Momcilovic

690

691

693

695

697

699

Effects of Lead on the Maturation of the Female Gamete and Fertility B. Lefèvre, J. Poupon, A. Pesty, V. Machelon, and J. Testart

701

Rhamnogalacturonan II (RG-II), a Prectic Polysaccharide Present in the Vegetable-Derived Products, Strongly Decreases Lead Tissue Accumulation: Potential Interest in Chronic Lead Exposure M. Tahiri, P. Pellerin, J. C. Tressol, T. Doco, Y. Rayssiguier, and C. Coudray

703

228.

Dietary Exposures to the Platinum Group Elements G. E. Ysart, P. F. Miller, H. Crews, P. Robb, M. Baxter, C. De L’argy, S. Lofthouse, C. Sargent, and N. Harrison

229.

Acute and Chronic Effects of Acid Phenol Compounds on Aluminum Retention in Rats Z. Deng, L. Gouzoux, C. Coudray, A. Mazur, Y. Rayssiguier, and D. Pépin

705

707

Contents

230.

xxxi

Determination of Total and Soluble Titanium in Soft Tissues Covering Titanium Microplates in Stomatology: An Analytical Study J. Poupon, J. P. Méningaud, C. Chenevier, M. Galliot-Guilley, and J. Ch. Bertrand IV.

231.

232.

233.

234.

235.

236.

237.

238.

239.

240.

709

TRACE ELEMENTS IN ANIMALS

Tissue Selenium Levels in Laying Hens Are Influenced by Dietary Oils and Fatty Acids Including Trans Fatty Acids Klaus Schäfer Dietary Mo as an Antagonist to Cu Absorption: Stable Isotope Measurements in Grazing Sheep S. O. Knowles, N. D. Grace, J. R. Rounce, A. Litherland, D. M. West, and J. Lee

711

717

Copper-Associated Cirrhosis in North Ronaldsay Sheep: A Possible Model for Idiopathic Copper Toxicosis S. Haywood, T. Müller, W. Müller, and Z. Dincer

723

Dietary Copper Affects Lipid and Cholesterol Metabolism in Finishing Steers T. E. Engle, J. W. Spears, C. L. Wright, and T. A. Armstrong

729

A Comparison of Two Methods of Copper Supplementation for Grazing Red Deer (Cervus Elaphus) D. V. Illingworth, D. W. Jackson, N. R. Kendall, and S. B. Telfer

733

Trace Elements and Vitamins in Blood of Alpacas (Lama pacos) and Sheep Grazing the Same Pasture G. J. Judson, B. A. McGregor, and A. M. Howse

737

A Comparison of the Efficacy of Proprietary Products in the Treatment of Molybdenum Induced Copper Deficiency N. R. Kendall, C. Middlemas, H. Maxwell, F. Birch, D. V. Illingworth, D. W. Jackson, and S. B. Telfer The Effect of a Copper, Cobalt, and Selenium Bolus on Sheep from Three Upland Scottish Farms A. M. Mackenzie, N. R. Kendall, D. V. Illingworth, D. W. Jackson, I. M. Gill, and S. B. Telfer

741

749

Selenium and Fluoride Toxicology—Water Quality Guidelines for Dohne Merino Sheep in Southern Africa J. A. Meyer and N. H. Casey

753

Effect of Dietary Copper Level and High Sulfate Water on Copper Metabolism and Growth in Cattle C. L. Wright, J. W. Spears, T. E. Engle, and T. A. Armstrong

759

xxxii

241.

242.

243.

244.

245.

246.

247.

248.

249.

250.

Contents

Apparent Trace Element Absorption in Growing Pigs Fed Rations of Increasing Calcium Carbonate Content Torben Larsen, José A. Fernandéz, and Ricarda M. Engberg

763

Heat Processing of Protein Sources on the Faecal Excretion of Their Selenium in Dairy Cows J. B. J. van Ryssen and G. E. Schroeder

767

Dietary Antagonists to Copper Absorption: Stable Isotope Measurements in Grazing Ruminants J. Lee, N. D. Grace, J. R. Rounce, and S. O. Knowles

768

The Effect of a Zinc, Cobalt, and Selenium Bolus on Ram Semen Quality and Trace Element Status N. R. Kendall, A. Green, S. McMullen, and R. G. Rodway

769

The Effect of Lead on the Rate of Fermentation of Herbage by Rumen Micro-Organisms S. T. Strojan and C. J. C. Phillips

772

Effects of Intraruminal Monensin and Selenium Capsules on Glutathion Peroxidase, Calcaemia, Phosphatemia, and Live Weight Gain in Heifers on Pasture with Solanum Malacoxylon Bruna E. Ruksan, Laura Marangunich, and L. Barry Lowe

775

Trace Element Content in Natural and Commercial Cat’s Food and Trace Element Status of Cats Depending on Sex and Disease S. Anke, H. Gürtler, and M. Anke

777

Validation and Normal Ranges of Plasma Ceruloplasmin Concentration in Cats and Dogs C. J. Charlton, S. L. Wattam, and N. D. Skinner

779

Current AAFCO and NRC Recommendations for Selenium Are Too Low for Puppies K. J. Wedekind, J. DeBraekeleer, and G. F. Combs

781

The Effect of a Zinc, Cobalt, and Selenium Bolus on Sheep from Nine Upland Scottish Farms N. R. Kendall, A. M. Mackenzie, D. V. Illingworth, D. W. Jackson, I. M. Gill, and S. B. Telfer

251.

Field Trials of A Copper, Cobalt, and Selenium Soluble Glass Bolus N. R. Kendall, A. M. Mackenzie, and S. B. Telfer

252.

Evaluation of the Trace Mineral Status of Lactating Dairy Cattle in Trans NZOIA District Western Kenya F. D. O. Oduor, I. O. Jumba, and S. O. Wandiga

782

784

786

Contents

253.

xxxiii

Canine Ferritin: Assay Validation and Normal Range for Serum N. D. Skinner, C. J. Charlton, and S. L. Ebbrell

787

V. INDIVIDUAL TRACE ELEMENTS Iron 254.

Iron in Infancy: Absorption, Erythrocyte Incorporation, Loss Robert E. Serfass

789

255.

Transport of Iron in Health and Diseases Andreas Rolfs and Matthias A. Hediger

795

256.

Benefits and Problems Associated with Iron Supplementation and Fortification Serge Hercberg

257.

258.

259.

260.

261.

Low Iron Diet and Cadmium Exposure Disrupt Steroidogenesis in the Rat Martina Piasek, John W. Laskey, Krista Kostial, Maja Blanuša, and Janet M. Ferrell

799

809

Peculiarities of Nonheme Iron Metabolism upon Experimental Modelling of Rat Glial Brain Tumour. Perspectives for Diagnosis and Treatment Olga M. Mykhaylyk, Natalie A. Dudchenko, Eugene A. Lebedev, and Bogdan S. Shurunov

813

Iron Bioavailability from Iron Amino Acid Chelate or Ferrous Sulfate for Treatment of Iron Deficiency Anemia Oscar Pineda, H. DeWayne Ashmead, and Alain Bourdonnais

819

The Effect of a High Intake of Whole Wheat Bread Produced with and without Phytase on Iron Status in Young Women M. Hansen, B. A. Jorgensen, and S. Sandström

820

Zinc Status in Pregnancy Assessed by Hair Analysis—Nutritional Influences U. Heins, C. Koebnick, and C. Leitzmann

821

262.

In Vitro Properties of Ferrous and Ferric Ions with Phytate D. Oberleas

263.

Iron Metabolism in Rats Consuming Olive Oil or Sunflower Oil Unused or Used in Repeated Potato Frying A. M. Pérez-Granados, M. P. Vaquero, and M. P. Navarro

823

825

xxxiv

264.

265.

Contents

The Ferritin Secreted by Cultured Hepatoma Cells in Response to Iron and Inflammatory Cytokines Strongly Resembles the Ferritin in Serum and Has Homology to Intracellular Ferritins as Well as Immunoglobulins L. Butcher, M. Hazegh-Azam, A. Nguyen, P. Nguyen, K. Vu, L. Rezaee, C. Juska, K. Schaffer, M. Zamany, L. Estevez, M. Hallock, and M. C. Linder Two Levels of Iron Supplementation and Developmental Outcome, Iron Nutrition, and Adverse Results in Low Birth Weight Infants J. K. Friel, P. F. Kwa, B. Simmons, K. Aziz, C. Mercer, A. MacDonald, and W. L. Andrews

827

829

Selenium 266.

Some Functions of the Essential Trace Element, Selenium Thressa C. Stadtman

267. Characteristics and Function of Selenoprotein P Raymond F. Burk and Kristina E. Hill 268.

Some Biochemical Functions of Selenium in Animals and Man J. R. Arthur and G. J. Beckett

269.

The Secis Element in Selenoprotein mRNAS. A Rosetta Stone for Decoding the UGA Selenocysteine Codon and a Molecular Tag for Unveiling New Selenoproteins Alain Lescure, Daniel Gautheret, Robert Walczak, Philippe Carbon, and Alain Krol

270.

271.

272.

273.

Prolonged Stimulation of Rats with Adrenocorticotrophic Hormone (ACTH) Alters Concentration of Selenium in the Kidney: Reversibility and Possible Mechanism B. Cheng, T. S. Srikumar, F. M. Al-Awadi, J. Thakkar, and F. Sequeira Dietary Selenium Intake and Antioxidant Defenses in Tissues of Peripuberal Rats Maria Cristina Santos, Jean Nève, Maria Leonor Pavão, and Ana Maria Viegas-Crespo Selenoprotein P in Plasma in Relation to Cancer Morbidity in MiddleAged Swedish Men Marie Persson-Moschos, Lars Stavenow, Björn Åkesson, and Folke Lindgärde Effect of Selenium Supplementation on Plasma Selenoprotein P of New Zealanders Christine D. Thomson, Anna J. Duffield, and Kristina E. Hill

831 837 843

849

855

859

865

869

Contents

274.

275.

276.

277.

278.

279.

280.

Tracking the Metabolism and Excretion of Selenium in Real Time in Humans G. Bellisola, M. Colombatti, G. Fracasso, F. Pasti, M. Valdes, and A. Torboli Changes in Indices of Selenium Status in Men on Low, Medium, and High Intakes T. Fox, C. Atherton, S. Fairweather-Tait, J. Dainty, J. Lewis, M. Baxter, and H. Crews

877

883

The Oral Intake of Antioxidant Nutrient Associated with Selenium Does Protect Skin against Actinic Exposure Jean Pierre Césarini and S. Demanneville

887

Bioavailability of Selenium from Untreated and Processed Selenomethionine-Enriched Fillets of Atlantic Salmon (Salmosalar) R. Oernsrud and M. Lorentzen

891

Stability of Glutathione Peroxidase Activity during Storage and Heat Treatment of Whey H. Lindmark Månsson, J. Chen, and B. Åkesson

892

Purification and Enzyme-Linked Immunoassay of Bovine Extracellular Glutathione Peroxidase H. Lindmark Månsson and B. Åkesson

893

Selenium Distribution in Liver of Hens Fed with Sunflower Oil I. Falnoga, V. Stibilj, D. Mazej, and M. Tušek Znidaric

282.

Selenium and Lipoproteins: Preliminary in Vivo and in Vitro Observations in Humans V. Ducros, F. Laporte, N. Belin, A. David, A.-M. Roussel, and A. Favier

284.

873

True Absorption, Excretion, and Tissue Retention of Selenium at Widely Varying Selenium Supply to Rats W. Windisch and M. Kirchgessner

281.

283.

xxxv

895

897

Transforming Growth Inhibits Selenoprotein P Expression in Cultured Human Liver Cells V. Mostert, I. Dreher, J. Köhrle, and J. Abel

899

Experimentally Induced Selenium and Vitamin E Deficiency in Growing Rabbits J. Pallauf, A. Müller, and Erika Most

901

xxxvi

285.

286.

287.

Contents

Identification of 5 Novel Selenoproteins Based on RNA Structural Tags A. Lescure, D. Gautheret, P. Carbon, and A. Krol

903

Tat Protein of HIV1 Decreases Selenoglutathione Peroxidase and Increases Uv-A Cytotoxicity Independently of Selenium Supplementation M. J. Richard, P. Guiraud, and A. Favier

904

Serum Selenium Concentrations and the Acute Phase Response T. M. T. Sheehan and Aleha Khatun

906

Copper 288.

Neurochemical Alterations following Perinatal Copper Deficiency in Rodents Joseph R. Prohaska

289.

Molecular Analysis of Wilson Disease Gene in French Patients I. Ceballos-Picot, A. Nicole, B. Aral, C. Franvel, C. Soulié, H. Lassal, F. Woimant, M. Haguenau, and P. Chappuis

290.

Defining Optimal Copper Status in Humans: Concepts and Problems J. J. Strain

291.

Dietary Copper, Vanadate, Lysyl Oxidase Activity, and Lysine Tyrosyl Quinone Formation Robert B. Rucker, Changtai Cui, Eskouhie H. Tchaparian, Alyson E. Mitchell, Michael Clegg, Janet Y. Uriu-Hare, Brian Rucker, Tracy Stites, and Carl L. Keen

292.

Copper Metabolism in the Rodent Kidneys Masaaki Kurasaki, Masashi Okabe, Mika Suzuki-Kurasaki, Shigeru Saito, Toshiyuki Hosokawa, Osamu Yamanoshita, and Takeshi Saito

293.

Effect of Copper in the Food Chain on Human Health (FOODCUE: Fair Ct95-0813) E. Rock, J. J. Strain, J. M. O’Connor, M. P. Bonham, Y. Rayssiguier, A. Mazur, B. Sandström, S. Hodjberg-Bugel, S. J. Fairweather-Tait, I. J. Harvey, G. Majsak-Newman, A. Flynn, K. Cashman, A. Baker, F. Ginty, J. H. Beattie, I. Bremner, M. D. Reid, G. Rotilio, M.-L. Scarino, Y. Sambuy, S. Ferruzza, M. R. Ciriolo, and A. DeMartino

909

917

923

929

933

937

Contents

294.

295.

296.

297.

298.

299.

xxxvii

The Effect of Copper Supplementation on Putative Indices of Body Copper Status and on Oxidative and Inflammatory Measures (FOODCUE Project) J. M. O’Connor, M. P. Bonham, E. Turley, C. Kehoe, J. S. Coulter, M. S. Faughnan, A. McKeown, V. J. McKelvey-Martin, E. Rock, Y. Rayssiguier, A. Mazur, A. Flynn, K. Cashman, A. Baker, and J. J. Strain

943

Role of Ceruloplasmin in Fe Efflux from Placenta; Identification of an Endogenous Cu Oxidase in Human Placental Cells (BeWo) Ruth Danzeisen and Harry J. McArdle

947

Dietary Molybdenum: Effect on Copper Absorption, Excretion, and Status in Young Men Judith R. Turnlund and William R. Keyes

951

Identity and Regulation of the Copper Transport Protein, Transcuprein N. Liu, L. Lo, T. Tran, L. Jones, and M. C. Linder

955

Response of Diamine Oxidase and Other Copper Status Biomarkers to Modifications in Dietary Copper in the Rat C. Feillet-Coudray, A. Mazur, C. Coudray, E. Rock, and Y. Rayssiguier

957

Effect of Dietary Copper Intake on Biochemical Indices Associated with Copper Metabolism L. Harvey, G. Majsak-Newman, S. Fairweather-Tait, A. Baker, K. Cashman, and A. Flynn

959

Zinc 300.

Metallothioneins: Their Cellular Function and Relationship with Zinc John H. Beattie and Ian Bremner

961

301.

Zinc as a Cellular Regulator of Apoptosis Alain Favier

969

302.

Zinc and Growth Michael Hambidge and Nancy Krebs

977

303.

Zinc Nutriture as Related to Brain Harold H. Sandstead, Christopher J. Frederickson, and James G. Penland

981

304.

Zinc in Preterm and Term Infants—Parallel Studies on Metabolic Balances and Plasma Concentrations E. Sievers, U. Schleyerbach, T. Arpe, D. Garbe-Schönberg, and J. Schaub

987

Contents

xxxviii

305. Activity of Calmodulin-Regulated Enzymes in Tissues of Zinc-Deficient

Rats H.-P. Roth, S. Schmidmayer, and M. Kirchgessner

306. Zinquin Ester—A Reagent for the Investigation of the Role of Available Zn(II) in Living Systems David Ward, Stephen Lincoln, Henry Betts, Peter Zalewski, Ian Forbes, Indumathy Mahadevan, Marc Kimber, and Kym Hendrickson 307.

308.

309.

310.

311.

312.

313.

314.

315.

Cognitive Effects of Adaptation to a Low Zinc Diet in Healthy Men M. J. Kretsch, A. K. H. Fong, J. G. Penland, B. Sutherland, and J. C. King Intracellular Zinc Chelation Induces Apoptosis, Caspases Activation, and Transcription Factors Degradation in Jurkat and HeLa Cells Sève Michel, Chimienti Fabrice, Richard Sandrine, Mathieu Jacques, and Favier Alain Effect of Zinc Supplementation on Biological Parameters of Bone Turnover in Healthy Men Anne Peretz, Pierre Bergmann, Théodore Papadopoulos, Vania Siderova, and Jean Néve

991

995

999

1003

1009

Betaine-Homocysteine S-Methyltransferase is an Abundant Zinc Metalloenzyme in Liver Andrew P. Breska III and Timothy A. Garrow

1013

Nourishment: To Moderate Constantly or to Indulge Occasionally—The Message of Zinc Dose-Rate Idiorrhythm to the Needy Berislav Momcilovic

1017

Concentration of Growth Hormone, IGF-1, Insulin and C-Peptide in Rat Serum during Development of Alimentary Zinc Deficiency H.-P. Roth and M. Kirchgessner

1021

Moderately High Zinc Intake Impairs Verbal Memory of Healthy Postmenopausal Women on a Low Copper Diet James G. Penland, David B. Milne, and Cindy W. Davis

1025

Identification of the Zinc Binding Protein in a Child with Hyperzincaemia as Calprotectin (MRP8/MRP14) B. Sampson, P. Richmond, B. E. Golden, M. K. Fagerhol, J. H. Beattie, and I. Z. Kovar Characterisation of the Different Steps during Zinc Chelation Induced Apoptosis in Jurkat and HeLa Cells F. Chimienti, M. Sève, S. Richard, J. Mathieu, and A. Favier

1031

1035

Contents

316.

317.

318.

319.

xxxix

Effect of Ion Zinc on the Metabolism of Phosphatidylcholine and Sphingomielin in Lung of Rat N. N. Gomez, M. R. Fernandez, M. S. Ojeda, and M. S. Gimenez

1036

Metallothionein, Calmodulin, and Trace Elements in Healing Skin Wounds B. Sampson, A. Lansdown, and A. Rowe

1038

Zinc Modulation of Macrophages Interleukin 1 Gene Expression and Secretion A. E. Aguilar, R. Pastelin, S. Pérez, and M. D. Lastra

1039

Assessment of Zn Role in Murine Experimental Cysticercosis Caused by Taenia crassiceps M. D. Lastra, E. Sciutto, R. Pastelin, A. E. Aguilar, and G. Fragoso

1040

Boron 320.

321.

322.

323.

The Dogged Path to Acceptance of Boron as a Nutritionally Important Mineral Element Forrest H. Nielsen

1043

Homing in on the Molecular Basis of Boron Essentiality Using Differential Display, Gene Arrays, and Northern Blots C. D. Eckhert, A. Bennett, K. Becker, and D. Luo

1049

Estimation of Dietary Boron Intake in Six Countries: Egypt, Germany, Great Britain, Kenya, Mexico, and the United States Charlene Rainey, Leslie Nyquist, Jennifer Casterline, and Dena Herman Nutritional Essentiality of Boron for Development, Maturation, and Reproduction in Frogs—A Review D. J. Fort, R. L. Rogers, E. L. Stover, P. L. Strong, and F. J. Murray

1053

1057

1061

324.

Effects of Boron in Wound Healing: Experiments on Nude Mice Brigitte Dousset, Mohamed Benderdour, Ketsia Hess, Rosine Mayap-Nzietchueng, Francine Belleville, and André Duprez

325.

Effect of Dietary Boron on Bone Characteristics and Plasma Parameters in Young Pigs T. A. Armstrong, J. W. Spears, T. E. Engle, and C. L. Wright

1067

Dietary Boron Is a Physiological Regulator of the Normal Inflammatory Response Curtiss D. Hunt

1071

326.

xl

Contents

327.

Boron Stimulated Yeast Differential Gene Expression A. Bennett, V. Gilman, N. Soch, D. Luo, and C. D. Eckhert

1077

328.

Identification by Gene Arrays of Boron Responsive Genes in Yeast K. Becker, A. Bennett, and C. D. Eckhert

1078

329.

Limb Teratogenesis Induced by Chronic Boron or Copper Deficiency in Xenopus D. J. Fort, E. L. Stover, F. J. Murray, and P. L. Strong

1079

An Assessment of Fertility in Boron-Exposed Turkish Subpopulations: An Epidemiological Approach M. Çöl, B. S. Sayli, Y. Genç, E. Erçevik, A. H. Elhan, and A. Keklik

1080

330.

331.

Photoreceptor Dystrophy in Boron Depleted Zebrafish S. Dohs and C. D. Eckhert

332.

Adverse in Vitro Development of Preimplantation Mouse Embryos Exposed to Low Boron Diet and Low Boron L. Lanoue, F. J. Murray, P. L. Strong, and C. L. Keen

333.

The Temporal Effects of Low Dietary Boron (B) on Tissue B Concentrations in Rats J. Y. Uriu-Adams, B. J. Horwath, F. J. Murray, G. Downing, P. Strong, and C. L. Keen

1082

1083

1085

334.

Boron Stimulates Yeast Growth A. Bennett, N. Soch, R. Rowe, and C. D. Eckhert

1087

335.

Does Boron Act on Transcription and Translation of Proteins? M. Dzondo-Gadet, R. Mayap Nzietchueng, K. Hess, P. Nabet, F. Belleville, and B. Dousset

1088

VI.

TRACE ELEMENT ANALYSIS

336.

New Sensitive Methods in the Determination of Trace Elements P. Schramel

337.

Distribution of Selenium, Arsenic, Iron, and Scandium in SeleniumDeficient Rats Using the Multitracer Technique Rieko Hirunuma, Shuichi Enomoto, Shizuko Ambe, and Fumitoshi Ambe

338.

Separation of Metallothionein Isoforms and Identification of Complexed Metals by Capillary Zone Electrophoresis Using Diode Array Detection V. Virtanen, G. Bordin, and A.-R. Rodriguez

1091

1099

1103

Contents

339.

340.

341.

342.

343.

344.

345.

346.

347.

Inductively Coupled Plasma Time of Flight Mass Spectrometry for Trace Element—and Speciation Analysis Håkan Emteborg, Xiaodan Tian, Monika Heisterkamp, and Freddy C. Adams Silicon Absorption from Stabilized Orthosilicic Acid and Other Supplements in Healthy Subjects M. Calomme, P. Cos, P. D’Haese, R. Vingerhoets, L. Lamberts, M. De Broe, C. Van Hoorebeke, and D. Vanden Berghe

xli

1107

1111

Preparation and ICP-MS Measurements of Magnesium Stable Isotopes in Human Samples M. Sabatier, W. R. Keyes, M. J. Arnaud, and J. R. Turnlund

1115

Characterization of Zinc Metallothioneins Using Electroanalytical Methods M. Dabrio and A. R. Rodríguez

1116

Hepatic Metallothionein Isoforms Induction by Cadmium and Its Detection Using Capillary Electrophoresis B. Ribas Ozonas, O. García Arribas, and M. Pérez Calvo

1117

Recent Developments in HPLC-ICP-MS for the “Difficult” Elements F. Abou-Shakra and P. Booker

1119

Determination of Heavy Metals in Calcium and Herbal Supplements Utilizing Inductively Coupled Plasma Mass Spectrometry (ICP-MS) Elzbieta (Ela) Bakowska

1120

Overview of Biomedical Applications of Inductively Coupled Plasma Mass Spectrometry (ICP-MS) Elzbieta (Ela) Bakowska

1121

Determination of Serum Cobalt at the Nanomolar Level by Direct Electrothermal Atomic Absorption Spectrometry J. Poupon, V. Gleizes, G. Saillant, and M. Galliot-Guilley

1122

List of Participants

1125

Author Index

1149

Subject Index

1157

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Trace Elements in Man and Animals 10

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1

INTEGRATIVE ASPECTS OF ZINC METABOLISM AND FUNCTION

Robert J. Cousins Center for Nutritional Sciences Food Science and Human Nutrition Department University of Florida Gainesville, Florida 32611-0370 USA

Over two decades have passed since Eric J. Underwood published the last edition of his highly regarded text, “Trace Elements in Human and Animal Nutrition” (Underwood, 1977). Since then, there have been tremendous advances in all aspects of the field of trace element research. Building upon research developed with radioisotopes and atomic absorption techniques, the 1980’s and 90’s have seen the application of techniques in cell and molecular biology, and experiments with stable isotopes, bring us closer to understanding the nutritional role played by trace elements in human and animal health. Zinc continues to be one of the most active areas of trace element research. In this brief review, I would like to highlight three specific areas. The first involves a discussion of the integrative aspects of zinc transporter proteins. Secondly, a discussion of the direct and indirect effects of zinc nutritional status on gene expression. Lastly, a discussion of the possible relationship between zinc, metallothionein and zinc-finger proteins. The following brief review summarizes some of our recent findings and those of others, and provides speculation as to future developments in these three areas of research on zinc metabolism and function.

ZINC TRANSPORTERS Kinetic studies using a variety of model systems, including intact animals, isolated organs, and isolated cells, have provided considerable background for approaching the area of zinc transporters. Specifically, zinc influx into cells has been shown to be a saturable process which may involve both rapid uptake and slower uptake/exchange processes (Pattison and Cousins, 1986). Now that a number of zinc transporters have been cloned, by hindsight, it is likely that the rapid uptake phase is a manifestation of Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000

1

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R. J. Cousins

the activity of intra-membrane zinc transporter proteins. A combination of rapid uptake and intracellular compartmentalization of zinc coupled with, until recently, the lack of a good indicator as to intracellular zinc concentrations has limited a clear definition of zinc uptake kinetics by cells. Similar limitations may also apply to zinc efflux studies. Nevertheless, there is evidence to suggest that, in situations where the extracellular zinc concentration is low, there is a downregulation of zinc transporters responsible for efflux. It should be emphasized that, while four zinc transporter genes have been cloned and some information about these membrane proteins is known, their function in zinc transport has yet to be demonstrated directly. The topic has been reviewed in detail previously (McMahon and Cousins, 1998a). The strategy used to clone zinc transporter-1 (ZnT-1), the first mammalian zinc transporter identified, was accomplished using a rescue approach for cells grown under mutagenic conditions in medium containing large amounts of zinc (Palmiter and Findley, 1995). To investigate the nutritional significance of ZnT-1, we cloned rat ZnT1 cDNA and used it as a probe to investigate ZnT-1 expression in rats that were fed deficient (5 mg/kg), adequate (30 mg/kg), and supplemental (180 mg/kg) levels of zinc (McMahon and Cousins, 1998b). Companion Western analysis studies were done with an affinity-purified polyclonal antibody raised against a C-terminal peptide from the ZnT-1 protein. It was shown that these dietary conditions markedly changed metallothionein expression. This response serves as an index of effectiveness of these dietary treatments. We found that ZnT-1 expression was significantly upregulated, but only in the intestine of animals fed supplemental zinc (180 mg/kg). Since ZnT-1 upregulation was observed in rats receiving supplemental zinc, we developed a hypothesis to examine ZnT1 expression at short intervals (2 and 6h) after an oral dose of zinc was administered in a fashion analogous to the zinc tolerance test for humans. Under these conditions, zinc produced robust upregulation of ZnT-1 as evidenced by both mRNA levels and ZnT-1 protein levels. An examination of ZnT-1 protein expression demonstrated conclusively that abundance is restricted to duodenum and jejunum, with virtually no expression in the lower small intestine or colon (McMahon and Cousins, 1998b). Furthermore, ZnT-1 protein was highly abundant in enterocytes from the villus tip, and was not detectable in cells derived from intestinal crypts. Immunofluorescence studies demonstrated ZnT-1 is localized primarily at the basolateral membrane surface of enterocytes. This orientation allows us to conclude that ZnT-1 functions as an exporter protein that may be responsible for zinc efflux from enterocytes. This function would be in line with a role in retention of body zinc. Similar immunofluorescence studies done with rat kidney indicated ZnT-1 localization was primarily at the basolateral surface of renal epithelial cells where it appears to co-localize with the Na/K ATPase transport system associated with sodium reabsorption (McMahon and Cousins, 1998a). Again, since ZnT-1 was not localized at the apical membrane, a role in body zinc retention by the kidney has been proposed. In all immunofluorescence experiments conducted with ZnT-1, there appears to be some punctate staining. This could indicate that there is localization of the transporter with vesicles. Future experiments will be required to evaluate that possibility. No experiments have been directed to date at the physiologic significance of ZnT2 (Palmiter, Cole, and Findley, 1996a). Significant progress has been made on the involvement of ZnT-3 in the handling of zinc by neurons (Palmiter et al., 1996b). ZnT-4 was identified accidentally in the search for the murine mutant pallid gene, a mutation responsible for congenital defects (Huang and Gitschier, 1997). A mutation in the ZnT-4 gene is responsible for the murine lethal milk (1m) syndrome, an autosomal recessive

Integrative Aspects of Zinc Metabolism and Function

3

condition. It is believed that, since the mammary epithelium is among the tissues where ZnT-4 is expressed, it is associated with zinc secretion into milk. The ZnT-4 transporter may be defective in lactating 1m dams and may account for the lack of zinc transfer into milk produced by 1m mothers (Lee, Shay, and Cousins, 1992). There is also evidence that a gene initially designated dri27, isolated from rat intestine (Barila et al., 1994), is the rat homolog of murine ZnT-4. While physiologic experiments designed to examine the zinc transporter family are only now being initiated, it is clear that, with respect to ZnT-1 and ZnT-4, these proteins could function in zinc acquisition. In the case of ZnT-1, this could involve basolateral transfer of zinc by enterocytes and reabsorption of filtered zinc by renal tubular epithelial cells, whereas in the case of ZnT-4, it is acquisition of zinc by nursing pups.

ZINC AND GENE EXPRESSION For decades, an important area of research on zinc has been the link between zinc and gene expression. Initially, this was viewed as a general requirement of zinc for metalloenzymes associated with growth. More recently, with the cloning of the metallothionein genes and subsequent identification of metal response elements (MREs) in the promoter and an MRE-binding transcription factor, an appreciation for the ability of zinc to regulate expression of individual genes has emerged (reviewed in Cousins, 1994). It is our view that changes in dietary zinc intake can have both direct and indirect influences on gene expression (Cousins, 1997; Cousins, 1998). A direct effect would be an influence on genes that are regulated by an MRE system. Presumably, these genes would decrease in activity in deficiency and increase in zinc excess, forming a positive loop of regulation. It cannot be ruled out, however, that there is negative regulation brought about by MRE sequences. This negative influence could be developed through a change in the distribution or structure of MRE-binding transcription factors. The indirect route to gene regulation by zinc is envisioned as one in which zinc is required for some physiologic process. If that process is not executed because of dietary zinc restriction or through consumption of excess zinc, secondary changes would lead to changes in altered transcription rates of specific genes, changes at the level of mRNA translation, or in post-translational modifications of the gene product. Our approach to examine this question has been to use the technique of differential mRNA display. This technique allows the systematic screening of mRNAs by various tissues through a polymerase chain reaction-based technique (described in Cousins, 1997). For these experiments, we now employ a Genomyx sequencer system, which uses 12 anchor primers and 20 arbitrary primers to screen the entire complement of expressed genes. RNA for differential display is obtained from rats that are fed zinc-deficient, normal, or a supplemental amount of zinc using the diet described above. Measures of zinc status include metallothionein mRNA levels in kidney and/or pancreas. Using differential mRNA display, it has been possible to identify zinc-regulated genes from the intestine and thymus (Blanchard and Cousins, 1996; Moore et al., 1999). Success in identifying specific genes is a function of ongoing worldwide genome projects. While a number of the sequences that have been shown to be regulated are expressed sequence tags, i.e., DNA sequences for yet unidentified genes and proteins, there have been a number of interesting determinations regarding previously identified genes. Two genes have been directly related to physiologic outcomes of zinc deficiency. Upregulation

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of cholecystokinin mRNA fits into a scenario where there is a reduction in food intake, which is one of the outcomes of zinc deficiency in the rat. Similarly, upregulation of the newly identified hormone uroguanylin in the intestine fits with a scenario that would involve increased intestinal fluid secretion, which is an outcome of severe zinc deficiency in humans and, under some conditions, in rodents. The association of zinc deficiency with secretory diarrhea in many areas of the world suggests that uroguanylin could be at least a contributing factor to this problem, arising from altered regulation of the uroguanylin gene during zinc deficiency (Blanchard and Cousins, 1997). Of equal interest is the recent identification that the selenium-dependent glutathione peroxidase-1 gene is increased in zinc deficiency, and decreased in zinc supplementation (Blanchard and Cousins, unpublished observation). The physiologic outcome of this finding has yet to be established. Nevertheless, there have been repeated reports of oxidative tissue damage associated with dietary zinc deficiency (reviewed in Cousins, 1996). Possibly related to this finding is recent in vitro evidence suggesting that glutathione peroxidase is involved in the catalytic removal of zinc from metallothionein (Jacob, Maret, and Vallee, 1999). Consequently, it is of interest that glutathione peroxidase is upregulated in zinc deficiency at a time when metallothionein synthesis and, consequently, the metallothionein-bound zinc pool is particularly low. Since a number of the genes regulated by dietary zinc are upregulated in conditions of zinc deficiency, it is unlikely that the direct MRE-regulated system is involved. As an explanation, we propose that, under normal conditions, the steady state levels of mRNAs for zinc-requiring gene products are produced in required amounts. However, in zinc deficiency, steady state is disturbed and those protein products may not be produced at a normal level, and/or the biologic responses resulting from those products may not be normal. Under these conditions, the cellular response is to compensate by attempting to increase the amount of protein product through increasing expression of the gene involved and, consequently, producing an increase in mRNA abundance.

ZINC, METALLOTHIONEIN, AND ZINC-FINGER PROTEINS The discovery of zinc-finger proteins has provided new insights into the biologic role of zinc in gene expression (Klug and Schwabe, 1995). The sheer number of zincfinger proteins that are being identified (>1% of the human genome) suggest that zinc has the capacity for having a profound effect on biologic responses if these zinc-finger proteins are influenced by dietary zinc intake. While the first zinc-finger protein was identified roughly 15 years ago, the zinc-binding protein metallothionein has been studied for over 40 years (reviewed in Kägi and Nordberg, 1979). Furthermore, the zinc-finger proteins are believed to be involved in DNA binding and/or protein-protein interaction, whereas, in contrast, no definitive function for metallothionein has been established. Possibilities of a function for metallothionein range from its involvement as an antioxidant and cellular redox regulator to involvement as a zinc-acceptor/donator molecule for zincfinger proteins. The complex nature of metallothionein function has led to a reliance on in vitro systems in attempts to elucidate a function. Unfortunately, it is frequently difficult to place these in vitro observations within a biologic context. The inducible nature of metallothionein by both hormones and cytokines, as well as changes in dietary zinc intake, merge with virtually any of these potential roles (Cousins, 1996), Of particular interest has been the recent suggestion that metallothionein and apo metallothionein (thionein) act as a conjugate pair to modulate DNA binding of the

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zinc-finger transcription factor called Tramtrack (Roesijadi, Bogumil, Vaák, and Kägi, 1998). In those experiments, it was envisioned that thionein inhibits DNA binding through zinc removal from this zinc-finger protein, with DNA-binding ability of the transcription factor protein restored as zinc is donated from metallothionein. Our approach to demonstrate the zinc donation properties of metallothionein has been to use metallothionein null mice to examine changes in the cellular concentration of an inducible zinc finger protein. We have conducted experiments with LPS-induced expression of the zinc-finger protein cysteine-rich intestinal protein (CRIP) to evaluate its cellular concentration in wild-type and metallothionein-null mice. Our data support the involvement of metallothionein in regulating the abundance of this zinc finger protein. Previous experiments showed that zinc associates with intestinal CRIP during transcellular movement (Hempe and Cousins, 1991). More recent experiments point to a role for CRIP in regulating the immune response, particularly upregulating the Th2 response (Hallquist, Khoo, and Cousins, 1996; Lanningham-Foster, Cottey, Bender, and Cousins, 1999). Both the CRIP and metallothionein genes are upregulated by lipopolysaccharide (LPS) in tissues where the genes are expressed, including intestine, spleen, and thymus. CRIP concentrations are measured by a sandwich ELISA method (Lanningham-Foster et al., 1999). These experiments showed the LPS-stimulated increase in tissue CRIP levels was not observed with metallothionein-null mice. There seems to be some specificity for the effect, as levels of the zinc-finger transcription factor Spl (measured by Western analysis) do not change under the same treatment conditions. CRIP has two zinc fingers, and is referred to as the LIM domain (Khoo et al., 1997). The LIM domain is found in many proteins. One finger is a CCCC, while the other has a CCHC motif. This is in contrast to Tramtrack, which has two CCHH motifs. The zinc binding constant for Tramtrack seems to be lower than that for metallothionein (Roesijadi et al., 1998), suggesting that, under appropriate conditions, metallothionein could donate zinc atoms to Tramtrack. Similarly, while the binding constant of zinc for CRIP is not known, metallothionein could act as the source of zinc for CRIP during upregulated LPS-induced synthesis. Finally, these separate studies may suggest that, at least with some zinc-finger proteins, individual association constants for zinc, as well as the protein’s cellular abundance coupled with the cellular abundance of metallothionein, which can be regulated by hormonal and dietary genes, could serve as an important system to regulate zinc-finger protein function.

CONCLUSION Evolution of our understanding of trace element metabolism and function has increased exponentially since TEMA-1 was held in Aberdeen, Scotland, three decades ago. The increase in knowledge clearly parallels the tremendous strides that have been made in our understanding of biologic processes. Nevertheless, much remains to be learned. In the case of zinc, we have three biologic functions: catalytic; structural; and regulatory. Yet, in each category, we have only begun to touch the surface with respect to understanding the basic biochemistry involved. Similarly, when we consider the important physiologic outcomes of zinc in regulating the immune system, cellular growth and differentiation, and neurologic behavior, we must proceed toward understanding these outcomes without, in most cases, having a clear understanding of the biochemical basis responsible. It is clear that the increasing complexity with which biological research is

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approached will require an ever-increasing reliance on collaborations with individuals from a variety of fields for research in the field of trace elements. Hopefully, as this knowledge base increases, we will have a better appreciation for optimal dietary intake levels of trace elements, based on sound assessment criteria, and an unequivocal answer to the value of trace element supplements in certain intervention and health promotion situations.

ACKNOWLEDGMENTS I would like to express gratitude to former and current students, postdoctoral associates, and research staff who have helped with these studies over a period of nearly 30 years. It is impossible to name them all individually because of space limitations. However, for this presentation, I would like to thank Virginia N. Mauldin and Walter M. Jones for their help. I would also like to express gratitude to the National Institutes of Health, who have supported my research throughout my professional career.

REFERENCES Barila, D., Murgia, C., Nobili, F., Gaetani, S., and Perozzi, G., 1994, Subtractive hybridization cloning of novel genes differentially expressed during intestinal development, Eur. J. Biochem. 223:701–709. Blanchard, R.K. and Cousins, R.J., 1996, Differential display of intestinal mRNAs regulated by dietary zinc, Proc Natl. Acad. Sci. USA 93:6863–6868. Blanchard, R.K. and Cousins, R.J., 1997, Upregulation of rat intestinal uroguanylin mRNA by dietary zinc restriction, Am. J. Physiol. 272:G972–G978. Cousins, R.J., 1994, Metal elements and gene expression, in: Annual Review of Nutrition (R.E. Olson, ed.), pp. 449–469, Annual Reviews Inc., Palo Alto, CA. Cousins, R.J., 1996, Zinc, in: Present Knowledge in Nutrition, 7th ed. (L.J. Filer and E.E. Ziegler, eds.), pp. 293–306, Internat. Life Sci. Inst.-Nutr. Foundation, Washington. Cousins, R.J., 1997, Differential mRNA display, competitive polymerase chain reaction and transgenic approaches to investigate zinc responsive genes in animals and man, in: Trace Elements in Man and Animals—9: Proceedings of the Ninth International Symposium on Trace Elements in Man and Animals (P.W.F. Fischer, M.R. L’Abbé, K.A. Cockell, and R.S. Gibson, eds.), pp. 649–652, NRC Research Press, Ottawa, Canada. Cousins, R.J., 1998, A role of zinc in the regulation of gene expression, Proc. Nutr. Soc. 57:307–311. Hallquist, N.A., Khoo, C., and Cousins, R.J., 1996, Lipopolysaccharide regulates cysteine-rich intestinal protein, a zinc-finger protein, in immune cells and plasma, J. Leukoc. Biol. 59:172–177. Hempe, J.M. and Cousins, R.J., 1991, Cysteine-rich intestinal protein binds zinc during transmucosal zinc transport, Proc. Natl. Acad. Sci. USA 88:9671–9674. Huang, L. and Gitschier, J., 1997, A novel gene involved in zinc transport is deficient in the lethal milk mouse, Nature Genetics 17:292–297. Jacob, C., Maret., W., and Vallee, B.L., 1999, Selenium redox biochemistry of zinc-sulfur coordination sites in proteins and enzymes. Proc. Natl. Acad. Sci. USA 96:1910–1914. Kägi, J.H.R. and Nordberg, M., 1979, Metallothionein, Birkhauser Verlag, Basel, Switzerland. Khoo, C., Blanchard, R.K., Sullivan, V.K., and Cousins, R.J., 1997, Human cysteine-rich intestinal protein: cDNA cloning and expression of recombinant protein and identification in human peripheral blood mononuclear cells, Protein Expression and Purification 9:379–387. Klug A. and Schwabe, J.W., 1995, Protein motifs 5. Zinc fingers, FASEB J. 9:597–604. Lanningham-Foster, L., Cottey, R.J., Bender, B.S., and Cousins, R.J., 1999, Overexpression of the zinc-finger protein cysteine-rich intestinal protein (CRIP) in CRIP transgenic mice increases sensitivity to lipopolysaccharide and influenza virus challenge, FASEB J. 13:A872 (abs. 659.5). Lee, D.-Y., Shay, N.F., and Cousins, R.J., 1992, Altered zinc metabolism occurs in murine lethal milk syndrome, J. Nutr. 122:2233–2238. McMahon, R.J. and Cousins, R.J., 1998a, Mammalian zinc transporters, J. Nutr. 128:667–670.

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McMahon, R.J. and Cousins, R.J., 1998b, Regulation of the zinc transporter ZnT-1 by dietary zinc, Proc. Natl. Acad. Sci. USA 95:4841–4846. Moore, J.B, Blanchard, R.K., and Cousins, R.J., 1999, Identification of dietary zinc regulated expressed sequence tags in murine thymus by mRNA differential display, FASEB J. 13:A241 (abs. 215.4). Palmiter, R.D. and Findley, S.D., 1995, Cloning and functional characterization of a mammalian zinc transporter that confers resistance to zinc, EMBO J. 14:639–649. Palmiter, R.D., Cole, T.B., and Findley, S.D., 1996a, ZnT-2, a mammalian protein that confers resistance to zinc by facilitating vesicular sequestration, EMBO J. 15:1784–1791. Palmiter, R.D., Cole, T.B., Quaife, C.J., and Findley, S.D., 1996b, ZnT-3, a putative transporter of zinc into synaptic vesicles, Proc. Natl. Acad. Sci. USA 93:14934–14939. Pattison, S.E. and Cousins, R.J., 1986, Kinetics of zinc uptake and exchange by primary cultures of rat hepatocytes, Am. J. Physiol. 250:E677–E685. Roesijadi, G., Bogumil, R., Va_ák, and Kägi, J.H.R., 1998, Modulation of DNA binding of a Tramtrack zinc finger peptide by the metallothionein-thionein conjugate pair, J. Biol. Chem. 273:17425–17432. Underwood, E.J., 1977, Trace Elements in Human and Animal Nutrition, 4th ed., Academic Press, New York.

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THE COPPER TRANSPORTING ATPASES IN HUMAN DISEASE

Jonathan D. Gitlin Edward Mallinckrodt Department of Pediatrics Washington University School of Medicine St. Louis, Missouri

Copper is an essential transition element which plays a fundamental role in biochemistry, permitting the fascile transfer of electrons in critical metabolic pathways. Menkes disease and Wilson disease are hereditary disorders of copper metabolism which underscore the essential role of copper in human biology. Each disease results from the absence or dysfunction of homologous copper transporting ATPases. The Menkes ATPase transports copper across the placenta, the gastrointestinal tract and the blood brain barrier and thus the clinical features of this X-linked disorder are the result of copper deficiency. The Wilson ATPase functions to transport copper from the hepatocyte secretory into the bile and the clinical features of this disorder are the result of hepatic copper overload. Despite the striking differences in the clinical presentation of these two diseases, the copper transporting ATPases function in precisely the same fashion within the cell. The unique phenotype of each disease is therefore the result of tissue specific expression of each ATPase. Elucidation of the basic defect in these rare disorders provides the opportunity for new approaches in the diagnosis and treatment of affected patients and permits novel insights into the cellular mechanisms of copper homeostasis. Copper is an essential trace element which plays a fundamental role in the biochemistry of all aerobic organisms. The unique electron structure of copper permits direct interaction with dioxygen, enabling this metal to serve as an essential cofactor in enzymatic reactions. In humans these include electron transport in the respiratory chain, antioxidant defense, neurotransmitter biosynthesis, connective tissue formation and iron metabolism. The unique properties which make copper biologically useful are also potentially highly toxic. For this reason, specific proteins have evolved for the

Address all correspondence to: Jonathan D. Gitlin, M.D., Washington University School of Medicine, Department of Pediatrics, One Childrenís Place, St. Louis, MO 63110; telephone: 314-454-6124; fax: 314-454-4861; email: [email protected] Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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compartmentalization and trafficking of copper within mammalian cells. Our understanding of the mechanisms of intracellular copper metabolism has increased greatly over the past several years in large part due to the elucidation of the basic genetic defect in the inherited disorders of copper metabolism, Menkes disease and Wilson disease (Culotta and Gitlin, 1999). Menkes disease is an X-linked recessive disorder which results in hypotonia, growth failure and fatal neurodegenerative disease in early childhood (Schaefer and Gitlin, 1999). Early studies revealed that affected infants were severely copper deficient accounting for the protean manifestations of abnormal hair, absence of pigmentation, laxity of the skin and joints, bony dysplasia and cerebellar degeneration. The Menkes disease gene was identified by physical mapping and cloning in affected female patients with balanced translocations (Vulpe et al., 1993; Chelly et al., 1993; Mercer et al., 1993). This analysis identified a gene encoding a predicted protein sequence highly homologous to a cation transporting P-type ATPase essential for prokaryotic copper homeostasis (Solioz and Vulpe, 1996). The Menkes ATPase transports copper across the placenta, the gastrointestinal tract and the blood brain barrier, accounting for the clinical features of profound copper deficiency in affected individuals. Milder forms of the disease in which the neurologic features are minimal or absent have been described and arise from allelic heterogeneity at the Menkes locus. Wilson disease is an autosomal recessive disorder resulting in hepatic cirrhosis and neuronal degeneration. Following cloning and characterization of the Menkes disease gene, the Wilson locus was identified and shown to encode an homologous member of this newly described family of copper transporting ATPases (Yamaguchi et al., 1993; Bull et al., 1993; Tanzi et al., 1993). The Wilson ATPase has 55% amino acid identity to the Menkes ATPase and is expressed predominantly within the liver. This protein transports copper into the secretory pathway of hepatocytes for subsequent incorporation into ceruloplasmin and excretion of copper into the bile. As copper homeostasis in humans is maintained entirely by intestinal absorption and biliary excretion, affected individuals develop hepatic copper overload which eventually results in hepatocellular necrosis and dissemination of excess copper to extrahepatic tissues including the limbus of the cornea (Kayser-Fleischer rings) and the basal ganglia of the brain (Schilsky, 1996; Cox, 1996). Sequence comparison and hydropathy plot analysis of the derived amino acid sequence of the copper transporting ATPases indicates the presence of a polytopic membrane protein predicted to transport copper across biological membranes in an ATP dependent fashion. Homologous proteins have been identified in a wide range of prokaryotic and eucaryotic species and where examined play an analogous role in copper transport (Solioz and Vulpe, 1996). Conserved amino acid motifs in these ATPases include the copper binding MXCXXC motif in the amino terminus as well as the invariant aspartate residue in the DKTGDT motif within the largest cytoplasmic loop. This aspartate residue is reversibly phosphorylated in the process of energy transduction consistent with the known mechanisms of all P-type ATPases. A CPC motif is located in the 6th transmembrane domain which by analogy with similar motifs in other metal transporters is likely essential for mediating copper transfer across biological membranes. A highly conserved histidine within an SEHPL motif also located in the large cytoplasmic loop is conserved in all copper transporting ATPases (Petrukhin et al., 1994). This histidine which is essential for Menkes and Wilson ATPase function (vide infra) is the site of the most common mutation (H1069Q) found in up to 40% of patients with Wilson disease (Cox, 1996; Petrukhin et al., 1994).

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Metabolic studies of the Wilson and Menkes ATPases reveal that each protein is synthesized as a single chain polypeptide (Yamaguchi et al., 1996; Hung et al., 1997). Cell biological studies utilizing immunofluorescence microscopy have revealed that the Wilson and Menkes ATPases are localized to the trans-Golgi network of cells (Yamaguchi et al., 1996; Petris et al., 1996; Dierick et al., 1997; Hung et al., 1997; Schaefer et al., 1999). In this location, these ATPases transport copper into the secretory pathway of cells for incorporation into specific cuproenzymes as well as export from the cell. Support for this model has come from studies in Saccharomyces cerevisiae deficient in the homologous copper transporting ATPase CCC2 (Yuan et al., 1995; Yuan et al., 1997). Restoration of copper incorporation into the ceruloplasmin homologue Fet3 can be accomplished in ccc2 stains by expression of either the Wilson or Menkes protein (Hung et al., 1997; Payne and Gitlin, 1998). These studies provide direct evidence of copper transport by these ATPases into the secretory pathway of the cell. Studies examining the Menkes ATPase with a point mutation in the conserved histidine to glutamine (H1086Q) homologous to the common H1069Q mutation in Wilson disease indicates that this mutation impairs copper transport when expressed in ccc2 yeast (Payne and Gitlin, 1998). The finding that the most commonly occurring mutation in Wilson disease compromises function of the Menkes protein provides compelling evidence for a commonality of transporter function. Expression studies which demonstrate that both the Wilson and the Menkes ATPases can rescue the phenotype of Menkes disease deficient cells also provides evidence that these ATPases work through common biochemical mechanisms (Payne et al., 1998; La Fontaine et al., 1998). Taken together these data support the concept that the clinical differences in the presentation of these two diseases are the result of tissue specific expression consistent with mRNA data indicating Wilson ATPases expression in liver and Menkes ATPase in a broader tissue distribution including placenta, GI tract and endothelium. Although the Menkes and Wilson ATPases are localized to the trans-Golgi network of cells under study state conditions, several studies have revealed that an increase in the copper concentration within cells results in the trafficking of these proteins to a cytoplasmic vesicular compartment (Hung et al., 1997; Petris et al., 1996). This trafficking response is rapid with movement from the trans-Golgi essentially is completed within 15 minutes. Furthermore, this process is reversible as evidenced by the rapid recycling of the Wilson ATPase back to the trans-Golgi network following copper chelation. This copper dependent trafficking of the Menkes and Wilson ATPases represents a novel posttranslational mechanism allowing for restoration of cellular copper homeostasis within minutes of A change in the copper concentration within the cell. Although the molecular mechanisms which result in this copper dependent trafficking have not been defined recent studies indicate that the conserved histidine residue in the cytoplasmic loop of the Wilson ATPase is essential for this process (Payne et al., 1998). The mechanisms which define the intracellular location of the copper transporting ATPases are beginning to be defined. Evaluation of a patient with a mild form of Menkes disease with a splice mutation eliminating exon 10 encoding the 3rd and 4th transmembrane domains indicates that this region is essential for the trans-Golgi network localization of the Menkes protein (Qi and Byers, 1998). This finding has been confirmed by expression of this mutant in COS cells (La Fontaine et al., 1998). Studies of the H1069Q mutant Wilson protein suggest that this conserved histidine residue is essential for localization to the trans-Golgi network. Metabolic labeling studies indicate that this H1069Q mutant is retained within the endoplasmic reticulum and rapidly degraded. The small amount of protein which does make its way to the trans-Golgi network is unresponsive

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to copper dependent recycling (Payne et al., 1998). Recent studies looking at site directed mutagenesis of the Menkes protein have revealed that a dileucine motif in the carboxy terminus is necessary for the response of this protein to intracellular copper (Petris et al., 1998). These findings suggest that trafficking signals common to the cytoplasmic domain of many intracellular cargo proteins may be utilized specifically by the copper transport ATPases to mediate the physiologic responses to copper. Several important issues remain for future study. The mechanisms of neuronal degeneration in Menkes disease are unclear as are the precise mechanisms of copper accumulation and neuronal degeneration within the basal ganglia in Wilson disease. Recent studies have identified a novel splice variant of the Wilson disease gene as a pineal night specific ATPase (PINA). Expression of this ATPase exhibits a dramatic diurnal rhythm in both pineal gland and retina with a 100-fold greater expression at night than during the day (Borjigin et al., 1999). Recent data from Saccharomyces cerevisiae reveal that the function of a CLC chloride channel Gef1 is essential for copper transport by ccc2 into the post-Golgi vacuolar compartment (Gaxiola et al., 1998). These data suggest that common physiological principles derived for the movement of cations across biological membranes may prove relevant to understanding the copper transporting function of these ATPases. The human disorders of copper metabolism underscore both the essential nature of copper in human biology as well as the toxicity of this metal when copper homeostasis is perturbed. These new studies highlight potentially important aspects of human copper metabolism for future investigations.

REFERENCES Borjigin, J., Payne, A.S., Deng, J., Li, X., Wang, M.W., Ovodenko, B., Gitlin, J.D., and Synder, S.H., 1999, A novel pineal night-specific ATPase encoded by the Wilson disease gene. J. Neurosci. 19:1018–1026. Bull, P.C., Thomas, G.R., Rommens, J.M., Forbes, J.R., and Cox, D.W., 1993, The Wilson disease gene is a putative copper transporting P-type ATPase similar to the Menkes gene. Nat. Genet. 5:327–337. Chelly, J., Tumer, Z., Tonnesen, T., Petterson, A., Ishikawa-Brush, Y., Tommerup, N., Horn, N., and Monaco, A.P., 1993, Isolation of a candidate gene for Menkes disease that encodes a potential heavy metal binding protein. Nat. Genet. 3:14–19. Cox, D.W., 1996, Molecular advances in Wilson disease. Prog. Liver Dis. 14:245–264. Culotta, V.C. and Gitlin, J.D., 1999, Disorders of copper transport. In: The Molecular and Metabolic Basis of Inherited Disease. (C.R. Scriver, A.L. Beaudet, W.S. Sly, and D. Valle, eds.) McGraw-Hill, (In press). Dierick, H.A., Adam, A.N., Escara-Wilke, J.F., and Glover, T.W., 1997, Immunocytochemical localization of the Menkes copper transport protein (ATP7A) to the trans-Golgi network. Hum. Mol. Genet. 6:409–416. Gaxiola, R.A., Yuan, D.S., Klausner, R.D., and Fink, G.R., 1998, The yeast CLC chloride channel functions in cation homeostasis. Proc. Natl. Acad. Sci. 95:4046–4050. Hung, I.H., Suzuki, M., Yamaguchi, Y., Yuan, D.S., Klausner, R.D., and Gitlin, J.D., 1997, Biochemical characterization of the Wilson disease protein and functional expression in the yeast Saccharomyces cerevisiae. J. Biol. Chem. 272:21461–21466. La Fontaine, S., Firth, S.D., Lockhart, P.J., Brooks, H., Parton, R.G., Camakaris, J., and Mercer, J.F., 1998, Functional analysis and intracellular localization of the human Menkes protein (MNK) stably expressed from a cDNA construct in Chinese hamster ovary cells (CHO-K1). Hum. Mol. Genet. 7:1293–1300. La Fontaine, S.L., Firth, S.D., Camakaris, J., Englezou, A., Theophilos, M.B., Petris, M.J., Howie, M., Lockhart, P.J., Greenough, M., Brooks, H., Reddel, R.R., and Mercer, J.F., 1998, Correction of the copper transport defect of Menkes patient fibroblasts by expression of the Menkes and Wilson ATPases. J. Biol. Chem. 273:31375–31380. Mercer, J.F., Livingston, J., Hall, B., Paynter, J.A., Begy, C., Chandrasekharappa, S., Lockhart, P., Grimes, A., Bhave, M., Siemieniak, D., and Glover, T.W., 1993, Isolation of a partial candidate gene for Menkes disease by positional cloning. Nat. Genet. 3:20–25.

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Payne, A.S. and Gitlin, J.D., 1998, Functional expression of the Menkes disease protein reveals common biochemical mechanisms among the copper-transporting P-type ATPases. J. Biol. Chem. 273:3765–3770. Payne, A.S., Kelley, E.J., and Gitlin, J.D., 1998, Functional expression of the Wilson disease protein reveals mislocalization and impaired copper-dependent trafficking of the common H1069Q mutation. Proc. Natl. Acad. Sci. 95:10854–10859. Petris, M.J., Mercer, J.F.B., Culvenor, J.G., Lockhart, P., Gleeson, P.A., and Camakaris, J., 1996, Ligandregulated transport of the Menkes copper P-type ATPase efflux pump from the Golgi apparatus to the plasma membrane: a novel mechanism of regulated trafficking. EMBO J. 15:6084–6095. Petris, M.J., Camakaris, J., Greenough, M., LeFontaine, S., and Mercer, J.F.B., 1998, A C-terminal di-leucine is required for localization of the Menkes protein in the trans-Golgi network. Hum. Mol. Genet. 7:2063–2071. Petrukhin, K., Lutsenko, S., Chernov, L., Ross, B.M., Kaplan, J.H., and Gilliam, T.C., 1994, Characterization of the Wilson disease gene encoding a P-type copper transporting ATPase: genomic organization, alternative splicing, and structure/function predicting. Hum. Mol. Genet. 3:1647–1656. Qi, M. and Byers, PH., 1998, Constitutive skipping of alternatively spliced exon 10 in the ATP7A gene abolishes Golgi localization of the Menkes protein and produces the occipital horn syndrome. Hum. Mol. Genet. 7:465-469. Schaefer, M. and Gitlin, J.D., 1999, Genetic Disorders of Copper Transport. Am. J. Physiol. 276:G311–G314. Schaefer, M., Hopkins, R.G., Failla, M.L., and Gitlin, J.D., 1999, Hepatocyte-specific localization, copperdependent trafficking and developmental expression of the Wilson disease protein in the liver. Am. J. Physiol. 276:G639–G646. Schilsky, M.L., 1996, Wilson disease: genetic basis of copper toxicity and natural history. Semin. Liver Dis. 16:83–95. Solioz, M. and Vulpe, C., 1996, CPx-type ATPases: a class of P-type ATPase that pump heavy metals. Trends Biochem. 21:237–241. Tanzi, R.E., Petrukhin, K., Chernov, I., Pellequer, J.L., Wasco, W., Ross, B., Romano, D.M., Parano, E., Pavone, E., Pavone, L., Brzustowicz, L.M., Devoto, M., Peppercorn, J., Bush, A.I., Sternlieb, I., Pirastu, M., Gusella, J.F., Evgratov, O., Penchaszadeh, G.K., Honig, B., Edelman, I.S., Soares, M.B., Scheinberg, T.H., and Gilliam, T.C., 1993, The Wilson disease gene is a copper transporting ATPase with homology to the Menkes disease gene. Nat. Genet. 5:344–350. Vulpe, C., Levinson, B., Whitney, S., Packman, S., and Gitschier, J., 1993, Isolation of a candidate gene for Menkes disease and evidence that it encodes a copper-transporting ATPase. Nat. Genet. 3:7–13. Yamaguchi, Y., Heiny, M.E., and Gitlin, J.D., 1993, Isolation and characterization of a human liver cDNA as a candidate gene for Wilson disease. Biochem. Biophys. Res. Commun. 197:271–277. Yamaguchi, Y., Heiny, M.E., Suzuki, M., and Gitlin J.D., 1996, Biochemical characterization and intracellular localization of the Menkes disease protein. Proc. Natl. Acad. Sci. 93:14030–14035. Yuan, D.S., Dancis, A., and Klausner, R.D., 1997, Restriction of copper export in Saccharomyces cerevisiae to a late Golgi or post-Golgi compartment in the secretory pathway. J. Biol. Chem. 272:25787–25793. Yuan, D.S., Stearman, R., Dancis, A., Dunn, T., Beeler, T., and Klausner, R.D., 1995, The Menkes/Wilson disease gene homologue in yeast provides copper to a ceruloplasmin-like oxidase required for iron uptake. Proc. Natl. Acad. Sci. 92:2632–2636.

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REGULATION AND FUNCTION OF THE COPPER ION TRANSPORT MACHINERY

Jaekwon Lee and Dennis J. Thiele Department of Biological Chemistry The University of Michigan Medical School Ann Arbor, Michigan 48109-0606, USA

1. INTRODUCTION The trace metal ion copper (Cu) is an essential nutrient in virtually all known life forms (Linder, 1991). Due to its ability to readily adopt two ionic states, Cu(I) and Cu(II), Cu serves as an essential redox co-factor for a wide variety of enzymes including cytochrome oxidase, involved in oxidative phosphorylation, dopamine involved in neurotransmitter maturation, Cu, Zn superoxide dismutase, which detoxifies superoxide anion, ceruloplasmin, involved in Fe mobilization and several other critical cellular enzymes listed in Table 1. Although the redox activity of Cu is essential for its function as a catalytic co-factor, this same property drives chemical reactions that generate hydroxyl radical, a highly reactive species that is known to cause direct damage to nucleic acids, proteins and lipids (Halliwell and Gutteridge, 1984). A major problem in the biology of Cu nutrition then is how do organisms accumulate sufficient Cu to serve as a co-factor for all of the Cu-dependent cellular enzymes, yet carefully control Cu accumulation to prevent the generation of highly damaging reactive oxygen species? Indeed, organisms use many levels of cellular regulation to orchestrate the fine balance of Cu sufficiency and Cu excess, including regulation at the level of Cu-dependent gene transcription, Cu-responsive protein trafficking and degradation and Cu-dependent proteinprotein interactions. Since Cu uptake represents the first stage at which cells encounter Cu ions, this is a critical step that must be tightly regulated to control Cu accumulation. In this chapter we summarize recent advances in the regulation and function of the Cu ion transport machinery.

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2. CU TRANSPORT IN YEAST CELLS Yeast cells have provided an outstanding eukaryotic model system with which to dissect the components involved in Cu ion transport, their mechanisms of action and their modes of regulation (Fig. 1). Yeast has proven to be such a convenient model system due to the ability to specifically manipulate the growth environment, its facile genetics and molecular biology, its well characterized biology and the recent sequencing of the entire yeast genome. Furthermore, it is clear that in their fundamental components and their mechanisms of action, yeast and human cells exhibit a striking similarity in such basic processes as gene expression, protein synthesis, processing and trafficking, DNA replication and cell cycle control. S. cerevisiae cells utilize the Frel/7 proteins to reduce Cu(II) to Cu(I), whereupon the Ctrl and Ctr3 high affinity Cu transporters facilitate Cu uptake. The cytosolic Cu chaperones Atxl, Ccs and Coxl7 deliver Cu to the secretory compartment, cytosolic Cu, Zn superoxide dismutase and mitochondrial cytochrome c oxidase, respectively. Delivery of Cu to the secretory compartment is essential for metallation of the Fet3 multicopper ferroxidase, a component of the high affinity Fe uptake complex with Ftrl. The Macl nuclear Cu metalloregulatory transcription factor binds to CuRE elements in the promoters of genes encoding components of the high affinity Cu transport machinery, regulating their transcription. Early studies using the yeast Saccharomyces cerevisiae for the analysis of Cu transport revealed that yeast cells contain a saturable, temperature dependent high affinity Cu uptake mechanism (Lin and Kosman, 1990). Furthermore, the reduced form of Cu [Cu(I)] was shown to be transported readily (Hassett and Kosman, 1995), consistent with the notion that cell surface Cu(II) reductases facilitate the transport of Cu(I) into the cell. Recent genetic studies in the area of Cu homeostasis in yeast have exploded, with a major thrust being the identification of yeast genes that encode Cu transport proteins and proteins that escort Cu, once transported into cells, to the appropriate intracellular destination. By chance, the hunt for yeast mutants that are defective in high affinity Fe

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uptake revealed the existence of a gene encoding a high affinity Cu ion transporter, and simultaneously provided a mechanistic explanation for the dependence on Cu for Fe uptake long ago observed in mammals. A yeast mutant defective in Fe uptake harbored a nonfunctional gene for a high affinity Cu transport protein, Ctrl (Dancis et al., 1994b). Ctrl is a plasma membrane protein that exists as a glycosylated multimeric protein, with a methionine-rich region thought to extend out to the periplasmic space in the extracellular region (Dancis et al., 1994a). This Met-rich domain contains several Met-X-X-Met or Met-X-Met regions and may provide a high concentration of Cu(I)-coordinate sites. Furthermore, at least seven genes in yeast encoding dual Fe(III) and Cu(II) reductase activities have been identified and isolated (Georgatsou and Alexandraki, 1994; Hassett and Kosman, 1995; Martins et al., 1998). Consistent with Cu(I) as the transported form of the metal ion, and reductases playing an important role in Cu(I) transport, the FRE1 and FRE7 yeast genes encode Cu(II) reductases and their transcription is repressed by Cu repletion and activated by Cu starvation (Georgatsou et al., 1997; Martins et al., 1998). Again using genetic selections in yeast, a gene encoding a second high affinity Cu(I) transporter, CTR3, was isolated as a dominant mutation which bypasses the requirement for the Ctrl transporter (Knight et al., 1996). Fortuitously, in the original strain used to isolate the CTR1 gene, the CTR3 gene had been transcriptionally silenced due to the presence of a transposable element in the CTR3 promoter region. Indeed, it appears that, although the Ctrl and Ctr3 proteins have very little sequence homology, they have at least partial overlap in Cu transport, since inactivation of both genes is required to observe Cu starvation defects in yeast. As could be predicted on the basis of the biochemistry of Cu-dependent enzymes, several phenotypes are presented in yeast cells defective in both Ctrl and Ctr3 (Dancis et al., 1994a; Dancis et al., 1994b; Knight et al., 1996). These include defective respiration (due to lack of incorporation of Cu into mitochondrial cytochrome c oxidase) and hypersensitivity to superoxide radicals

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(due to lack of Cu incorporation into Cu, Zn superoxide dismutase. Furthermore, the mechanistic link between Cu transport and Fe uptake became clear with the identification of the yeast Fet3 protein, a multi-copper ferroxidase required for the assembly of an active Fe transport complex with the Ftrl Fe permease, as the plasma membrane (Askwith et al., 1994; Stearman et al., 1996). Therefore, yeast cells defective in high affinity Cu transport, and defective in Fet3 Cu loading, are also defective in high affinity Fe transport. How are the genes involved in high affinity Cu transport in yeast regulated? As described above for the FRE-encoded Cu(II) reductases, the CTR1, CTR3 and FRE1, 7 genes are regulated in opposite directions as a function of Cu ion availability (Georgatsou et al., 1997; Hassett and Kosman, 1995; Labbe et al., 1997; Pena et al., 1998). When S. cerevisiae cells are starved for Cu ions, FRE1/7, CTR1 and CTR3 are transcriptionally activated via conserved cis-acting promoter elements called CuREs (Cu Responsive Elements) with the consensus sequence 5'-TTTGCKCR-3' (where K = G or T and R = A or G). These same elements are responsible for the down regulation of high affinity Cu transport gene expression under Cu replete conditions, which require as little as 100pM Cu added to liquid growth medium. Recently, several laboratories have identified the S. cerevisiae Macl protein as the direct Cu sensing transcription factor that binds to CuREs to activate or repress CTR1, CTR3 and FRE1/7 gene expression (Labbe et al., 1997; Martins et al., 1998; Pean et al., 1998; Yamaguchi-Iwai, Serpe, Haile, Yang, Kosman, Klausner and Dancis, 1997). Although the biochemistry of Macl Cu sensing has not been completely elucidated, evidence for both allosteric changes in the carboxylterminal Macl trans-activation domain, as well as Cu-dependent inhibition of Macl DNA binding suggest that Macl is a direct sensor of fluctuations in nutritional Cu levels (Jensen and Winge, 1998).

3. COPPER TRANSPORT IN MAMMALIAN CELLS The structural and mechanistic similarity between the baker’s yeast, S. cerevisiae, and mammals is further supported in studies of Cu ion transport. Recently, the use of S. cerevisiae cells defective in high affinity Cu transport as recipients for a human complementary DNA library resulted in the isolation of a cDNA encoding a putative high affinity Cu transporter, denoted hCtrl (Zhou and Gitschier, 1997). Yeast cells expressing hCtrl can take up Cu ions, respire and incorporate Cu into Cu, Zn superoxide dismutase. It is interesting that the human Ctrl protein exhibits sequence homology to both the S. cerevisiae Ctrl and Ctr3 proteins, suggesting that mammals may have combined the activities of these two distinct yeast transporters into a single protein molecule. From RNA blotting analysis hCtrl appears to be highly expressed in liver, heart, pancreas and prostate, however, it is expressed at readily detectable levels in virtually all human tissues examined. Where the hCtrl protein functions in human tissues, and the directionality of its Cu transporting activity, as well as how hCtrl is regulated are important issues that remain to be resolved.

4. INTRACELLULAR COPPER DISTRIBUTION The propensity of Cu ions to engage in redox chemistry that generates hydroxyl radical demands that once Cu is transported into cells, its distribution must be carefully

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controlled. Recent exciting advances have broken new ground in this mechanism. Genetic experiments in S. cerevisiae have demonstrated that small, cytosolic Cu ion binding proteins usher Cu to specific compartments, or protein targets within the cell. One such Cu “chaperone” Atxl, delivers Cu to the secretory compartment, where Cu is handed off to a P-type ATPase, Ccc2, which pumps Cu into the secretory compartment for incorporation into the Fet3 multi-copper ferroxidase (Lin et al., 1997; Pufahl et al., 1997). Another Cu chaperone, Ccs, loads Cu onto the cytosolic Cu, Zn superoxide dismutase (Culotta et al., 1997). Both Atxl and Ccs make use of a Cu binding motif, GMTCXXC, also found in Ccc2 and mammalian orthologs of Ccc2 defective in Menkes disease and Wilson disease, MNK and WND, respectively (Bull and Cox, 1994; Casareno et al., 1998; DiDonato and Sarkar, 1997; Lin and Culotta, 1995; Yuan et al., 1995). A third Cu chaperone of yeast, Cox 17, is thought to deliver Cu to a receptor in the mitochondrial intermembrane space (Glerum et al., 1996), where it is ultimately transferred to cytochrome c oxidase. Candidates for these mitochondrial Cu receptors are the Sco1/2 proteins, however, the exact mechanisms by which Coxl7, and the other Cu chaperones transfer Cu to target proteins or compartments have not been completely elucidated. Again, human orthologs for Atx1, Ccs and Cox 17 have been isolated and are functional in yeast cells (Amaravadi et al., 1997; Culotta et al., 1997; Klomp et al., 1997).

5. SUMMARY Impressive arrays of highly structurally and functionally conserved proteins have dedicated roles in Cu ion transport and distribution. Many questions remain to be answered in the field of Cu ion homeostasis. Currently, it is unclear exactly what mechanisms are used by the plasma membrane Cu transport proteins to safely move Cu ions across biological membranes. Are these proteins functioning alone or in a large metal ion-transporting complex? Further, once Cu is imported, how do Cu chaperones obtain their Cu cargo for delivery to proteins and cellular compartments? These and related questions are of great importance in formulating a comprehensive understanding of the molecular basis for Cu ion transport and distribution in all cells.

REFERENCES Amaravadi, R., Glerum, D.M., and Tzagoloff, A., 1997, Isolation of a cDNA encoding the human homolog of COX17, a yeast gene essential for mitochondrial copper recruitment, Hum. Genet. 99:329–333. Askwith, C., Eide, D., Ho, A.V., Bernard, P.S., Li, L., Davis-Kaplan, S., Sipe, D.M., and Kaplan, J., 1994, The FET3 gene of S. cerevisiae encodes a multicopper oxidase required for ferrous iron uptake, Cell 76:403–410. Bull, P.C. and Cox, D.W., 1994, Wilson disease and Menkes disease: new handles on heavy-metal transport, Trends in Genet. 10:246–252. Casareno, R.L.B., Waggoner, D., and Gitlin, J.D., 1998, The copper chaperone CCS directly interacts with copper/zinc superoxide dismutase, J. Biol. Chem. 273:23625–23628. Culotta, V.C., Klomp, L.W.J., Strain, J., Casareno, L.B., Krems, B., and Giltin, J.D., 1997, The Copper chaperone for superoxide dismutase, J. Biol. Chem. 272:23469–23472. Dancis, A., Haile, D., Yuan, D.S., and Klausner, R.D., 1994a, The Saccharomyces cerevisiae copper transport protein (Ctrlp). Biochemical characterization, regulation by copper, and physiologic role in copper uptake, J. Biol. Chem. 269:25660–25667. Dancis, A., Yuan, D.S., Haile, D., Askwith, C., Eide, D., Moehle, C., Kaplan, J., and Klausner, R.D., 1994b, Molecular characterization of a copper transport protein in S. cerevisiae: An unexpected role for copper in iron transport, Cell 76:393–402.

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DiDonato, M. and Sarkar, B., 1997, Copper transport and its alterations in Menkes and Wilson diseases, Biochem. Biophy. Acta 1360:3–16. Georgatsou, E. and Alexandraki, D., 1994, Two distinctly regulated genes are required for ferric reduction, the first step of iron uptake in Saccharomyces cerevisiae, Mol. Cell. Biol. 14:3065–3073. Georgatsou, E., Mavrogiannis, L.A., Fragiadakis, G.S., and Alexandraki, D., 1997, The yeast Frelp/Fre2p cupric reductase facilitate copper uptake and are regulated by the copper-modulated Macl activator, J. Biol. Chem. 272, 13786–13792. Glerum, D.M., Shtanko, A., and Tzagoloff, A., 1996, Characterization of COX17, a yeast gene involved in copper metabolism and assembly of cytochrome oxidase, J. Biol. Chem. 271:14504–14509. Halliwell, B. and Gutteridge, J.M.C., 1984, Oxygen toxicity, oxygen radicals, transition metals and disease, Biochem. J. 219:1–14. Hassett, R. and Kosman, D.J., 1995, Evidence of Cu(II) reduction as a component of copper uptake by Saccharomyces cerevisiae, J. Biol. Chem. 270:128–134. Jensen, L.T. and Winge, D.R., 1998, Identification of a copper-induced intramolecular interaction in the transcription factor Macl from Saccharomyces cerevisiae, EMBO 17:5400–5408. Klomp, L.W.J., Lin, S.-J., Yuan, D.S., Klausner, R.D., Culotta, V.C., and Gitlin, J.D., 1997, Idnetification and functional expression of HAH1, a novel human gene involved in copper homeostasis, J. Biol. Chem. 272:9221–9226. Knight, S.A.B., Labbe, S., Kwon, L.F., Kosman, D.J., and Thiele, D.J., 1996, A widespread transposable element masks expression of a yeast copper transport gene, Genes & Dev. 10:1917–1929. Labbe, S., Zhu, Z., and Thiele, D.J., 1997, Copper-specific transcriptional repression of yeast genes encoding critical components in the copper transcription pathway, J. Biol. Chem. 272:15951–15958. Lin, S.-J. and Culotta, V.C., 1995, The ATX1 gene of Saccharomyces cerevisiae encodes a small metal homeostasis factor that protects cells against reactive oxygen toxicity, Proc. Natl. Acad. Sci. USA 92:3784–3788. Lin, C.M. and Kosman, D.J., 1990, Copper uptake in wild type and copper metallothionein-deficient Saccharomyces cerevisiae. Kinetics and mechanism, J. Biol. Chem. 265:9194–9200. Lin, S.-J., Pufahl, R.A., Dancis, A., O’Halloran, TV., and Culotta, V.C., 1997, A role for the Saccharomyces cerevisiae ATX1 gene in copper trafficking and iron transport, J. Biol. Chem. 272:9215–9220. Linder, M.C., 1991, Biochemistry of copper, Plenum press, New York. Martins, L.J., Jensen, L.T, Simons, J.R., Keller, G.L., and Winge, D.R., 1998, Metalloregulation of FRE1 and FRE2 homologs in Saccharomyces cerevisiae, J. Biol. Chem. 273:23716–23721. Pena, M.M.O., Koch, K.A., and Thiele, D.J., 1998, Dynamic regulation of copper uptake and detoxification genes in Saccharomyces cerevisiae, Mol. Cell Biol. 18:2514–2523. Pufahl, R.A., Singer, C.P., Peariso, K.L., Lin, S.-J., Schmidt, P.J., Fahrni, C.J., Culotta, V.C., Penner-Hahn, J.E., and O’Halloran, T.V., 1997, Metal ion chaperone function of the soluble Cu(I) receptor Atx1, Science 278:853–856. Stearman, R., Yuan, D.S., Yamaguchi-Iwai, Y., Klausner, R.D., and Dancis, A., 1996, A permease-oxidase complex involved in high-affinity iron uptake in yeast, Science 271:1552–1557. Yamaguchi-Iwai, Y., Serpe, M., Haile, D., Yang, W., Kosman, D., Klausner, R.D., and Dancis, A., 1997, Homeostatic regulation of copper uptake in yeast via direct binding of MAC1 protein to upstream regulatory sequences of FRE1 and CTR1, J. Biol. Chem. 272:17711–17718. Yuan, D.S., Stearman, R., Dancis, A., Dunn, T., Beeler, T., and Klausner, R.D., 1995, The Menkes/Wilson disease gene homologue in yeast provides copper to a ceruloplasmin-like oxidase required for iron uptake, Proc. Natl. Acad. Sci. USA 92:2632–2636. Zhou, B. and Gitschier, J., 1997, hCTRl: a human gene for copper uptake identified by complementation in yeast, Proc. Natl. Acad. Sci. USA 94:7481–7486.

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CONTROL OF GENE EXPRESSION OF GLUTATHIONE PEROXIDASE-1 AND OTHER SELENOPROTEINS IN RATS AND CULTURED CELLS

Roger A. Sunde and Jacqueline K. Evenson Departments of Nutritional Sciences and Biochemistry University of Missouri Columbia, Missouri 65211 USA

1. INTRODUCTION Selenium-dependent glutathione peroxidase-1 (GPX1, EC 1.11.1.9) is but one of a number of Se-dependent parameters in mammals. We and others have found that GPX1: activity, protein and mRNA decrease exponentially, dramatically, and coordinately in progressive Se deficiency (Sunde et al., 1989). This regulation makes GPX1 the most sensitive parameter to changes in Se status over the deficient to adequate range (Sunde, 1997), and has made GPX1 arguably the parameter of choice for assessment of Se status and Se requirements. We have been engaged in the past decade in a series of experiments to understand the molecular basis of this regulation and to exploit this regulation to accurately assess nutrient requirements using a molecular approach. We hypothesize that the Se regulation of GPX1 expression is a major component of GPX1 function (Sunde, 1997), that this is linked to the mechanism that cells use to assess and regulate their Se status, and that this can be used accurately to assess Se status and requirements under conditions which are intractable to more traditional means. The objectives here are to review these experiments and what we have learned about the control points of gene expression of GPX1.

2. CONTROL POINTS OF GENE EXPRESSION The genome provides a fixed blueprint for development and metabolism, but control of this expression by environmental factors such as dietary Se plays an important role in modulating the impact of the genome. Nutrients in general and Se Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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specifically can potentially impact gene expression at six points: transcription, nuclear processing, nuclear export, translation, mRNA stability, and protein turnover. Our data shows that GPX1 expression is regulated significantly at least at three of these levels: transcription, mRNA stability and translation.

3. REGULATION OF TRANSCRIPTION There are order-of-magnitude differences in the level of GPX1 activity in different tissues in the same individual as well as in different species, in spite of having the same gene (two copies of autosomal genes) in each cell. For example, GPX1 mRNA is relatively high in rat liver and heart but low in testes whereas phospholipid hydroperoxide glutathione peroxidase (GPX4, EC 1.11.1.12) mRNA is low in liver and heart but high in testes (Lei et al., 1995). At this point, this tissue-specific control of selenoperoxidase expression is inferred to be transcriptional, just as for most other genes. There is no experimental evidence that Se status has any effect on rate of initiation of transcription for any Se-dependent gene, and there is no evidence for altered rate of nuclear processing or for export of GPX1 mRNA from the nucleus (Moriarty et al., 1998). Gender regulation of GPX1 expression can be striking. Liver GPX1 mRNA levels in female rats are twice the level found in male littermates fed the same diet (Fig. 1). And GPX1 activity levels parallel these differences. This doesn’t mean that male rats are Se deficient; in most other rat tissues, in contrast, male and female levels of activity and mRNA are similar. Interestingly, liver GPX1 mRNA levels in mice are not affected by gender (Prohaska and Sunde, 1993), indicating that this difference is not universal. Decreases in GPX1 mRNA and activity are also found in copper-deficient (Prohaska et al., 1992) rats, and most likely reflect transcriptional down-regulation. Most recently,

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we have found that vitamin E deficiency does not alter any aspect of GPX1 gene expression (Sunde et al., 1999a). These examples and other examples of apparent regulation (see below) show that it is critical to know that a marker of nutrient status is not modulated by these conditions before assuming that a lowered level of the marker indicates insufficient dietary intake of the nutrient.

4. REGULATION OF TRANSLATION In eukaryotes as well as prokaryotes, Se incorporation into selenoproteins occurs co-translationally. The selenocysteine (Sec) used in translation is synthesized while esterified to its cognate tRNA using inorganic selenophosphate and , and the position of Sec in the peptide backbone of selenoproteins is encoded by in-frame UGA codons in the mRNA (reviewed in Sunde, 1997). Availability of the rather than free Sec or inorganic Se compounds, controls translation of selenoproteins as the UGA will be interpreted as a stop codon when the concentration of is limited. This translational mechanism thus offers more possibilities for Se control of gene expression as compared to post-translational binding of a metal to a protein. The fall in GPX1 activity in Se-deficient rats was one of the supporting pieces of evidence in the original report that GPX1 is a Se-dependent enzyme (Rotruck et al., 1973). Following this, we showed that a minimal level of 0.1 µg Se/g diet elicits maximum, plateau levels of liver GPX1 activity in rats fed graded levels of dietary Se (Hafeman et al., 1974). In the intervening years, we found that GPX1 mRNA levels can also be used effectively to determine Se requirements. Starting with Se-adequate rat pups, we found in these young rapidly growing rats (Lei et al., 1995; Weiss et al., 1996) that the Se requirement for plateau levels of GPX1 mRNA is 0.05 µg Se/g diet, and that the minimum Se requirement for plateau levels of GPX1 activity (plateau breakpoint) is 0.1 µg Se/g diet (see Fig. 1). At 0.05 µg Se/g diet and above, GPX1 mRNA levels are at the plateau, so the reduced level of GPX1 activity reflects control of GPX1 translation due to limited Table 1 (Sunde et al., 1999b) shows the percent decrease in eight Se-dependent parameters in weanling Se-deficient female rats as compared to rats fed 0.2 µg Se/d diet. Also shown is the minimum dietary requirement for each of these parameters as determined by plateau breakpoint (Weiss et al., 1996); diet is sufficient to raise levels of all eight Se-dependent parameters to plateau levels in weanling rats. mRNA levels for GPX4 and selenoprotein P in these experiments reach plateau levels at diet (unpublished data). In 4-week Se deficiency in our rats, the levels of mRNA for the other selenoproteins are little affected. The impact is that translational control by Se availability is a major control point for expression of the other selenoproteins. Please note that growth was not affected in these experiments starting with Seadequate pups, showing that the Se requirement for growth is <0.007 µg Se/g diet; using crystalline amino acid diets, we have shown that the Se requirement for growth is <0.002 µg Se/g diet (Lei et al., 1995). Pups from Se-dencient dams, however, grow at 50% of the rate of Se-supplemented littermates, showing that there is an absolute Se requirement for growth (Thompson et al., 1995). We are now focusing on Se requirements in later periods of the life-cycle that are less amenable to traditional approaches. To evaluate the National Research Council

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(1995) recommendation of at least 0.4 for pregnant and lactating rats, female weanling rats were fed a Se-deficient diet supplemented with graded levels of dietary Se from 0 to 0.31 as and mated at >10 weeks (Sunde et al., 1998). At all times throughout pregnancy and lactation, liver Se and liver GPX1, plasma GPX3 and RBC GPX1 activities reached plateau levels at diet, and ribonuclease protection assay (RPA) showed that 0.05gSe/g diet was necessary for maximal GPX1 mRNA levels (Table 1). Clearly Se requirements in the pregnant and lactating rat do not increase above the requirements for rapidly growing young rats. In year-old female rats, as shown in table 1, Se requirements assessed with most parameters are further reduced as compared to young rats. The data in table 1, however, are not the whole story; we found that the magnitude of the plateau level for liver GPX1 activity in lactating rats decreases to 60% of the level in non-pregnant females, and the plateau level of GPX1 mRNA in late pregnancy and in lactation also decreases (Sunde et al., 1998). Additional dietary Se will not raise the activity above this level, indicating that availability of is not controlling translation under these conditions. Instead, control of gene expression, presumably at the transcriptional level, is reducing GPX1 mRNA levels. This experiment clearly shows the importance of understanding the control of expression of a marker used for determining dietary requirements.

5. CONTROL OF GPX1 MRNA STABILITY The regulation of GPX1 expression is unique and specific relative to other selenoproteins because of the dramatic down regulation of GPX1 mRNA level relative to levels of mRNA for other selenoproteins (Table 1). For instance, rat liver GPX4 mRNA is little affected by Se deficiency in the same animals that have GPX1 mRNA levels at 6% of Seadequate levels (Lei et al., 1995). The mechanism for this regulation is unknown. We have hypothesized that this regulation is part of the function of GPX1 and that GPX1 acts as a “biological selenium buffer” (Sunde, 1994).

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To directly study the cis-acting elements necessary for Se regulation of GPX1 mRNA levels, Weiss (1998) transfected CHO cells with GPX1 DNA constructs in which specific regions of the GPX1 gene were deleted, mutated or replaced by comparable regions from unregulated monomeric GPX4. Substitution of the GPX1 3'UTR with the GPX4 3'UTR did not impair Se regulation as measured by RPA of chimeric construct mRNA levels. Substitutions in the coding region indicate that Se regulation of GPX1 requires a UGA codon followed by an intron as well as a functional 3'UTR SECIS element. Maquat and co-workers (1998) have demonstrated recently that this degradation occurs only in the cytoplasm and that this is likely to occur via nonsense-mediated decay of mRNA. In Se deficiency, insufficient results in interpretation of UGA as a nonsense codon rather than the Sec codon. In summary, concentrations control gene expression of GPX1 not only by limiting translation but also by modulating GPX1 mRNA stability. The final control point for gene expression is protein degradation. There is no solid evidence, to date, for Se deficiency increasing the degradation of GPX1 or any other selenoprotein.

6. OTHER FACTORS CONTROLLING TRANSLATION The domain swapping experiment of Weiss and Sunde (1998) indicate that GPX1 mRNA is uniquely sensitive to degradation in Se deficiency compared to other selenoproteins, but the underlying cause for these differences is not understood. Berry and colleagues (1993) have swapped 3'-UTRs to show that changing SECIS elements can alter the efficiency of translation of deiodinase. To systematically study the role of UGA position, Wu et al. (1998) constructed a genomic GPX1 expression vector that, in COS-7 cells, over-expressed recombinant protein at levels greater than endogenously encoded selenoproteins. Mutation of ten separate codons to UGA indicated that UGA codons located in the middle of the open reading frame efficiently direct Sec incorporation. Codons located close to the 5'start or 3'-termination codons were much less efficient, but insertion of a green fluorescent protein coding region as a spacer increased the efficiency of Sec incorporation at these positions to that of the wild-type UGA position. These studies suggest that for optimum Sec incorporation, the UGA must be >21 nt from the AUG-start and >204nt from the SECIS element. In these studies, one particular UGA codon position, normally restricted in Sec insertional efficiency because it was located 81 nt from the SECIS element, had its Sec insertional efficiency increased 10-fold when this distance was increased by 711 nt. This suggest that local UGA context or RNA secondary structure may be part of the mechanism that modulates Sec insertion during translation.

7. CONCLUSIONS In summary, there are six control points for gene expression. Studies with rats clearly indicate that tissue type, gender, and pregnancy/lactation all can have dramatic effects on transcription, but there is no evidence for Se regulation of transcription. Severe mineral deficiencies can elicit modest down-regulation of GPX1 transcription, apparently secondary to the primary deficiency. Expression of GPX1 does not appear to be controlled by nuclear RNA processing or rate of nuclear export. In the cytoplasm, the spe-

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cific and unique control point for GPX1 expression is the regulation of GPX1 mRNA stability by Se status. In addition, the availability of for translation clearly regulates protein synthesis of GPX1; this step in translation is the major control point for regulation of synthesis of the other selenoproteins, as the mRNA levels for the other selenoproteins are not appreciably down-regulated when Se status is marginal. Lastly, it appears that Se status has no effect on rates of selenoprotein turnover.

ACKNOWLEDGMENTS Supported by USDA 98-35200-6051 and by University of Missouri Food for the 21st Century and MO Agricultural Experiment Station.

REFERENCES Berry, M.J., Banu, L., Harney, J.W., and Larsen, P.R. 1993 Functional characterization of the eukaryotic SECIS elements which direct selenocysteine insertion at UGA codons. EMBO J. 12:3315–3322. Hafeman, D.G., Sunde, R.A., and Hoekstra, W.G. 1974 Effect of dietary selenium on erythrocyte and liver glutathione peroxidase in the rat. J. Nutr. 104:580–587. Lei, X.G., Evenson, J.K., Thompson, K.M., and Sunde, R.A. 1995 Glutathione peroxidase and phospholipid hydroperoxide glutathione peroxidase are differentially regulated in rats by dietary selenium. J. Nutr. 125:1438–1446. Moriarty, P.M., Reddy, C.C., and Maquat, L.E. 1998 Selenium deficiency reduces the abundance of mRNA for Se-dependent glutathione peroxidase 1 by a UGA-dependent mechanism likely to be nonsense codon-mediated decay of cytoplasmic mRNA. Mol. Cell. Biol. 18:2932–2939. National Research Council 1995 Nutrient Requirements of Laboratory Animals. Pp. 1–173. National Academy Press, Washington, DC. Prohaska, J.R. and Sunde, R.A. 1993 Comparison of liver glutathione peroxidase activity and mRNA in female and male mice and rats. Comp. Biochem. Physiol. [B]. 105:111–116. Prohaska, J.R., Sunde, R.A., and Zinn, K.R. 1992 Livers from copper-deficient rats have lower glutathione peroxidase activity and mRNA levels but normal liver selenium levels. J. Nutr. Biochem. 3:429–436. Rotruck, J.T., Pope, A.L., Ganther, H.E., Swanson, A.B., Hafeman, D.G., and Hoekstra, W.G. 1973 Selenium: biochemical role as a component of glutathione peroxidase. Science 179:588–590. Sunde, R.A. 1994 Intracellular glutathione peroxidases—structure, regulation and function. In: Selenium in Biology and Human Health (Burk, R.F., ed.), pp. 45–77. Springer-Verlag, New York, NY. Sunde, R.A. 1997 Selenium. In: Handbook of Nutritionally Essential Mineral Elements (O’Dell, B.L. and Sunde, R.A., eds.), pp. 493–556. Marcel Dekker, New York. Sunde, R.A., Saedi, M.S., Knight, S.A.B., Smith, C.G., and Evenson, J.K. 1989 Regulation of expression of glutathione peroxidase by selenium. In: Selenium in Biology and Medicine (Wendel, A., ed.), pp. 8–13. Springer-Verlag, Heidelberg, Germany. Sunde, R.A., Thompson, K.M., Evenson, J.K., and Weiss, S.L. 1998 Selenium requirements based on glutathione peroxidase-1 (GPX1) activity and mRNA levels and other selenium-dependent parameters are not increased by pregnancy and lactation in rats. FASEB J. 12:A824. Sunde, R.A., Thompson, K.M., Evenson, J.K., and Weiss, S.L. 1999b Use of glutathione peroxidase-1 activity and mRNA levels and other selenium-dependent parameters to assess selenium requirements in female rats throughout the life-cycle. Proc. Nutr. Soc. (In press). Sunde, R.A., Thompson, K.M., Xia, Y., Fritsche, K.L., and Evenson, J.K. 1999a Vitamin E deficiency does not alter activities and mRNA levels of glutathione peroxidase-1 (GPX1) and other selenoproteins in severely selenium-deficient rats. FASEB J. 13:A875. Thompson, K.M., Haibach, H., and Sunde, R.A. 1995 Growth and plasma triiodothyronine concentrations are modified by selenium deficiency and repletion in second-generation selenium-deficient rats. J. Nutr. 125:864–873. Weiss, S.L., Evenson, J.K., Thompson, K.M., and Sunde, R.A. 1996 The selenium requirement for glutathione peroxidase mRNA level is half of the selenium requirement for glutathione peroxidase activity in female rats. J. Nutr. 126:2260–2267.

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Weiss, S.L., Evenson, J.K., Thompson, K.M., and Sunde, R.A. 1997 Dietary selenium regulation of glutathione peroxidase mRNA and other selenium-dependent parameters in male rats. J. Nutr. Biochem. 8:85–91. Weiss, S.L. and Sunde, R.A. 1998 Cis-acting elements are required for selenium regulation of glutathione peroxidase-1 mRNA levels. RNA 4:816–827. Wen, W., Weiss, S.L., and Sunde, R.A. 1998 UGA codon position affects the efficiency of selenocysteine incorporation into glutathione peroxidase-1. J. Biol. Chem. 273:28533–28541.

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5

CELLULAR AND SUBCELLULAR DISTRIBUTION OF SELENIUM AND SELENIUM-CONTAINING PROTEINS IN THE RAT

Dietrich Behne, Henning Pfeifer, Doris Röthlein, and Antonios Kyriakopoulos Hahn-Meitner-Institut Berlin Glienicker Str. 100, D -14109 Berlin Germany

Although it has been known for over more than forty years that selenium is essential for the mammalian organism, our knowledge of the metabolism and the functions of the element is still incomplete. In order to investigate some of the still unanswered questions, several studies have been carried out on rats. The animals were either supplied with adequate amounts of selenium or were depleted by feeding with a seleniumdeficient diet over longer periods of time, sometimes for several generations. By combining methods for trace element analysis, tracer techniques and various biochemical procedures, the distribution of selenium was determined among tissues, tissue fractions, certain types of cells, subcellular compartments and proteins. In this way information was obtained on the regulation of selenium and on new selenium-containing proteins and their sites of action. Our main findings in these two areas of research will be presented and discussed.

STUDIES ON THE REGULATION OF SELENIUM METABOLISM Selenium was found to be very unevenly distributed among the different cells. This was shown, for instance, by the analysis of the selenium concentrations in the tissues of rats which had been fed a diet with the normal level of (to be published). The values expressed as mgSe/kg dry mass were high in the kidney (5.3ppm) and in the testis (6.1 ppm) and epididymis (11.2ppm). In the latter two organs this was mainly due to the very high selenium concentrations of about 25ppm in the spermatozoa. At the Address all correspondence to: Prof. Dr. Dietrich Behne, Hahn-Meitner-Institut Berlin, Department “Trace Elements in Health and Nutrition”, Glienicker Str. 100, D-14109 Berlin, Germany; telephone: 0049-3080622784; fax: 0049-30-80622781; e-mail: [email protected] Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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other end of the scale there are tissues such as the brain, the spinal marrow and the seminal vesicle for which levels below 0.5ppm were found. However, there are not only large variations in the concentrations but also large differences in the metabolic behavior of the element in the different cells from which it can be concluded that selenium might also be of special importance in tissues with low concentrations such as the brain and the spinal marrow. The first information on these differences was obtained in an experiment in which rats were fed a selenium-deficient diet for 70 days (Behne and Höfer, 1984). After that period the selenium content in the liver and in the blood cells had decreased considerably while the changes in the testis and adrenals were relatively small. When the animals were then given a small amount of selenite, it was not the depleted tissues which retained a large proportion of the dose but retention was highest in the organs which had lost little of the element. These differences in the behavior of the tissues were even more striking when a very small dose of labeled selenite was administered to severely selenium-depleted rats of the 2nd generation and to adequately supplied controls (Behne et al., 1988). Here the deficient animals retained about 20 to 50 times more of the dose in the adrenals, the brain, the corpora lutea, the ovaries, the pituitary and the testes than the control animals. In other tissues, however, such as the blood fractions, the heart, the liver and the skeletal muscle there were only slight differences in retention between the two groups. From these findings it could be concluded that the selenium metabolism is regulated and that in periods of insufficient selenium intake the organism will strive to maintain the selenium level in certain types of cells. This is achieved by preferential supply of these cells with the element from the amounts taken up with the diet, but also by a redistribution of the metabolized element which in deficiency is excreted to a much lesser extent and is instead transported back to the priority target cells. In order to obtain information on the effects of this hierarchy on the concentrations of selenium in the different tissues during insufficient intake, we measured these levels in rats fed a low selenium diet for six generations and compared them with those of adequately supplied control animals (to be published). The selenium content of the deficient diet was less than 2% of that of the control diet and without the existence of regulatory mechanisms the selenium levels in all parts of the organism should have decreased to the same degree. It was found, however, that with some tissues the losses were even greater and led to a drastic selenium depletion, while with others the remaining concentrations were still astonishingly high. In the blood cells, the diaphragm, the seminal vesicle and the skeletal muscle the selenium levels had dropped to values below the limit of detection. In the epididymis, in the liver and also in the blood plasma they were below 1%, and in several other tissues such as the heart, the lung, the prostate and the stomach less than 3% of the levels in the control animals. In the testis, too, the protective mechanisms, which work during the first stages of deficiency, had broken down. On the other hand, as was to be expected from the results of the tracer experiments, the brain showed the smallest changes and still contained 60% of the concentration found in the control animals, followed by the spinal marrow, the pituitary, the thyroid, the ovaries and the adrenals. All these organs are thus well protected against losses during periods of insufficient selenium supply. With these cells it seems to be virtually impossible to produce a state of severe selenium deficiency, even after severe experimental depletion for six generations.

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Besides these intercellular mechanisms which control the supply of selenium to the different types of cells and are responsible for the hierarchy between the tissues, there is also an intracellular regulation of the distribution of the element. This became evident from an experiment in which we administered a small dose of labeled selenium to deficient male rats of the 4th generation and to control animals and measured the retention of the element in the subcellular fractions of the liver (Behne et al, 1990). As already found in the previous experiments, the overall retention in the liver cells was very similar in the two groups, but there were differences between the subcellular compartments. The results showed that in the deficient animals the element was preferentially incorporated into the microsomes and nuclei, while the cytosol was last in this hierarchy. This effect of the selenium status on the distribution of the element among the subcellular compartments was also observed in several other tissues. By measuring the distribution of the element among the selenium-containing proteins in the different tissues of deficient and control animals, we could then show that there is also a hierarchy at a molecular level and that with insufficient selenium intake there is a priority supply of the element to certain selenoproteins (Behne et al., 1988). In most of the tissues investigated the element was preferentially incorporated into an 18 kD-selenoprotein (to be published), while the cellular glutathione peroxidase was at the end of this hierarchy. The main findings of these studies are summarized in the scheme in Fig. 1 which shows several levels of priority with regard to the selenium distribution among the different compartments. We now know that there is an intercellular regulation and that mechanisms exist by which the organism strives to maintain the selenium concentrations in the tissues of the highest priority. Regulation also exists at a molecular level as is schematically shown by the differently shaded areas which represent different levels of priority with regard to the incorporation of the element into the selenoproteins. During the first stages of selenium depletion there is above all a decrease in the selenium concentrations in the compartments with the lowest priority level such as the blood compo-

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nents, the liver, the skeletal muscle and the heart muscle and here first a decrease in glutathione peroxidase. With progressing selenium depletion these pools are emptied almost completely. The existence of this hierarchy might explain the fact that in selenium deficiency or a combined low selenium and low vitamin E state lesions are found first in the liver, the heart and the skeletal muscle. As the selenium concentrations and the glutathione peroxidase activity of the blood fractions decrease considerably during the first stages of depletion, these parameters can serve as markers for an indadequate selenium intake but not for the selenium status and the selenium concentrations of most of the tissues. The findings also show that it is not possible to obtain information on the significance and the effects of the high priority selenoproteins from pathological changes during selenium depletion, because these proteins still have relatively high concentrations, even in severe experimental selenium deficiency. We therefore have to use other approaches and to carry out specific investigations in order to identify these proteins and to determine their biochemical functions and their sites of action.

INVESTIGATION OF NEW SELENOPROTEINS By labeling rats in vivo with 75Se, separation of the tissue proteins by SDSpolyacrylamide gel electrophoresis (SDS-PAGE) and autoradiographic determination of the tracer distribution 13 selenium-containing proteins had been found in several tissues of the rat (Behne et al., 1988). After improving these techniques, mainly by using selenite with a very high specific activity and severely selenium-depleted rats, we were able to determine selenium compounds present in the organism at very low concentrations. In this way 28 selenium-containing protein bands were detected. (Behne et al., 1996). By applying two-dimensional SDS-PAGE / isoelectric focusing some of these bands could be resolved into several spots with different isoelectric points. So far it has been possible to distinguish about 35 labeled proteins or protein subunits. Some of them were found to be present in only a few tissues or strongly enriched in an organ. Fractionation by ultracentrifugation showed that the selenium compounds were unevenly distributed among the subcellular compartments, which indicated that they are involved in different parts of intracellular metabolism. Based on the findings from these distribution studies, which provided information on some characteristics and possible sites of action, several selenium-containing proteins were chosen from the different subcellular fractions for further studies. The first compound to be investigated in more detail was a microsomal 28 kD-protein found in the thyroid, liver and kidney. We were able to show that it was identical to the subunit of the type I iodothyronine deiodinase, and in this way we could identify the deiodinase as a selenoenzyme (Behne et al., 1990). Selenium compounds from the other subcellular compartments which are at present under investigation include a nuclear 34 kD-protein, which was only localized in the sperm cells, a mitochondrial 18 kD-protein, which was detected in several tissues and a 15 kD-protein enriched in the cytosol of the prostate gland. They were all found to contain selenium in the form of selenocysteine, which is a characteristic of a genuine, genetically encoded selenoprotein.

The 18 kD-selenoprotein A selenium-containing 18 kD-band was found in the mitochondrial fraction of several tissues. In the kidney, liver and brain it was localized in the mitochon-

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drial membranes (Kyriakopoulos et al., 1996). Two-dimensional separation of the membrane proteins showed that it consists of a single protein with an isoelectric point of about 4.6–4.8. Its biological function is still unknown. However, an interesting characteristic is the fact that in the hierarchy of selenium it was found to be one of the most preferentially supplied proteins, which can be taken as an indication of its biological importance. Procedures for its purification and further investigation are at present being developed.

The 15 kD-selenoprotein After administration of to selenium-deficient rats and protein separation by SDS-PAGE a 15 kD-selenoprotein was found which was strongly labeled in the prostate gland (Kalcklösch et al., 1995, Behne et al., 1997). It showed glutathione peroxidase activity, had an apparent native molecular mass of about 250 kD and a pI value around 4.5. This selenoprotein was found to be enriched in the cytosol of the epithelial cells but was not released into the prostatic secretion. The fact that with insufficient selenium intake the incorporation of the element into this compound has priority over that into the cellular glutathione peroxidase implies an important biological function. Its further investigation is of special interest with regard to the findings of epidemiological studies which suggest an inverse relationship between the incidence of prostate cancer and the selenium status of the patients.

The 34 kD-selenoprotein In the testis of rats a 34 kD-selenoprotein was found which could not be detected in any other tissues (Behne et al., 1988). It was not present in immature animals but appeared with the onset of puberty. In several distribution studies it was localized in the nuclei of the sperm cells (Behne et al., 1997). It first appears in the nuclei of the spermatids where it is the main selenoprotein and contains about 80% of the total selenium present. The 34 kD-selenoprotein was not only found in the rat but also in the spermatid nuclei of other mammalian species including man. A purification procedure has been developed in order to isolate sufficient amounts for sequence analysis and biochemical characterization. In these studies it was identified as a further glutathione peroxidase (to be published). It is unique in that it is a nuclear selenoenzyme which is present only in nuclei of the sperm cells and most probably has a specific role in the reproductive processes. To summarize. By combining methods for trace element analysis, tracer techniques and various biochemical procedures, the distribution of selenium was investigated in the organism of the rat. In this way information was obtained on the regulation of selenium and on new selenium-containing proteins and their sites of action. Selenium was found to be unevenly distributed among tissues, certain types of cells, subcellular compartment and proteins. Regulation mechanisms were shown to exist which during insufficient intake ensure the preferential supply of selenium to certain priority target tissues such as the central nervous system and the endocrine and reproductive organs and within the cells to certain selenoproteins. In most of the tissues investigated an 18 kDselenoprotein was preferentially supplied whereas the cellular and the plasma glutathione peroxidases had lowest priority. After labeling of rats with and gel electrophoretic separation of the proteins in tissues and subcellular fractions, about 35 selenium-

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containing proteins or protein subunits could be distinguished. Of those a 15 kDselenoprotein enriched in the cytosol, a mitochondrial 18 kD-selenoprotein and a nuclear 34 kD-selenoprotein were investigated in more detail. The latter was identified as a further glutathione peroxidase which is located in the nuclei of the sperm cells.

REFERENCES Behne, D. and Hofer-Bosse, T., 1984, Effects of a low selenium status on the distribution and retention of selenium in the rat, J. Nutr. 114:1289–1296. Behne, D., Hilmert, H., Scheid, S., Gessner, H., and Elger, W., 1988, Evidence for specific selenium target tissues and new biologically important selenoproteins, Biochim. Biophys. Acta 966:12–21. Behne, D., Scheid, S., Kyriakopoulos, A., and Hilmert, H., 1990, Subcellular distribution of selenoproteins in the liver of the rat, Biochim. Biophys. Acta 1033:219–225. Behne, D., Kyriakopoulos, A., Meinhold, H., and Kohrle, J., 1990, Identification of type I iodothyronine 5'-deiodinase as a selenoenzyme, Biochem. Biophys. Res. Commun. 173:1143–1149. Behne, D., Kyriakopoulos, A., Weiss-Nowak, C., Kalcklösch, M., Westphal, C., and Gessner, H., 1996, Newly found selenium-containing proteins in the tissues of the rat, Biol. Trace Elem. Res. 35:99–110. Behne, D., Kyriakopoulos, A., Kalcklosch, M., Weiss-Nowak, C., Pfeifer, H., Gessner, H., and Hammel, C., 1997, Two new selenoproteins found in the prostatic glandular epithelium and in the spermatid nuclei, Biomed. Environ. Sci. 10:340–345. Kalcklösch, M, Kyriakopoulos, A., Hammel, C., and Behne, D., 1995, A new selenoprotein found in the glandular epithelial cells of the rat prostate, Biochem. Biophys. Res. Commun. 217:162–170. Kyriakopoulos, A., Hammel, C., Gessner, H., and Behne, D., 1996, Characterization of an 18 kDselenium-containing protein in several tissues of the rat, Am. Biotech. Lab. 14:22.

6

THE MECHANISM AND REGULATION OF ZINC TRANSPORT IN YEAST

David J. Eide Department of Nutritional Sciences University of Missouri-Columbia Columbia, Missouri 65211 USA

Zinc plays an amazing number of critical roles in cellular biochemistry. Analysis of the S. cerevisiae genome sequence indicated that almost 3% of all yeast proteins (i.e. >150 of approximately 6,000 total genes) contain potential zinc binding domains. Of these, 109 yeast genes encode transcriptional regulators containing either the zinc finger domain (Bohm et al., 1997) or the zinc cluster motif (Schjerling and Holmberg, 1996). Although zinc is an essential nutrient, it can be toxic if excess amounts are accumulated. The precise cause of this toxicity is unknown but the metal may bind to inappropriate intracellular ligands or compete with other metals for enzyme active sites, transporter proteins, etc. Therefore, in the face of fluctuating extracellular zinc levels, cells must maintain an adequate intracellular zinc level to meet cellular requirements while preventing metal ion accumulation. In this report, I describe our current understanding of these processes in the yeast Saccharomyces cerevisiae. A diagram representing an overview of this discussion is shown in Fig. 1.

1. ZINC UPTAKE IN YEAST Zinc uptake in S. cerevisiae is time-, temperature-, and concentration-dependent and saturable (Fuhrmann and Rothstein, 1968; Mowll and Gadd, 1983; White and Gadd, 1987). Kinetic studies of zinc uptake by cells grown with different amounts of zinc in the medium suggested the presence of at least two uptake systems. One system has a high affinity for zinc with an apparent of and is active in zinc-limited cells (Zhao and Eide, 1996a). The second system has a lower affinity for zinc (apparent of

Address all correspondence to: Dr. David Eide, Department of Nutritional Sciences, 217 Gwynn Hall, University of Missouri-Columbia, Columbia, MO 65211 USA telephone: 573-882-9686; fax: 573-882-0185; email: [email protected] Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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and is detectable in zinc replete cells (Zhao and Eide, 1996b). These apparent Km values are overestimates of the true Km values because they don’t consider the chelation properties of the uptake assay media. Computer-assisted thermodynamic equilibrium calculations suggest that the true are approximately 10 and 100nM for the high and low affinity uptake systems, respectively. The ZRT1 gene encodes the transporter protein of the high affinity system (Zhao and Eide, 1996a). The level of ZRT1 mRNA correlated with activity of the high affinity system. Overexpressing ZRT1 increased high affinity uptake whereas disrupting the ZRT1 gene eliminated high affinity activity and resulted in poor growth of the mutant on zinc-limiting media. In similar studies, it was determined that the ZRT2 gene encodes the transporter of the low affinity uptake system (Zhao and Eide, 1996b). More recently, the ZRT1 protein has been found to be glycosylated and localized to the plasma membrane of the cell (Gitan et al., 1998). Additional, as yet uncharacterized zinc uptake systems are also present in S. cerevisiae as demonstrated by the observation that the zrt1 zrt2 mutant is viable (Zhao and Eide, 1996b). These other systems are unlikely to be major sources of zinc under any but the most zinc replete conditions given that zrt1 zrt2 mutant cells require more zinc to grow than wild type cells (Zhao and Eide, 1996b). ZRT1 and ZRT2 share 44% sequence identity and 67% similarity. They each contain eight potential transmembrane domains and have a similar predicted membrane topology in which the amino- and carboxy-terminal ends of the protein are located on the outside surface of the plasma membrane. These proteins are also similar in sequence and predicted topology to the IRT1 transporter from Arabidopsis (Eide et al., 1996). Expression of the IRT1 gene in yeast suppresses the iron-limited growth defect of a fet3 fet4 mutant. Our current hypothesis is that IRT1 is an transporter that takes up iron from the soil, a proposal that is consistent with the observation that yeast expressing IRT1 possess a novel uptake activity. Moreover, in Arabidopsis, IRT1 mRNA is expressed in roots and is induced by iron-limiting growth conditions. In addition to

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sharing sequence similarity and numbers of potential transmembrane domains, ZRT1, ZRT2, and IRT1 each have a potential metal-binding domain between transmembrane domains three and four that is predicted to be cytoplasmic. For example, in ZRT1, this sequence is HDHTHDE and in IRT1, this motif is HGHGHGH. While the function of this motif is currently unknown, its conserved location in these three proteins and its potential for metal binding suggested that it plays an important role in metal ion uptake or its regulation. Through DNA sequence database comparisons and additional expression cloning studies, it is now clear that these three metal ion transporters are members of a family of proteins found in a diverse array of eukaryotic organisms (Eng et al., 1998). This family is referred to collectively as the ZIP family for “ZRT, IRT-like Protein.” At this time, 26 ZIP members have been identified including ten in plants (eight from Arabidopsis, one from rice, and one from pea), two in S. cerevisiae (i.e. ZRT1 and ZRT2), five in nematodes, one in Drosophila, one in mice, and two in humans. All but two of these proteins contain the putative metal-binding domain described above. Four of the Arabidopsis proteins, ZIP1- 4, may be involved in zinc transport (Grotz et al., 1998). Expressing ZIP1, ZIP2, or ZIP3 in yeast confers increased uptake activity with distinct biochemical properties. Furthermore, ZIP1, ZIP3, and ZIP4 mRNAs are induced in zinclimited plants. Based on the studies of IRT1, ZRT1, ZRT2, and ZIP1-4, it seems likely that the other proteins in this family are also metal ion transporters. This is an exciting hypothesis given that mammalian transporters responsible for zinc uptake have not yet been identified. It is currently unclear what force is used by these transporters to drive zinc uptake. Zinc uptake requires metabolic energy yet these transporters do not contain ATP binding sites nor do they have similarity to the ubiquitous P-type ATPase family of transport proteins. This observation suggests that uptake may be driven by indirect coupling to energy metabolism, perhaps through the electrical potential generated across the plasma membrane by the plasma membrane Alternatively, uptake may be driven by a transmembrane gradient of another ion such as

2. TRANSCRIPTIONAL CONTROL OF ZINC UPTAKE Zinc uptake in yeast is controlled at the transcriptional level in response to intracellular zinc levels. The high affinity system is induced more than 30-fold in zinc limited cells and results from increased transcription of the ZRT1 gene (Zhao and Eide, 1996a). The low affinity system is also regulated through the control of ZRT2 transcription (Zhao and Eide, 1997). Regulation of these genes in response to zinc is mediated by the product of the ZAP1 gene (Zhao and Eide, 1997). ZAP1 is likely to encode a transcriptional activator; the carboxy-terminal end of the protein contains seven zinc finger domains and the amino terminus has two potential activation domains (Fig. 2). ZAP1 was also found to regulate its own transcription through a positive autoregulatory mechanism. This type of regulatory circuitry would allow a rapid, amplified response to changes in zinc levels and ZAP1 activity under progressively zinc-limiting conditions. We recently described the characterization of zinc-responsive elements (ZREs) in the promoters of the ZRT1, ZRT2, and ZAP1 genes (Zhao et al., 1998). A ZRE consensus sequence, 5'-ACCYYNAAGGT-3', was identified and found to be both necessary and sufficient for zinc-responsive transcriptional regulation. We also demonstrated that ZREs are DNA binding sites for ZAP1. First, a dominant ZAP1 mutation,

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which causes increased expression of ZAP1-regulated genes in zinc-replete cells, exerted its effects specifically through the ZREs. Second, electrophoretic mobility shift assays and in vitro DNase I footprint analyses indicated that ZAP1 binds to ZREs in a sequencespecific fashion. These studies demonstrate that ZAP1 plays a direct role in controlling zinc-responsive gene expression in yeast by binding to zinc-responsive elements in the promoters of genes that it regulates. Our studies also demonstrated that there is differential zinc responsiveness among the ZRT1, ZRT2, and ZAP1 genes; i.e., significantly more zinc is required to repress the ZRT2 promoter than is required to repress the ZRT1 or ZAP1 promoters. This differential sensitivity to zinc is also consistent with the different functions of these proteins and leads us to propose the following scenario: Basal (i.e., ZAP1-independent) expression of the ZRT2 low affinity transporter is sufficient to supply zinc to cells under zincreplete conditions (Zhao and Eide, 1997). As cells first become zinc-limited, their initial response is to increase the activity of the ZRT2 transporter. If zinc limitation becomes more severe, the ZRT1 high affinity transporter is induced to provide high affinity uptake activity for zinc accumulation. Increased expression of the ZAP1 gene, which would allow maximum expression of its target genes, would only be needed under conditions of extreme zinc-limitation. The mechanism underlying the differential regulation of these ZAP1 target genes is not yet known. If zinc controls the affinity of ZAP1 for its ZRE binding sites, one possible model is that other proteins bind to the ZRT2 promoter and help stabilize binding of ZAP1 to the ZREs, thus increasing the affinity of ZAP1 for these sites. This and other possible models will be addressed in future studies. Given the size of the ZRE sequence, we propose that this site is bound by a single ZAP1 polypeptide. Our current understanding of how zinc fingers bind to DNA comes largely from x-ray crystal structures of protein-DNA complexes. In all of these structures, there are contiguous zinc finger interactions with base pairs in the major groove. In Zif268, for example, each of three fingers binds to a four bp site that overlaps the adjacent finger’s site by a single bp. We predict that three consecutive zinc fingers of ZAP1 would bind to a 10 bp sequence, similar in size to the 11 bp ZRE. Given that there are five potential zinc fingers in the DNA-binding, C-terminal 194 amino acids of ZAP1, and only three may be required for site-specific binding, we propose that the two

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additional fingers play roles in non-specific DNA binding interactions and/or in proteinprotein interactions. We have also identified two additional zinc finger motifs at amino acids 581–604 and 618–641 and our data indicate that these upstream fingers are not required for DNA binding. The functions of each of the zinc finger domains in ZAP1 are currently under investigation. An intriguing question that remains to be answered is precisely how zinc regulates ZAP1 activity. The characterization of the ZAP1 binding site in its target promoters is a critical step toward understanding this regulation. For example, in vitro and in vivo studies are now possible to determine if zinc alters ZAP1 DNA binding. The characterization of a ZRE consensus sequence also provides us with a powerful tool to identify other zinc-responsive genes in the yeast genome, made possible by the recent completion of the Saccharomyces genome sequence. Using the consensus ZRE sequence, sequence database analysis (PatMatch software; http://genome-www.stanford.edu/ Saccharomyces/) identified a total of 20 genes in the yeast genome that contain one or more ZRE-like sequences in their promoters. This list of potential ZAP1 target genes is an exciting resource for the future analysis of how eukaryotic cells respond to zinc limitation and maintain zinc homeostasis.

3. POST-TRANSLATIONAL CONTROL OF ZINC UPTAKE We have also recently identified a second, post-translational mechanism that regulates zinc transporter activity. In zinc-limited cells, ZRT1 is a stable, N-glycosylated plasma membrane protein. Exposure to high levels of extracellular zinc triggers a rapid loss of ZRT1 uptake activity and protein. This inactivation occurs through zinc-induced endocytosis of the protein and its subsequent degradation in the vacuole. Mutations that inhibit the internalization step of endocytosis also inhibited zinc-induced ZRT1 inactivation and the major vacuolar proteases were required to degrade ZRT1 in response to zinc. Furthermore, immunofluorescence microscopy showed that ZRT1 is localized to the plasma membrane in zinc-limited cells and that the protein is transferred to the vacuole via an endosome-like compartment upon exposure to zinc. ZRT1 inactivation is a relatively specific response to zinc; cadmium and cobalt ions trigger the response but less effectively than zinc. Moreover, zinc does not alter the stability of several other plasma membrane proteins. Therefore, zinc-induced ZRT1 inactivation is a specific regulatory system to shut off zinc uptake activity in cells exposed to high extracellular zinc levels thereby preventing overaccumulation of this potentially toxic metal. The zinc-induced endocytosis mechanism that we have characterized raises a number of exciting new questions. First, while it is clear that zinc induces endocytosis of ZRT1, it is unknown if this response is induced by a mechanism that senses intracellular or extracellular metal ion levels. Second, it is unclear if the signal being monitored is ions per se, the activity of a zinc-dependent or zinc-inhibited enzyme, or a more indirect consequence of high metal accumulation. The observation that and may also induce endocytosis of ZRT1 is potentially instructive. Both and have similar coordination chemistries to Zn2+ and will bind to protein ligands in a similar fashion but generally cannot replace zinc as a functional enzymatic cofactor. Therefore, the simplest hypothesis is that ions trigger endocytosis directly and that and mimic that signal. The lower activity of and in triggering the response may be due to a greater specificity of the sensing mechanism for different uptake efficiencies for different metal ions, or some other factor. A third unanswered question

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is how the zinc signal is transmitted to ZRT1. This could occur through the metal binding directly to the transporter or through an indirect signal transduction pathway.

4. THE RELATIONSHIP BETWEEN TRANSCRIPTIONAL AND POST-TRANSLATIONAL CONTROL OF ZINC UPTAKE The post-translational ZRT1 regulatory mechanism is clearly separate from the transcriptional control system given that inactivation of ZRT1 uptake activity occurs normally in a zap1 deletion mutant. However, these two systems undoubtedly work together to maintain the homeostatic control of intracellular zinc levels. It is interesting to note that the transcriptional control system exerts its greatest effect on ZRT1 expression when cell-associated zinc levels vary between 0.01 and cells (i.e., Approximately 90% repression of a ZRTl-lacZ fusion was observed when cell-associated zinc levels rose to cells (Zhao et al., 1998). In contrast, the post-translational response is triggered only at cell-associated zinc levels of greater than cells. Thus, we envision a two-tiered regulatory system in which the transcriptional control can respond to moderate changes in zinc availability and the post-translational control responds to more extreme variations. A likely scenario in which the post-translational control would be important for maintaining zinc homeostasis is when zinc-limited cells are suddenly exposed to high levels of zinc. The rapid downregulation of zinc uptake by ZRT1 endocytosis helps to prevent overaccumulation of zinc and this would not be possible solely through the transcriptional control of a stable plasma membrane protein. During inactivation of zinc uptake activity, other systems may be induced to facilitate storage of the excess zinc or mediate its efflux from the cell. In summary, the transport systems in yeast responsible for the accumulation of metal ions like zinc have been well-characterized thanks to the sophisticated genetic and molecular biological techniques available to study this organism. An overview of the recent findings in this field illuminates several consistent themes that will undoubtedly apply to metal ion transport in mammals. For example, one general feature of metal ion uptake in yeast is the presence of two or more relatively substrate-specific transport systems for the uptake of any single metal ion. High affinity systems are active in metallimiting conditions whereas low affinity systems play an important role when the substrate is more abundant. A second important theme coming from yeast studies is that metal ion uptake systems are tightly regulated through both transcriptional and posttranscriptional mechanisms of regulation. Third, and perhaps most importantly, these studies have helped us recognize the existence and importance of new families of transport proteins that appear to play related roles in all eukaryotes.

REFERENCES Bohm, S., Frishman, D., and Mewes, H.W., 1997, Variations of the C2H2 zinc finger motif in the yeast genome and classification of yeast zinc finger proteins, Nucl. Acids Res. 25:2464–2469. Eide, D., Broderius, M., Fett, J., and Guerinot, M.L., 1996. A novel iron-regulated metal transporter from plants identified by functional expression in yeast, Proc. Natl Acad. Sci. U.S.A. 93:5624–5628. Eng, B.H., Guerinot, M.L., Eide, D., and Saier, M., 1998, Sequence analyses and phylogenetic characterization of the ZIP family of metal ion transport proteins, J. Memb. Biol. 166:1–7. Fuhrmann, G.F. and Rothstein, A., 1968, The transport of Zn2+, Co2+ and Ni2+ into yeast cells, Biochim. Biophys. Acta 163:325–330.

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Gitan, R., Luo, H., Rodgers, J., Broderius, M., and Eide, D., 1998, Zinc-induced inactivation of the yeast ZRT1 zinc transporter occurs through endocytosis and vacuolar degradation, J. Biol. Chem. 273: 28617–28624. Grotz, N., Fox, T., Connolly, E., Park, W., Guerinot, M.L., and Eide, D., 1998, Identification of a family of zinc transporter genes from Arabidopsis thaliana that respond to zinc deficiency, Proc. Natl. Acad. Sci. USA 95:7220–7225. Mowll, J.L. and Gadd, G.M., 1983, Zinc uptake and toxicity in the yeast Sporobolomyces roseus and Saccharomyces cerevisiae, J. Gen. Microbiol, 129:3421–3425. Schjerling, P. and Holmberg, S., 1996, Comparative amino acid sequence analysis of the C6 zinc cluster family of transcriptional regulators, Nucl. Acids Res. 24:4599–4607. White, C. and Gadd, G.M., 1987, The uptake and cellular distribution of zinc in Saccharomyces cerevisiae, J. Gen. Microbiol. 133:727–737. Zhao, H. and Eide, D., 1996a, The yeast ZRT1 gene encodes the zinc transporter of a high affinity uptake system induced by zinc limitation, Proc. Natl. Acad. Sci. USA 93:2454–2458. Zhao, H. and Eide, D., 1996b, The ZRT2 gene encodes the low affinity zinc transporter in Saccharomyces cerevisiae, J. Biol. Chem. 271:23203–23210. Zhao, H. and Eide, D.J., 1997, Zaplp, a metalloregulatory protein involved in zinc-responsive transcriptional regulation in Saccharomyces cerevisiae, Mol. Cell. Biol. 17: 5044–5056. Zhao, H., Butler, E., Rodgers, J., Spizzo, T., Duesterhoeft, S., and Eide, D., 1998, Regulation of zinc homeostasis in yeast by binding of the ZAP1 transcriptional activator to zinc-responsive promoter elements, J. Biol. Chem. 273: 28713–28720.

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METALLOTHIONEIN FACILITATES ZINC ABSORPTION IN ZINC DEFICIENT MICE BUT LIMITS ABSORPTION IN ZINC REPLETE MICE

P. Coyle, J. C. Philcox, and A. M. Rofe Clinical Biochemistry Institute of Medical & Veterinary Science Adelaide, Australia

1. INTRODUCTION Zn absorption is thought to be facilitated by carrier mediated processes, although the transport ligands remain to be identified (Hoadley et al., 1987; Hoadley et al., 1988; Steel and Cousins, 1985; Lönnerdal, 1989). Many studies have demonstrated that Zn deficiency increases the efficiency of Zn absorption and it has been suggested that this may result from increased absorption at low Zn intakes, decreased secretion of endogenous Zn or a balance between the two (reviewed by Lönnerdal, 1989; Vallee and Falchuk, 1993). The contribution of intestinal metallothionein (MT) to the processes involved in Zn absorption and secretion is ill defined (Cousins, 1985; Lönnerdal, 1989; Davis et al., 1998). MT synthesis in mucosal cells is triggered by both fasting and high luminal Zn concentrations (Cousins, 1985; Tran et al., 1998) but is not significantly induced at normal dietary Zn intakes (Cousins, 1985; Lönnerdal, 1989; Vallee and Falchuk, 1993). It has been argued that when dietary Zn is excessive, MT sequesters Zn in the intestinal wall, transiently reducing the absorption of Zn and favouring Zn transfer back into the gut lumen (Cousins, 1985). However increased intestinal MT levels have not always been found to be associated with greater mucosal Zn accumulation, indicating that rather than act as a Zn sequestrant, MT may provide a labile Zn pool for maintaining mucosal-toluminal Zn flux (Davis et al., 1998). In this report, we examine the influence of MT on Zn absorption in response to changes in dietary Zn intake, by determining the uptake of in normal and MT-knockout mice fed Zn-replete or Zn-deficient diets.

2. MATERIALS AND METHODS MT-null (MT–/–) and normal C57BL6 (MT+/+) mice were housed together for 7 days and given free access to water and a purified egg-white diet (Coyle et al., 1999) that Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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was either Zn-replete or Zn-deficient. The Zn content of the Zn-replete and Zn-deficient diets was 100 and 0.7mgZn/kg, respectively. Mice were deprived of food for 20h prior to oral gavage with (37kBq; contained in 154 (low), 770 (normal) or 1540nmol (high) Zn as Four hours later blood was taken from each mouse by cardiac puncture and the mice were killed while under light halothane anaesthesia. Radioactivity was determined in the tissue samples and the tissue Zn uptakes corrected for the appropriate specific activities. The term “Zn transfer” is defined here as the fraction of the gavaged dose absorbed into all non-gut tissues. The recovery of the oral dose, including that in the gastrointestinal tract was 96.3 ± 0.9% (n = 132). MT was determined on homogenised samples of liver, pancreas and washed small intestine by a Cd/Haem assay (Eaton and Toal, 1982). Plasma Zn concentrations were determined by atomic absorption spectrometry. Data are presented as the mean ± SEM (n = 6–28). Significant differences (at p < 0.05) between groups were determined by the Mann-Whitney test.

3. RESULTS Plasma Zn concentrations increased in proportion to the oral Zn dose, and in MT–/– mice were approximately double those in MT+/+ mice, at the highest Zn dose

MT Facilitates Zinc Absorption in Zinc Deficient Mice but Limits Absorption in Zinc Replete Mice

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(Table 1). In general, plasma Zn levels were significantly higher in Zn-deficient than Zn-replete mice. In Zn-replete mice gavaged the highest Zn dose, the transferred dose was greater (18.4%) in MT–/– than MT+/+ mice. In Zn-deficient mice however, the presence of MT was associated with an increased Zn transfer of 40% at the normal dose. The MT+/+ mice had significantly higher Zn uptakes in liver and to a lesser extent the pancreas. In the presence of MT, the liver in the Zn-replete mice sequestered (36–40%) more Zn, and this increased by a further 50% at the lower two Zn intakes in the Zn-deficient state. In the pancreas, Zn-replete MT+/+ mice had higher Zn uptakes than MT–/– mice given the low Zn dose, and MT+/+ Zn deficient mice had uptakes 138% and 55% higher than MT–/– mice at the low and normal Zn intakes, respectively. In muscle and skin, Zn uptakes were generally higher in MT–/– mice being double those in MT+/+ mice at high Zn intakes, regardless of diet. In muscle of MT+/+ mice, Zn uptakes were higher in Zndeficient than Zn-replete mice at low and normal Zn intakes. The uptake by skin was also higher in Zn-deficient MT+/+ mice than in Zn-replete mice given the two lowest Zn doses. In MT–/– mice, non-specific binding in small intestine, liver and pancreas was <3nmol Cd bound/g wetwt. In MT+/+ mice, liver MT increased in proportion to the oral Zn dose with higher levels in Zn deficient mice at the lowest Zn dose (Table 2). At the highest Zn intake, MT in the small intestine of mice fed the Zn-deficient diet was 48% higher than levels in Zn-replete mice. Pancreatic MT levels of mice fed Zn-deficient diets were 33% lower at the lowest intake but 38% higher with the highest oral dose than their counterparts fed Zn-replete diets.

4. CONCLUSIONS It is concluded that Zn absorption is enhanced by Zn deficiency and this is in part facilitated by MT. The effect of MT to limit Zn absorption in the Zn replete state but enhance absorption in Zn deficiency appears to result from MT sequestering Zn in the gut and pancreas as well as in the liver. The higher levels of induction of pancreatic and small intestinal MT found in Zn deficient mice may be important to sequestering Zn, limiting its secretion.

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REFERENCES Cousins, R.J., 1985, Absorption, transport and hepatic metabolism of copper and zinc: special reference to metallothionein and caeruloplasmin. Physiol. Rev. 65:238–309. Coyle, P., Philcox, J.C., and Rofe, A.M., 1999, Metallothionein-null mice absorb less Zn from an egg-white diet, but a similar amount from solutions, although with altered intertissue Zn distribution. J. Nutr. 129:372–379. Davis, S.R., McMahon, R.J., and Cousins, R.J., 1998, Metallothionein knockout and transgenic mice exhibit altered intestinal processing of zinc with uniform zinc-dependent zinc transporter-1 expression. J. Nutr. 128:825–831. Eaton, D.L. and Toal, B.F., 1982, Evaluation of the Cd/hemoglobin assay for the rapid determination of metallothionein in biological tissues. Toxicol. Appl. Pharmacol. 66:134–142. Hoadley, J.E., Leinart, A.S., and Cousins, R.J., 1987, Kinetic analysis of zinc uptake and serosal transfer by vascularly perfused rat intestine. Am. J. Physiol. 252:g825–g831. Hoadley, J.E., Leinart, A.S., and Cousins, R.J., 1988, Relationship of 65Zn absorption kinetics to intestinal metallothionein in rats: Effects of zinc depletion and fasting. J. Nutr. 118:497–502. Lönnerdal, B., 1989, Intestinal absorption of zinc, in: Zinc in human biology (C.F. Mills, ed.), pp. 33–55, Springer-Verlag, London. Steel, L. and Cousins, R.J., 1985, Kinetics of zinc absorption by luminally and vascularly perfused rat intestine. Am. J. Physiol. 248:G46–G53. Tran, C.D., Butler, R.N., Philcox, J.C., Rofe, A.M., Howarth G.S., and Coyle, P., 1998, Regional distribution of metallothionein and zinc in the mouse gut. Comparison with Metallothionein-null mice. Biol. Trace Elem. Res. 63:239–251. Valee, B.L. and Falchuk, K.H., 1993, The biochemical basis of zinc physiology. Physiol. Rev. 73:79–118.

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METALLOTHIONEIN AND INNATE ACTIVATION OF PRIMARY HUMAN AND MOUSE MONOCYTES James Koropatnick1, Suzanne Dale1, and Rudolfs K. Zalups2 1

University of Western Ontario London, Ontario, Canada, and 2 Mercer University School of Medicine Macon, Georgia, USA

Group IIB metal ions exert a wide range of toxicities in human tissues and organs. They also induce a variety of genes (including those encoding metallothionein [MT]), through signal transduction mechanisms that do not require overt toxicity for effect. Monocyte/macrophages undergo activation in response to extracellular signals that are received, transduced to the nucleus, and processed to trigger transcription and/or repression of genes. We hypothesized that zinc (Zn) ions can affect the capacity of primary monocytes to respond to activation signals, and that they do so at concentrations that have no discernible toxic effect. Zn is required for normal leukocyte development and function. Zinc-deficient animals and humans have reduced levels of markers of macrophage activation (Wirth et al., 1989; Vruwink et al., 1991; Briggs et al., 1982). On the other hand, zinc supplementation has been reported to have inhibitory effects: macrophage and monocyte activation, measured by a variety of parameters, has been reported to be suppressed by in vitro zinc treatment (Nakamura et al., 1987; Chvapil et al., 1977; Kiremidjian-Schumacher et al., 1981; Kazimierczak et al., 1974; Marone et al., 1981). Immunomodulation, and not only cytotoxicity, may be a consequence of exposure to metal salts. We tested the hypothesis that treatment of primary mouse and human monocytes with low, non-toxic doses of affects the ability of the cells to be activated, as assessed oxygen free radical production and induction. We have reported that metallothionein (which is induced by metal pretreatment in primary and immortalized monocytes) is required for activation of immortalized human Address all correspondence to: Dr. James Koropatnick, Cancer Research Laboratories, London Regional Cancer Centre, 790 Commissioners Road East, London, Ontario, CANADA N6A 4L6; telephone 519-6858654; fax: 519-685-8646; email: [email protected] Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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monocytes by LPS or PMA (Leibbrandt et al., 1994). Disregulation of metallothionein— the major intracellular binder of transition metals in eukaryotic cells—leads to altered ability to respond to activation signals in human monocyte/macrophages. A role for metallothionein in supplying essential zinc and/or copper to metalloproteins (including transcription factors, hormone receptors, metalloproteinases, superoxide dismutase, catalase, among many others), or in sequestering zinc to inactivate metal-dependent molecules has been suggested (Zeng et al., 1991). To determine whether MT plays a physiological role in activation of primary monocytes, we tested the capacity of leukocytes isolated from peripheral blood of mice with genetically-ablated MT-1 and MT-2 genes to undergo activation in response to PMA.

RESULTS AND DISCUSSION Non-Toxic Zinc Treatment and Immune Cell Activation Primary mouse monocytes were isolated from the pooled blood of ten 1 month old CD-1 mice (Leibbrandt and Koropatnick, 1994) and incubated with different concentrations of zinc chloride for 45 minutes prior to activation with PMA (50 ng per ml for 45 minutes, as described previously (Leibbrandt and Koropatnick, 1994; Koropatnick and Zalups, 1997). At the end of the PMA treatment, respiratory burst was measured by a luminol chemiluminescence assay (Sozzani et al., 1995). (of which increased levels are a measure of monocyte activation) was assessed in total cellular RNA isolated from primary monocytes. The data are shown in Figure 1 and Table 1. pretreatment of primary mouse monocytes in vitro inhibited PMA-induced respiratory burst, and basal levels. inhibited PMA-induced levels. The zinc levels used had no toxic effect on the cells, as assessed by their capacity to exclude trypan blue dye and to incorporate radiolabeled CTP in a run-on transcription assay in isolated nuclei (data not shown). The results indicate that low levels of zinc that have no discernible cytotoxic effects on primary mouse monocytes can still inhibit in vitro

Metallothionein and Innate Activation of Primary Human and Mouse Monocytes

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activation (as measured by respiratory burst and induction) by phorbol ester. Therefore, alteration in immune cell behaviour can be caused by low, physiological zinc levels that have no measurable effect on cell viability, an observation that is consistent with previously reported results using immortalized human monocytes (Koropatnick and Zalups, 1997). We have previously reported that antisense downregulation of MT expression in immortalized human monocytes abolishes their capacity to undergo activation in response to LPS (Leibbrandt et al., 1994). To extend these experiments into the in vivo situation, and to assess signal responsiveness in the complete absence of metallothionein, we investigated the capacity of primary monocytes isolated from MT-1/MT-2 gene knockout mice. Primary mouse monocytes were isolated from the pooled blood of ten 1 month old (5 male plus 5 female) MT knockout mice and genetic background-matched wild-type mice (Masters et al., 1994), obtained from Jackson Laboratories (Bar Harbor, Maine). Monocytes were immediately activated with PMA (but without metal pretreatment), as described for Figure 1. Respiratory burst was measured by the luminol chemiluminescence method. Data is shown in Figure 2. There was a greater than 50% decrease in respiratory burst in the MT knockout monocytes compared to wild-type monocytes. Over 4 independent experiments, the PMA-activated respiratory burst varied from 50% of control down to 18% of control.

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Therefore, monocytes isolated from MT knockout mice were severely deficient in activation potential in response to phorbol ester. Metallothionein appears to be necessary for the innate immune response in leukocyte subpopulations. The mechanism by which it mediates responses is under investigation.

REFERENCES B, W.A., Pedersen, M.M., Mahajan, S.K., Sillix, D.H., Prasad, A.S., and McDonald, F.D. (1982). Lymphocyte and granulocyte function in zinc-treated and zinc-deficient hemodialysis patients. Kid Int. 21, 827–832. Chvapil, M., Stankova, L., Zukoski IV, C., and Zukoski III, C. (1977). Inhibition of some functions of polymorphonuclear Leukocytes by in vitro zinc. J. Lab. Clin. Med. 89, 135–141. Kazimierczak, W. and Maslinski, C. (1974). Histamine release from mast cells by compound 48/80. The membrane action of zinc. Agents Actions 4, 320–323. Kiremidjian-Schumacher, L., Stotzky, G., Likkite, V., Schwartz, J., and Dickstein, R.A. (1981). Influence of cadmium, lead and zinc on the ability of guinea pig macrophages to interact with macrophage migration inhibitory factor. Env. Res. 24, 106–116. Koropatnick, J. and Zalups, R.K. (1997). Effect of non-toxic mercury, zinc or cadmium pretreatment on the capacity of human monocytes to undergo lipopolysaccharide-induced activation. Br. J. Pharmacology 120, 797–806. Leibbrandt, M.E.I., Khokha, R., and Koropatnick, J. (1994). Antisense down-regulation of metallothionein in a human monocytic cell line alters anherence, invasion, and the respiratory burst. Cell Growth Differ. 5, 17–25. Leibbrandt, M.E.I, and Koropatnick, J. (1994). Activation of human monocytes with lipopolysaccharide induces metallothionein expression and is diminished by zinc. Toxicol. Appl. Pharmacol. 124, 72–81. Marone, G. et al. (1981). Modulation of histamine release from human basophils in vitro by physiological concentrations of zinc. J. Pharmacol. Exp. Ther. 217, 202–298. Masters, B.A. et al. (1994). Targeted disruption of metallothionein I and II genes increases sensitivity to cadmium. Proc. Nat’l Acad. Sci. (USA) 91, 584–588. Nakamura, T., Shiraishi, N., and Aono, K. (1987). Effects of in vitro and in vivo supplementation with zinc on superoxide anion production in leukocytes. Physiol. Chem. Phys. Med. NMR 19, 147–151. Sozzani, P. et al. (1995). Interleukin-13 inhibits protein kinase C-triggered respiratory burst in human monocytes. Role of calcium and cyclic AMP. J. Biol Chem. 270, 5084–5088. Vruwink, K.G., Fletcher, M.P., Keen, C.L., Golub, M.S., Hendrickx, A.G., and Gershwin, M.E. (1991). Moderate zinc deficiency in rhesus monkeys. An intrinsic defect on neutrophil chemotaxis. J. Immunol. 146, 244–249 (1991). Wirth, J.J., Fraker, P.J., and Kierszenbaum, F. (1989). Zinc requirement for macrophage function: effect of zinc deficiency on uptake and killing of a protozoan parasite. Immunol. 68, 114–119. ZENG, J. et al. (1991). Zinc transfer from transcription factor IIIA fingers to thionein clusters. Proc. Nat’l Acad. Sci. (USA) 90, 8013–8017.

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ROLE OF METALLOTHIONEIN ON ZN, CU, CD, AU, AND AG ACCUMULATION IN HEPATIC CYTOSOL OF HEAVY METAL-INJECTED RATS

S. Saito and K. Yoshida Department of Preventive Medicine St. Marianna University School of Medicine 2-16-1, Sugao, Miyamae-Ku Kawasaki, 216-8511, Japan

SUMMARY To examine the role of metallothionein (MT) on heavy metal accumulation in hepatic cytosol of rats, this study was carried out to determine the relative Zn, Cu, Cd, Au and Ag-binding capacities of MT in hepatic cytosol of Zn, Cu, Cd, Au and Aginjected rats, respectively. Our results demonstrated that approx. 60% of the Zn, Cu or Ag increments in the hepatic cytosol of Zn, Cu or Ag-injected rats was bound to MT, while approx. 80 or 4% of the Cd or Au increment in the hepatic cytosol of Cd or Auinjected rats was bound to MT. Therefore we suggested the order of the relative capacity in vivo of MT was determined for several metals (Cd > Zn = Ag > Cu > Au). These results suggested that the role of MT in Zn, Ag or Cu accumulation in the liver of Zn, Ag or Cu-injected rats was different from that of MT in Cd or Au accumulation in the liver of Cd or Au-injected rats.

1. INTRODUCTION Metallothionein (MT) is characterized by a low molecular weight (6,500–7,000 Da), a high affinity for heavy metals such as zinc (Zn), cadmium (Cd), copper (Cu) and silver (Ag), a high cysteine content and a lack of aromatic amino acids (Kägi, 1993). A remarkable feature of MT is its inducibility by several heavy metals, hormones, cytotoxic agents, various physiological conditions associated with physical or chemical stresses and X-ray irradiation. The physiological function of MT is not completely understood but appears Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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to be associated mainly with detoxification of heavy-metal ions, e.g. Cd (Kojima and Kägi, 1978), and homeostasis of essential metals, e.g. Zn and Cu (Hamer, 1986; Kägi and Schaffer, 1988). In order to estimate the role of MT in heavy metal accumulation in hepatic cytosol of rats, this study was carried out to determine the relative Zn, Cd, Cu, Au and Ag-capacities of MT (the ratio of heavy metal content associated with MT to increased heavy metal content in hepatic cytosol) of Zn, Cd, Cu, Au and Ag-injected rats, respectively.

2. MATERIALS AND METHODS Fifty-one male Sprague-Dawley rats were injected i.p. with a single dose of (1, 5, 10, and 20mgZn/kg b.w.), (2, 4, and 6mgCu/kg b.w.), CdC12 (1, 2 and 3mg Cd/kg b.w.), (10, 2 0 and 40mg Ag/kg b.w.) or (5, 10 and 20mg Au/kg b.w.), respectively. They were killed 14h after injection by anesthesia with diethylether. The livers were removed immediately and stored at –20°C until use. One gram of each liver was digested with mixed acids (1ml 5ml and 10ml The Zn, Cu, Ag and Au were measured with a flame atomic absorption spectrophotometer and the Cd was assayed using an inductively coupled argon plasma-atomic emission spectrometer. Five grams of fronzen liver were thawed at 4°C, cut into pieces and homogenized (2:1 = v: w) in ice-cold 50mM Tris/HCl, pH 8.1, with a polytron three times for 30sec. The homogenate was centrifuged at 10,000 × g for 30min at 4°C. The supernatant was centrifuged at 110,000 × g for 60min at 4°C. The cytosol was applied to a Sephadex G-75 column, equilibrated with 10mM Tris/HCl, pH 8.1, and eluted with the same buffer at a flow rate of approx. 30ml/h at 4°C. The eluent was collected in 10ml fractions and assayed for Zn, Cu, Cd, Ag and Au concentrations with a flame atomic absorption spectrophotometer. The data with 95% confidence interval from the comparison of two regression slopes were statistically analysed using the Statview II program on a Macintosh computer. If 95% confidence intervals did not overlap, the difference between the values was considered significant at P < 0.05. The data were also compared by an unpaired Student’s t-test, and a probability value of P < 0.05 was accepted as significant.

RESULT AND DISCUSSION The amounts of the Zn, Cu and Ag increments were attributable to MT and high molecular weight proteins, while most of the Cd or Au increment was ascribed to MT or high molecular weight proteins (Saito and Kojima, 1997; Saito et al., 1998; Saito and Yoshida, 1998; Saito et al, 1999). There were close relationships between heavy metal contents of the cytosol and MT in all heavy metal-injected rats (Table 1). Our results demonstrated that approx. 60% of the Zn, Cu or Ag increment in the hepatic cytosol of Zn, Cu or Ag-injected rats was bound to MT, while approx. 80 or 4% of the Cd or Au increment in the hepatic cytosol of Cd or Au-injected rats was bound to MT. Therefore we suggested the order of the relative capacity in vivo of MT was determined for several metals These results suggested that the role of MT in Zn, Ag or Cu accumulation in the liver of Zn, Ag or Cu-injected rats was different from that of MT in Cd or Au accumulation in the liver of Cd or Au-injected rats.

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REFERENCES Kägi, J.H.R., 1993, Overview of metallothionein. Methods Enzymol. 205:613–626. Kojima, Y. and Kägi, J.H.R., 1978, Metallothionein. Trends Biochem. Sci. 3:90–93. Hamer, D.H., 1986, Metallothionein. Annu. Rev. Biochem. 55:913–951. Kägi, J.H.R. and Schaffer, A., 1988, Biochemistry of metallothionein. Biochemistry 27:8509–8515. Saito, S. and Kojima, Y, 1997, Differential role of metallothionein on Zn, Cd and Cu accumulation in hepatic cytosol of rats. Cell Mol. Life Sci. 53:267–270. Saito, S., Okabe, M., Kurasaki, M., Saito, T., and Saito, K., 1998, Role of metallothionein on Zn, Cu and Cd accumulation in rat liver after heavy metal injection. Trace Elements Electrolytes 15:58–64. Saito, S. and Yoshida, K., 1998, The effect of gold on zinc in liver and in metallothionein. Res. Commun. Mol. Pathol Pharmacol. 100:83–91. Saito, S., Okabe, M., Yoshida, K., and Kurasaki, M., 1999, Role of metallothionein on Ag accumulation in hepatic and renal cytosol after Ag injection to rats, Pharmacol. Toxicol. in press.

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MOLECULAR CLONING OF A HUMAN CDNA REGULATED BY HEAVY METALS M. G. Pagliuca, S. Cigliano, R. Lerose, and A. Leone Department of Pharmaceutical Sciences Via ponte Don Melillo Fisciano 84080, Salerno Italy

Metals induce in eukaryotes the transcription of two major gene families: the Metallothioneins and the Heat Shock Proteins (Hsp). Metallothioneins constitute a class of low-molecular-weight, cysteine-rich metalbinding stress proteins which are biosyntheticaly regulated at level of gene transcription in response to metals, hormones, cytokines and other physiological and enviroment stresses (Tamai et al., 1994) Induction by metals of MTs and Hsp proteins, mainly of the Hsp 70 form has been related to the protection of the cellular machinery from the metal-induced cellular injury, in association with intracellular glutathione (GSH) levels (Abe et al., 1998). With the aim to isolate new genes involved in the cellular stress response to metals, we isolated from a zinc-induced HeLa cell cDNA library, enriched for long transcripts, some cDNA clones, using as probe the coding region of the monkey MTI cDNA. Sequence analysis of some of them showed high homology to MT sequences; among the isolated clones, we focused our attention on F23 clone, due to its responsiveness to metals (see below). This clone, about l,500 nt long, contained at the 5’end a region highly homologus to human hMTI-e and at the 3’end sequences not present in the EMBL data bank.The nucleotide and amino acid sequences of F 23 clone are showed in Fig. 1. Further analyses demostrated that during the construction of the library the hMTIe sequences were aberrantly ligated to another fragment of cDNA, which contained sequences non present in computer data bank. This fragment, denominated F23, lenght 1,000 nt, was further characterized in detail. Southern blot experiments carried out on EcoRI digested HeLa genomic DNA showed that this gene appeared to be present in single copy. Furthermore, the expression of F23 was strongly dependent upon metal induction with zinc (250mM), copper (250mM and 500mM) and cadmium (10mM) (Fig. 2). In

Address all correspondence to: Pagliuca Maria Gabriella, Via Pansini 5, Napoli. Telephone: 0039-081-7463205, Fax 0039-O81-7463150; email: [email protected]

Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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human tissues the F23 mRNA is mainly accumulated in the skeletal and cardiac muscle and less in testicle, lung and placenta. The deduced amino-acid sequence of F23 is very similar (63%) to the skigene product. This gene is mainly involved in the differentiation of skeletal muscle (Claycom and Lanson, 1987). Moreover we found that through tridimensional computer analysis, the F23 deduced protein sequence was also similar to the SOOD protein, SOOD means: silencer of death domains, which binds the death domains of TNF-R1, controlling the

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signaling of apotosis and NF-KB activation (Jang et al., 1999). Experiments are in progress to obtain the full-size cDNA and to define its complete deduced aminoacid sequence.

ACKNOWLEDGMENTS This work was supported by C.N.R., PF “Biotechnology”.

REFERENCES Abe, T., Yamamura K., Gotoh, S., Kashimura M., and Higashi K., 1998, Concentration-dependent differential effects of N-acetyl-L-cysteine on the expression of HSP70 and metallothionein genes induced by cadmium in human amniotic cells, Biochimica et biophhysica—Acta Vol 1380, pp. 123–132.

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Claycomb, W.C. and Lanson, N.A., 1987, Proto-oncogene expression in proliferating and differentiating cardiac and skeletal muscle, Biochem.J., Vol 247, pp. 701–706. Jiang Y., Woronicz, J.D., Liu, W., and Goeddel, D.V., 1999, Prevention of constitutive TNF Receptor 1 Signaling by Silencer of Death Domains, Science, Vol 283, pp. 543–546. Tamai, K.T., Liu, X., Silar P., Sosinowski, T., and Thiele D.J., 1994, Heat Shock Transcription Factor Activates Yeast Metallothionein Gene Expression in Response to Heat and Glucose Starvation via Distinct Signalling Pathways, Molecular and Cellular Biology, Vol 14 no.12 pp. 8155–8165.

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DISTRIBUTION OF COPPER TRANSPORTED ATP7B IN EMBRYO AND NEW BORN RAT T. Hosokawa1, M. Okabe2, M. Kurasaki2, A. Hata3, F. Endo4, K. Nagano4, I. Matsuda4, K. Urakami5, and T. Saito6 1

Research Division for Higher Education Center for Research and Development in Higher Education Hokkaido University Sapporo 060-0809 Japan 2 Department of Environmental Medicine and Informatics Graduate School of Environmental Earth Science Hokkaido University Sapporo 060-0811 Japan 3 Department of Public Health Asahikawa Medical School Asahikawa 078-8510 Japan 4 Department of Pediatrics Kumamoto University School of Medicine Kumamoto 860 Japan 5 Terumo Corporation Research and Development Center Terumo Corporation Kanagawa 259-01 Japan 6 Department of Hygiene and Preventive Medicine Hokkaido University School of medicine Sapporo 060-8638 Japan

1. INTRODUCTION Copper (Cu) is an essential trace element and is reported to play remarkable roles in living organisms (Agarwal et al., 1989). For studies on abnormal Cu metabolism in mammals, the Long-Evans Cinnamon (LEC) rat is used as an animal model of Wilson disease, a genetic disorder of the Cu metabolism in humans (Li et al., 1991; Saito et al., Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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1995; Wilson, 1912). A Cu transported protein, ATP7B is recognized to be responsible gene for Wilson disease. However the timing of expression of the ATP7B is not known. The functions of ATP7B in development have not been elucidated, and it is reasonable to consider that such functions might be different from that of the protein in adults. In the present study, we investigated the histochemical distribution of ATP7B in the embryo and newborn of the Long-Evans agouti (LEA) rat, which is a control strain of the LEC rat, using an confocal laser microscopy. Furthermore, the obtained results were compared with the distribution of ATP7B in adult rat which were given by other researchers.

2. METHOD Day 18 and 20 embryos (El 8, E20) and newborn (NB) were obtained from LEA rats. After receiving transcardial perfusion they were stocked at –80 °C. All procedures were performed in accord with the regulations defined by the NIH guides for the care and use of laboratory animals. The obtained El8, E20 and NB were cut in the cryostat microtome. The sections were mounted on glass slides and fixed with methanol. The immunohistochemistry of ATP7B was carried out by the method previously described (Saito et al., 1999). In briefly, monoclonal antibody against ATP7B as the first antibody and biotinilated anti mouse IgG as the secondary antibody were used. Avidine conjugated FITC was employed for detection of immunohistochemical signals with a confocal microscope.

3. RESULTS AND DISCUSSION The immunoreactivity against an ATP7B monoclonal antibody was detected in liver, heart, kidney, brain and intestine. In the liver, although the signals of ATP7B were observed in El8 and E20, the expression amount of ATP7B was estimated to be lower than that of the protein in NB as shown in Fig. 1. The similar results were obtained in the case of the intestine. It was reported that ATP7A and ATP7B was detected in liver of embryo and adult, respectively (Kuo et al., 1997; Yamaguchi et al., 1994). In contrast, ATP7A in normal animal liver was not detected (Murata et al., 1997). In the heart, there

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is no major difference of ATP7B signal between embryos (E18, E20) and NB. In all cases ATP7B signals were localized at the endocardiac wall. In adission, signal of ATP7B in the kidney of NB was detected more intensely than that of adult rat (Yamaguchi et al., 1994). It is noted that intense signals of ATP7B were detected in neurons in El8 and NB. Cu transporting was speculated to contribute the important role on the neuronal development. In conclusion, The obtained distribution of ATP7B in both new born and embryo was slightly different from that in adult LEA rat. On the other hand, there are no major difference of the distribution between embryo and new born. The results suggested that the ATP7B functioned primarily in the homeostatic maintenance and/or in biosynthesis of distinct Cu-protein in these organs. Further investigation will be needed to elucidate the function of ATP7B in each organ during the development.

REFERENCES Agarwal, K., Sharma, A., and Talukdar, G., 1989, Effects of copper on mammalian cell components, Chem. Biol Interact. 69:1–16. Kuo, Y.M., Gitschier, J., and Packman, S., 1997, Developmental expression of the mouse mottled and toxic milk genes suggests distinct functions for the Menkes and Wilson disease copper transporter, Hum. Mol. Genet. 6:1043–1049. Li, Y, Togashi, Y, Saito, S., Emoto, T., Kang, J.H., Takeichi, N., Kobayashi, H., Kojiima, Y, Une, Y, and Uchino, J., 1991, Sopntaneous hepatic copper accumulation in LEC rats with hereditary hepatitis: a model of Wilson’s disease, J. Clin. Invest. 87:1858–1861. Murata, Y, Kodama, H., Abe, T., Ishida, N., Nishimura, M., Levinson, B., Gitschier, J., and Packman, S., 1997, Mutation analysis and expression of the mottled gene in the macular mouse model of Menkes disease, Pediat. Res. 42:436–442. Saito, T., Itoh, T., Fujimura, M., and Saito, K., 1995, Age-dependent and region-specific differences in the distribution of trace elements in 7 brain regions of Long-Evans Cinnamon (LEC) rats with hereditary abnormal copper metabolism, Brain Res. 695:240–244. Saito, T., Okabe, M., Hosokawa, T., Kurasaki, M., Hata, A., Endo, F., Nagano, K., Matsuda, I., Urakami, K., and Saito, K., 1999, Immunohistochemical determination of the Wilson copper-transporting P-type ATPase in the brain tissues of the rat, Neuroscience Letters, in print. Wilson, S.A.K., 1912, Progressive leticular degeneration: a familial nervous disease associated with cirrhosis of the liver, Brain 34:295–509. Yamaguchi, Y, Heiny, M.E., Shimizu, N., Aoki, T., and Gitlin, J.D., 1994, Expression of the Wilson disease gene is deficient in the Long-Evans Cinnamon rat, Biochem. J. 301:1–4.

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INTERACTION BETWEEN COPPER AND IRON METABOLISM IN THE HUMAN INTESTINAL CACO-2 CELLS

I. Gabrielli, Y. Sambuy, S. Ferruzza, and M. L. Scarino Istituto Nazionale della Nutrizione Via Ardeatina 546 00179 Rome, Italy

It has long been known that copper deficiency impairs iron absorption, leading to an iron-resistant anemia. In recent years, links between iron uptake and cellular copper homeostasis have been established in yeast, by cloning iron transporters which require copper. However, in higher eucaryotes like mammals, such direct relationship has yet to be defined. We used the human intestinal in vitro model, the Caco-2 cell line, to investigate how modifications in cellular copper homeostasis could modulate intestinal iron absorption. The Caco-2 cell line is a human cultured cell line derived from a human colon adenocarcinoma, that undergoes in culture a process of spontaneous differentiation that leads to the formation of a monolayer of epithelial cells displaying several morphological and functional characteristics of the mature enterocytes. This cell model has extensively been utilized for the study of trans-epithelial passage and intracellular metabolism of several nutrients, including trace elements. Caco-2 cells were grown and allowed to differentiate on filters to reproduce in vitro the conditions of the intestinal mucosal cells in vivo. In copper loading experiments was added to complete basolateral medium for 18h. Under these conditions a ten fold increase of intracellular copper content was achieved, without modifications in the integrity of the monolayer, as monitored by measuring trans-epithelial electrical resistance (TEER) after copper loading. Apical ferrireductase activity, apical uptake of either as Fe(II) ascorbate or Fe(III)NTA and iron transport across the cells to the basolateral compartment were studied on Cu-loaded cells. Results showed that copper loading did not alter AP ferrireductase activity nor AP uptake of either Fe(II) or Fe(III). However, transport of iron,

* Email [email protected]

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either given to the cells as Fe(II) or Fe(III) was significantly enhanced by copper treatment as compared to control. In order to remove copper from the Caco-2 cells, increasing concentrations of a high affinity Cu(II) cage chelator (DIAMSAR) were added to the AP serum-free medium and tight junctions integrity was monitored by TEER measure. Intracellular copper concentrations were also measured by atomic absorption spectrometry. Treatment of the Caco-2 cells for 28 h with DIAMSAR resulted in a depletion of 50% as compared to control cells grown in complete medium. Under these conditions no alterations of tight junction permeability, as measured by TEER, were observed. Since data in literature have shown that copper depletion in HeLa cells is able to modify iron uptake, we are planning to use the Cu-depleted Caco-2 cells to clarify the effects of copper deficiency on iron absorption in the intestinal cells. This work was supported by European Community, Contract FAIR CT 95 0813 “FOODCUE” and by COST action D8/0006/97.

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INDUCTION OF METALLOTHIONEINS BY ZINC (Zn) SUPPLEMENTATION QUANTIFICATION IN YOUNG RAT TISSUES

G. S. Henriques and S. M. F. Cozzolino Faculdade de ciencias farmaceuticas (School of Pharmacy) University of Sao Paulo Sao Paulo Brazil

The induction of metallothioneins (MTs) has been done with relatively high doses of zinc added to diet and other sources. This work intends to quantify MTs in tissues of rats fed with different concentrations of zinc, in order to check animal nutritional response to the metal. Therefore, 32 Wistar newly-weaned male rats were distribute into 4 experimental groups and housed in individual cages. They had free access to tap water and diets with different concentrations of Zn (DC and DH were unsupplemented and DCS and DHS supplemented). After four weeks, the animals were sacrificed and liver, kidneys femurs and testis were isolated to assay the content in MTs, by Cd/Hb affinity assay. Zn content was determined by flame absorption spectroscopy (FAAS). The results showed that zinc supplementation did not change Mts’s profile in the tissues under analysis, in the doses studies and during the time therein adopted, MTs’s greater concentration in the liver stands out its central function in zinc metabolism and metalloproteins that coordinate it. Significant increases in zinc concentrations in liver tissue DCS and tissue DHS) and femurs tissue DCS and 86.57 tissue DHS) of young supplementad rats point to a distribution model for zinc between its storage tissues and those with great metabolic activity.

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IRON, OXIDATIVE STRESS, THE HFE GENE, AND LUNG CANCER

J. M. McCord, S. K. Bose, and B. Gao Webb-Waring Institute University of Colorado Denver, Colorado 80262, USA

1. INTRODUCTION Iron is physiologically essential, yet biochemically dangerous. The most common cause of infant death in the U.S. by accidental poisoning is the ingestion of ferrous sulfate tablets. After the recognition of iron as an essential nutrient, the faulty assumption followed that “more is better”. This assumption persists today, even among some physicians and nutritionists. Accordingly, for several decades most nations have produced “ironfortified” foods in an attempt to eliminate iron-deficiency (which afflicts less than 10% of the U.S. population) with little appreciation of what this supplementation might do to the remaining 90% of the populace who are in a state of iron excess. For most of us, iron supplementation results in ever increasing stores of excess iron in the body. Evolution has given us no mechanism for the elimination of excess iron. As a result, cells store excess iron in a complex with the protein ferritin. This protein is found in all tissues, but especially in liver and spleen. If the body experiences a sudden and significant loss of blood, these stores are drawn upon for the synthesis of new hemoglobin. In modern society, transfusion has supplanted even this need. Hence, excessive iron stores serve no known useful function to an otherwise healthy body. Iron metabolism and superoxide metabolism are clearly interactive, especially under pathological conditions.1 Each can exacerbate the toxicity of the other. A major mechanism by which superoxide contributes to oxidative stress appears to involve its ability to liberate redox active iron from storage proteins2 or from proteins containing Fe/S centers.3 This liberated iron may then repeatedly initiate chains of lipid peroxidation by redox cycling. Iron overload has been seen to amplify the damaging effects of superoxide overproduction in a broad spectrum of inflammatory,4 ischemia-related,5 or cancer-related conditions.6 Furthermore, chronic oxidative stress may encourage increased iron uptake and storage,7 leading to an environment more likely to produce cytotoxic and potentially mutagenic events. Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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While iron status is known to be a factor affecting survival time in patients with cancer, it has been less clear whether high iron stores per se predispose for cancer. Homozygotes for the C282Y mutation in the hemochromatosis gene (known as HFE or HLA-H) have a clearly increased risk of liver cancer8 (as well as cardiomyopathy, diabetes, and cirrhosis), but the association between iron overload caused by heterozygosity at this locus (reflected by transferrin saturation level) and various other types of cancer has been inconsistent.9,10 Using restriction fragment length polymorphism (RFLP) analysis, we have determined the HFE genotype at the 282 locus for 44 patients with lung cancer to determine whether these patients show a higher than expected incidence of heterozygosity at this locus.

2. MATERIALS AND METHODS Samples of purified genomic DNA from 44 patients with lung cancer were generously provided by the Tissue Procurement Core of the University of Colorado Cancer Center. A 400 bp segment of the HFE gene containing the C282Y mutation site was amplified using the oligoprimers described by Feder et al.11 The polymerase chain reaction (PCR) was performed using Taq polymerase (Gibco-BRL) with 1 mM The initial denaturation was at 94°C for 5min, followed by 30 cycles of 94°C for 1min, 55 °C for 1 min, 73 °C for 1 min, and a final extension step of 73 °C for 10min. The C282Y mutation substitutes an A for a G at nucleotide 845 in the exonic sequence, thereby creating a second recognition site for the restriction endonuclease Rsa I in the amplified region. Thus, the PCR products were digested with Rsa I (Gibco-BRL) as recommended by the manufacturer, and the products electrophoresed in 3% NuSieve GTG agarose (FMC BioProducts) gels. After staining with ethidium bromide, the gels were photographed under UV light. From wild type DNA, the 400 bp PCR product was cut only once by Rsa I, yielding two products of 260 bp and 140 bp. In PCR products of alleles containing the C282Y mutation, the 140 bp product was cut again, yielding products of 110bp and 30bp. The 30bp product was too small to be seen on these gels.

3. RESULTS We examined 88 allelic copies of the HFE gene (44 patients). Alleles containing the C282Y mutation were readily detected in agarose gels by the appearance of the 110bp band as seen in lanes 3 and 4 of Fig. 1. We found 6 instances of the C282Y mutation, all in heterozygous individuals. This frequency of the C282Y mutation (6.8%) is not significantly different from that seen in the population at large, suggesting that moderate iron overload does not, in itself, create a substantial predisposition for lung cancer.

4. DISCUSSION Superoxide is produced biologically under a very broad spectrum of pathological circumstances, including all infectious diseases,12 all inflammatory diseases,13 and all diseases that involve ischemia and reperfusion.14 Phagocytes such as neutrophils and macrophages possess an NADPH oxidase that produces superoxide radical when the cell is activated as a component of the bactericidal armamentarium.12 Many tissues contain

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the enzyme xanthine dehydrogenase, which may be converted to the superoxideproducing xanthine oxidase following ischemia and reperfusion.14 In addition, it appears that ischemically injured mitochondria become a major source of superoxide radical during post-ischemic reoxygenation.15 Superoxide radical is not the highly reactive species some expect it to be, but its reactions show considerable versatility. It can serve as a mild oxidant, a fairly strong reductant, or as an initiator or terminator of free radical chain reactions. Even so, many believe that the most generally destructive action of superoxide radical may be bringing about the reductive release of iron from ferritin.16 It has been proposed that enters the ferritin-core through the hydrophilic channels, followed by reduction of Fe(III) to Fe(II). This enables the release of iron from the ferritin-core. Iron is a redox-active transition metal, catalyzing a variety of damaging reactions within the cell. Because of this redox activity, iron is normally handled very carefully by cells and organisms. In the healthy state, there is never an appreciable concentration of “free” iron (or iron chelated by low molecular weight compounds). Any released Fe(II) is immediately chelated by compounds such as citrate or ADP, but these complexes readily participate in redox reactions, catalyzing the formation of HO× which can cause DNA strand breaks.17 The macromolecular chelators of iron such as transferrin and ferritin, on the other hand, provide binding sites of such rigid specificity that Fe(III) is bound extremely tightly, but Fe(II) is not bound at all. Due to kinetic restrictions as well as the thermodynamics of binding, the iron is very difficult to reduce in transferrin and in ferritin by the usual cellular reductants, and is thus shielded from release and from unwanted redox participation.18 While our understanding of the regulation of iron uptake is incomplete, recent studies by Sly and coworkers19,20 have shed new light on the process. An HFE gene knockout mouse displays characteristics remarkably similar to human hemochromatosis. HFE modulates uptake of transferrin-bound iron from plasma by crypt enterocytes, providing a signal that regulates the uptake of dietary iron by mature villus enterocytes. Thus, the mutant HFE fails to provide the feedback that iron stores are adequate, and uptake proceeds at a maximal rate. For decades we have been told that our bodies absorb dietary iron with great difficulty, and that we need supplemental iron to be healthy. Clearly, this is not the case. Our bodies have a very sophisticated mechanism for regulating iron uptake, and are, in the healthy state, limiting iron uptake. Any attempt to override this mechanism should be considered with serious deliberation. Fortunately, the excess iron stores that result from the C282Y mutation in HFE are almost completely confined to the liver. While excessive iron stores pose a genotoxic and

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mutagenic threat to the liver, with an increased risk of hepatocellular carcinoma, the present study suggests that the same threat does not extend to the lung.

REFERENCES McCord, J.M., 1998, Iron, free radicals, and oxidative injury, Semin.Hematol. 35:5–12. Harris, L.R., Cake, M.H., and Macey, D.J., 1994, Iron release from ferritin and its sensitivity to superoxide ions differs among vertebrates, Biochem. J. 301:385–389. Gardner, P.R., Raineri, I., Epstein, L.B., and White, C.W., 1995, Superoxide radical and iron modulate aconitase activity in mammalian cells, J. Biol.Chem. 270:13399–13405. Morris, C.J., Earl, J.R., Trenam, C.W., and Blake, D.R., 1995, Reactive oxygen species and iron—a dangerous partnership in inflammation., Int.J. Biochem.Cell Bio. 27:109–122. Koster, J.F. and Sluiter, W., 1995, Is increased tissue ferritin a risk factor for atherosclerosis and ischaemic heart disease?, Br. Heart J. 73:208–208. Okada, S., 1996, Iron-induced tissue damage and cancer: the role of reactive oxygen species-free radicals, Pathol. Int. 46:311–332. Martins, E.A., Robalinho, R.L., and Meneghini, R., 1995, Oxidative stress induces activation of a cytosolic protein responsible for control of iron uptake, Arch. Biochem. Biophys. 316:128–134. Aslam, S. and Standen, G.R., 1997, Rapid diagnosis of asymptomatic hereditary haemochromatosis by detection of the Cys282Tyr mutation in the HLA-H gene, Postgrad. Med. J. 73:573–574. Knekt, P., Reunanen, A., Takkunen, H., Aromaa, A., Heliovaara, M., and Hakulinen, T., 1994, Body iron stores and risk of cancer, Int. J. Cancer 56:379–382. Herrinton, L.J., Friedman, G.D., Baer, D., and Selby, J.V., 1995, Transferrin saturation and risk of cancer, Am. J. Epidemiol. 142:692–698. Feder, J.N., Gnirke, A., Thomas, W., Tsuchihashi, Z., Ruddy, D.A., Basava, A., Dormishian, F., Domingo, R., Jr., Ellis, M.C., Fullan, A., Hinton, L.M., Jones, N.L., Kimmel, B.E., Kromnal, G.S., Lauer, P., Lee, V.K., Loeb, D.B., Mapa, F.A., McClelland, E., Meyer, N.C., Mintier, G.A., Moeller, N., Moore, T., Morikang, E., Wolff, R.R., and et al., 1996, A novel MHC class I-like gene is mutated in patients with hereditary haemochromatosis, Nature. Genet. 13:399–408. Babior, B.M., 1978, Oxygen-dependent microbial killing by phagocytes, N. Engl. J. Med. 298:659–668 and 721–725. McCord, J.M., 1987, Oxygen-derived radicals: a link between reperfusion injury and inflammation, Fed. Proc. 46:2402–2406. McCord, J.M., 1985, Oxygen-derived free radicals in post-ischemic tissue injury, N. Engl. J. Med. 312:159–163. McCord, J.M. and Turrens, J.F., 1994, Mitochondrial injury by ischemia and reperfusion, Curr. Topics Bioenerg. 17:173–195. Biemond, P., Swaak, A.J., van Eijk, H.G., and Koster, J.F., 1988, Superoxide dependent iron release from ferritin in inflammatory diseases, Free Radical Biol. Med. 4:185–198. Halliwell, B. and Gutteridge, J.M.C., 1992, Biologically relevant metal ion-dependent hydroxyl radical generation—an update, FEBS Lett. 307:108–112. Thomas, C.E., Morehouse, L.A., and Aust, S.D., 1985, Ferritin and superoxide-dependent lipid peroxidation, J. Biol. Chem. 260:3275–3280. Zhou, X.Y., Tomatsu, S., Fleming, R.E., Parkkila, S., Waheed, A., Jiang, J., Fei, Y., Brunt, E.M., Ruddy, D.A., Prass, C.E., Schatzman, R.C., O’Neill, R., Britton, R.S., Bacon, B.R., and Sly, W.S., 1998, HFE gene knockout produces mouse model of hereditary hemochromatosis, Proc. Natl. Acad. Sci. U.S.A. 95:2492–2497. Fleming, R.E., Migas, M.C., Zhou, X., Jiang, J., Britton, R.S., Brunt, E.M., Tomatsu, S., Waheed, A., Bacon, B.R., and Sly, W.S., 1999, Mechanism of increased iron absorption in murine model of hereditary hemochromatosis: Increased duodenal expression of the iron transporter DMT1, Proc. Natl. Acad. Sci. U.S.A. 96:3143–3148.

15

SELENIUM AND THE PROTECTION AGAINST PEROXYNITRITE

Helmut Sies, Lars-Oliver Klotz, Stefan M. Schieke, Karlis Briviba, and Gavin E. Arteel

1. PEROXYNITRITE AND OXIDATIVE STRESS Peroxynitrite is produced by the diffusion-limited reaction of nitric oxide and superoxide anion. Peroxynitrite is stable, but upon protonation to peroxynitrous acid (pKa 6.8) it decays to nitrate with a rate constant of at 25 °C. Peroxynitrous acid is highly reactive, yielding oxidizing and nitrating species (see Beckman et al., 1990; Koppenol et al., 1992). Peroxynitrite can cause oxidation, nitration, as well as nitrosation reactions. At the level of the whole organism, the reactive chemistry of peroxynitrite can be considered beneficial. For example, peroxynitrite is cytotoxic to bacteria (Zhu et al., 1992) or other invading organisms. Inflammatory cells, such as macrophages and neutrophils, produce large amounts of both nitric oxide and superoxide which, in turn, rapidly form peroxynitrite (Ischiropoulos et al., 1992). However, excessive production can damage normal tissue. Indeed, the formation of protein 3-nitrotyrosine, an index of reactive nitrogen species (Ohshima et al., 1990), has been shown in a number of inflammatory conditions (Ischiropoulos, 1998).

2. REDUCTION OF PEROXYNITRITE BY SELENIUM-CONTAINING COMPOUNDS AND PROTEINS Interception of damaging species, once formed, so as to exclude it from further activity is a major form of antioxidant defense in organisms. For determining the detoxifying capacity of a direct reaction of a given compound with a prooxidant, it is useful to consider the rate constant of this reaction. Table 1 lists the rate constants of the reaction of some selenium-containing compounds and proteins and/or the concentration required to inhibit the oxidation of dihydrorhodamine 123 by peroxynitrite by 50%.

Address all correspondence to: Prof. Dr. Helmut Sies; Institut für Physiologische Chemie I; Heinrich-HeineUniversität Düsseldorf; Postfach 101007; D-40001 Düsseldorf, Germany; telephone: +49-211-811-2707; fax: +49-211-811-3029; email: [email protected] Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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Ebselen The organoselenium compound ebselen rapidly reacts with peroxynitrite (Briviba et al., 1996; Masumoto & Sies, 1996; Sies & Masumoto, 1997). The rate constant of the reaction of ebselen with peroxynitrite is (25 °C, pH 7.4; Masumoto et al., 1996), and ebselen protects against DNA damage caused by peroxynitrite more effectively than their sulfur analogs (Roussyn et al., 1996). Peroxynitrite is reduced to nitrite by these compounds; the resulting selenoxide is subsequently reduced by GSH, establishing a catalytic cycle so that the defense can be maintained in a peroxynitrite reductase reaction. This reaction applies generally to ebselen or to selenocysteine in free or protein-bound form, such as in glutathione peroxidase (see below). Clinically, ebselen has been found to be protective in patients with delayed neurological deficits and aneurysmal subarachnoid hemorrhage (Saito et al., 1998), in acute ischemic stroke (Yamaguchi et al., 1998), and in acute middle cerebral artery occlusion (Ogawa et al., 1999). Since increased nitrotyrosine levels were found to be associated with such neurological disorders, these protective effects of ebselen could be due in part to peroxynitrite defense.

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Selenomethionine Selenomethionine is oxidized to the selenoxide by peroxynitrite with a second-order rate constant approximately 100-fold higher than that for the reaction of methionine with peroxynitrite (Padmaja et al., 1996). Methionine selenoxide is effectively and rapidly reduced to selenomethionine by glutathione, permitting a catalytic reaction by selenomethionyl residues in proteins (Fig. 2; Assmann et al., 1998). In contrast, methionine sulfoxide is not reduced by glutathione; the enzyme methionine sulfoxide reductase is necessary for the reduction of methionine sulfoxide to methionine (Levine et al., 1996). Since selenomethionine can occur in proteins such as hemoglobin (Beilstein & Whanger, 1986), these residues may play a defensive role against peroxynitrite.

Glutathione Peroxidase The selenocysteine-containing glutathione peroxidase (GPx) can act as a peroxynitrite reductase, preventing oxidation and nitration reactions caused by peroxynitrite (Sies et al., 1997). Glutathione peroxidase reduces peroxynitrite to nitrite using GSH in a catalytic reaction (Fig. 2), similar to that described above for ebselen, and also for Selenomethionine. Increases in nitrite during exposure to peroxynitrite were observed with GPx (Sies et al., 1997), indicating two-electron reduction of peroxynitrite; however, the nitrite yield was less than complete (~50%). The second-order rate constant for the reaction of glutathione peroxidase (tetrameric) with peroxynitrite is (Briviba et al., 1998a). While there is no net loss of GPx activity when GPx is maintained in the reduced state by supplying reductants (Sies et al., 1997; Briviba et al., 1998a), GPx is inactivated in the absence of GSH (Padmaja et al., 1998) or upon exposure to nitric oxide donors (Asahi et al., 1997). These data suggest that inside the cell, GPx outcompetes thiols for the direct reaction with peroxynitrite (Table 1). Increasing the level of selenoproteins (e.g. GPx 14-fold) by selenium supplementation attenuated mitogen-activated protein kinase (p38, JNK1/2 and ERK1/2) activation by peroxynitrite in WB-F344 rat liver cells (Schieke et al., 1999). Thus, the reaction of GPx with peroxynitrite is considered a biologically efficient detoxication pathway in vivo.

Selenoprotein P and Thioredoxin Reductase Selenoprotein P in human plasma also protects against peroxynitrite (Arteel et al., 1998), suggesting that it may serve as a protectant against peroxynitrite in human blood. The heparin-binding domains of Selenoprotein P enable surface coating of cellular membranes (e.g. endothelial cells; Wilson & Tappel, 1993; Burk et al., 1997). Recent work with surface plasmon resonance (BIAcore) has indicated that Selenoprotein P has two heparin-binding sites, one in the low nM range and one in the mid nM range (Arteel et al., unpublished observations). This coating may serve as a protective barrier against peroxynitrite. Thioredoxin reductase can function in the reduction of peroxynitrite by selenocysteine or ebselen (Arteel et al., 1999b).

Organotellurium Compounds In addition to organoselenium compounds, there exist organotellurium compounds that also protect against oxidation and nitration reactions caused by peroxynitrite

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(Briviba et al., 1998b; Briviba et al., 1999); bis[4-aminophenyl] telluride protects against peroxynitrite-mediated oxidation of dihydrorhodamine 123 more efficiently than its selenium analogue or ebselen (Briviba et al., 1998b). This could also become a promising area of future research.

3. INDIRECT REACTIONS In addition to direct interception of prooxidants, selenium-containing compounds and proteins can also indirectly play a role in defense against peroxynitrite. For example, hydroperoxides and lipid hydroperoxides are often products formed during oxidative stress, e.g. during excessive peroxynitrite production (Radi et al., 1991b). In this context, glutathione peroxidase and phospholipid hydroperoxide glutathione peroxidase can play another role in defense (Flohé and Brand, 1969; Chance et al., 1979). Ebselen and organotellurium compounds also reacts with hydroperoxides and can protect the organism at this level (Muller et al., 1984; Briviba et al., 1998b). Further, selenoprotein P was also shown to serve a function as an extracellular phospholipid hydroperoxide glutathione peroxidase using thiols as reductants (Saito et al., 1999); this function may also make selenoprotein P important in removal of potential products of nitric oxide/peroxynitrite. While the mammalian thioredoxin system (NADPH, thioredoxin reductase, and thioredoxin) has been shown to directly reduce lipid hydroperoxides (Mitsui et al., 1992; Björnstedt et al., 1995), the reaction rate is relatively slow. However, the ability of thioredoxin reductase to reduce more potent antioxidants (Björnstedt et al., 1994, Cha & Kim, 1995, Mendiratta et al., 1998) may be biologically significant. Further, the thioredoxin system also helps maintain cellular thiol pools in the reduced state at the expense of NADPH (Björnstedt et al., 1997).

4. CONCLUDING REMARKS AND SUMMARY The general strategies of antioxidant defense employed by organisms can be categorized as prevention, interception and repair (for review, see Sies, 1993). Defense against peroxynitrite by organoselenium compounds and selenium-containing proteins as detailed above falls under the category of interception of peroxynitrite and oxidant species derived from the reaction of peroxynitrite with biological molecules. Coupled with other compounds and proteins that assist in defense against peroxynitrite (for review, see Arteel et al., 1999a), organoselenium compounds and seleniumcontaining proteins contribute to the “antioxidant network”, helping to protect the organism from irreversible damage caused by overproduction of peroxynitrite. Understanding of these general principles of antioxidant defense, as well as mechanisms of defense specific against certain reactive species, can be used as a predictive tool for pharmacologic measures that may be useful in models of overproduction of nitric oxide/peroxynitrite.

ACKNOWLEDGMENTS Support by the Deutsche Forschungsgemeinschaft, SFB 503, Project B1, and by the National Foundation for Cancer Research, Bethesda, MD, is gratefully acknowledged.

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G. E. Arteel is a Research Fellow of the Alexander von Humboldt Foundation, Bonn, Germany.

REFERENCES Arteel, G.E., Briviba, K., and Sies, H., 1999a, Protection against peroxynitrite. FEBS Lett. 445:226–230. Arteel, G.E., Briviba, K., and Sies, H., 1999b, Function of thioredoxin reductase as a peroxynitrite reductase using selenocystine or ebselen. Chem. Res. Toxicol. 12:264–269. Arteel, G.E., Mostert, V., Oubrahim, H., Briviba, K., Abel, J., and Sies, H., 1998, Protection by selenoprotein P in human plasma against peroxynitrite-mediated oxidation and nitration. Biol. Chem. 379:1201–1205. Asahi, M., Fujii, J., Takao, X, Kuzuya, T., Hori, M., Shimonishi, Y., and Taniguchi, N., 1997, The oxidation of selenocysteine is involved in the inactivation of glutathione peroxidase by nitric oxide donor. J. Biol. Chem. 272:19152–19157. Assmann, A., Briviba, K., and Sies, H., 1998, Reduction of methionine selenoxide to selenomethionine by glutathione. Arch. Biochem. Biophys. 349:201–203. Beckman, J.S., Beckman, T.W., Chen, J., Marshall, P.A., and Freeman, B.A., 1990, Apparent hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide. Proc. Natl. Acad. Sci. U. S. A. 87:1620–1624. Beilstein, M.A. and Whanger, P.D., 1986, Deposition of dietary organic and inorganic selenium in rat erythrocyte proteins. J. Nutr. 116:1701–1710. Björnstedt, M., Hamberg, M., Kumar, S., Xue, J., and Holmgren, A., 1995, Human thioredoxin reductase directly reduces lipid hydroperoxides by NADPH and selenocystine strongly stimulates the reaction via catalytically generated selenols J. Biol. Chem. 270:11761–11764. Björnstedt, M., Xue, J., Huang, W., Åkesson, B., and Holmgren, A., 1994, The thioredoxin and glutaredoxin systems are efficient electron donors to human plasma glutathione peroxidase. J. Biol. Chem. 269: 29382–29384. Briviba, K., Kissner, R., Koppenol, W.H., and Sies, H., 1998a, Kinetic study of the reaction of glutathione peroxidase with peroxynitrite. Chem. Res. Toxicol. 11:1398–1401. Briviba, K., Klotz, L.-O., and Sies, H., 1999, Defenses against peroxynitrite. Methods Enzymol. 301:301–310. Briviba, K., Roussyn, I., Sharov, VS., and Sies, H., 1996, Attenuation of oxidation and nitration reactions of peroxynitrite by selenomethionine, selenocystine and ebselen. Biochem. J. 319:13–15. Briviba, K., Tamler, R., Klotz, L.-O., Engman, L., Cotgreave, I.A., and Sies, H., 1998b, Protection by organotellurium compounds against peroxynitrite-mediated oxidation and nitration reactions. Biochem. Pharmacol. 55:817–823. Burk, R.F., Hill, K. E., Boeglin, M.E., Ebner, F.F., and Chittum, H.S., 1997, Selenoprotein P associates with endothelial cells in rat tissues. Histochem. Cell Biol. 108:11–15. Cha, M.-K. and Kim, I.-H., 1995, Thioredoxin-linked peroxidase activity from human red blood cell: evidence for the existence of thioredoxin and thioredoxin reductase in human red blood cell. Biochem. Biophys. Res. Commun. 217:900–907. Chance, B., Sies, H., and Boveris, A., 1979, Hydroperoxide metabolism in mammalian organs. Physiol. Rev. 59:527–605. Flohé, L. and Brand, I., 1969, Kinetics of glutathione peroxidase. Biochim. Biophys. Acta. 191:541–549. Ischiropoulos, H., 1998, Biological tyrosine nitration: a pathophysiological function of nitric oxide and reactive oxygen species. Arch. Biochem. Biophys. 356:1–11. Ischiropoulos, H., Zhu, L., and Beckman, J.S., 1992, Peroxynitrite formation from macrophage-derived nitric oxide. Arch. Biochem. Biophys. 298:446–451. Koppenol, W.H., Moreno, J.J., Pryor, W.A., Ischiropoulos, H., and Beckman, J.S., 1992, Peroxynitrite: a cloaked oxidant from superoxide and nitric oxide. Chem. Res. Toxicol. 5:834–842. Lee, J.L., Hunt, J.A., and Groves, J.T., 1997, Rapid decomposition of peroxynitrite by manganese poryphyrinantioxidant redox couples. Bioorgan. Med. Chem. Lett. 7:2913–2918. Levine, R.L., Mosoni, L., Berlett, B.S., and Stadtman, E.R., 1996, Methionine residues as endogenous a ntioxidants in proteins. Proc. Natl. Acad. Sci. U. S. A. 93:15036–15040. Masumoto, H., Kissner, R., Koppenol, W.H., and Sies, H., 1996, Kinetic study of the reaction of ebselen with peroxynitrite. FEBS Lett. 398:179–182. Masumoto, H. and Sies, H., 1996, The reaction of ebselen with peroxynitrite. Chem. Res. Toxicol. 9:262–267. Mendiratta, S., Qu, Z.-C, and May, J.M., 1998, Enzyme-dependent ascorbate recycling in human erythrocytes: role of thioredoxin reductase. Free Radic. Biol. Med. 25:221–228.

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Mitsui, A., Hirakawa, T., and Yodoi, J., 1992, Reactive oxygen-reducing and protein-refolding activities of adult T cell leukemia-derived factor/human thioredoxin. Biochem. Biophys. Res. Commun. 186: 1220–1226. Müller, A., Cadenas, E., Graf, P., and Sies, H., 1984, A novel biologically active seleno-organic compound– I. Glutathione peroxidase-like activity in vitro and antioxidant capacity of PZ 51 (Ebselen). Biochem. Pharmacol. 33:3235–3239. Ogawa, A., Yoshimoto, T., Kikuchi, H., Sano, K., Saito, I., Yamaguchi, T., and Yasuhara, H., 1999, Ebselen in acute middle artery occlusion: a placebo-controlled, double-blind clinical trial. Cerebrovasc. Dis. 9:112–118. Ohshima, H., Friesen, M., Brouet, I., and Bartsch, H., 1990, Nitrotyrosine as a new marker for endogenous nitrosation and nitration of proteins. Food Chem. Toxicol. 28:647–652. Padmaja, S., Squadrito, G.L., Lemercier, J.-N., Cueto, R., and Pryor, W.A., 1996, Rapid oxidation of DL-selenomethionine by peroxynitrite. Free Radic. Biol. Med. 21:317–322. Padmaja, S., Squadrito, G.L., and Pryor, W.A., 1998, Inactivation of glutathione peroxidase by peroxynitrite. Arch. Biochem. Biophys. 349:1–6. Pryor, W.A., Jin, X., and Squadrito, G.L., 1994, One- and two-electron oxidations of methionine by peroxynitrite. Proc. Natl. Acad. Sci. U. S. A. 91:11173–11177. Radi, R., Beckman, J.S., Bush, K.M., and Freeman, B.A., 1991a, Peroxynitrite oxidation of sulfhydryls. The cytotoxic potential of superoxide and nitric oxide. J. Biol. Chem. 266:4244–4250. Radi, R., Beckman, J.S., Bush, K.M., and Freeman, B.A., 1991b, Peroxynitrite-induced membrane lipid peroxidation: the cytotoxic potential of superoxide and nitric oxide. Arch. Biochem. Biophys. 288: 481–487. Roussyn, I., Briviba, K., Masumoto, H., and Sies, H., 1996, Selenium-containing compounds protect DNA from single-strand breaks caused by peroxynitrite. Arch. Biochem. Biophys. 330:216–218. Saito, I., Asano, T., Sano, K., Takakura, K., Abe, H., Yoshimoto, T., Kikuchi, H., Ohta, T., and Ishibashi, S., 1998, Neuroprotective effect of an antioxidant, ebselen, in patients with delayed neurological deficits after aneurysmal subarachnoid hemorrhage. Neurosurgery, 42:269–278. Saito, Y, Hayashi, T., Tanaka, A., Watanabe, Y., Suzuki, M., Saito, E., and Takahashi, K., 1999, Selenoprotein P in human plasma as an extracellular phospholipid hydroperoxide glutathione peroxidase. Isolation and enzymatic characterization of human selenoprotein P. J. Biol. Chem. 274:2866–2871. Schieke, S.M., Briviba, K., Klotz, L.-O., and Sies, H., 1999, Activation pattern of mitogen-activated protein kinases elicited by peroxynitrite: Attenuation by selenium supplementation. FEBS Lett. 448:301–303. Sies, H., 1993, Strategies of antioxidant defense. Eur.J.Biochem. 215:213–219. Sies, H. and Masumoto, H., 1997, Ebselen as a glutathione peroxidase mimic and as a scavenger of peroxynitrite. Adv. Pharmacol. 38:229–246. Sies, H., Sharov, V.S., Klotz, L.-O., and Briviba, K., 1997, Glutathione peroxidase protects against peroxynitrite-mediated oxidations. A new function for selenoproteins as peroxynitrite reductase. J. Biol. Chem. 272:27812–27817. Wilson, D.S. and Tappel, A.L., 1993, Binding of plasma selenoprotein P to cell membranes. J. Inorg. Biochem. 51:707–714. Yamaguchi, T., Sano, K., Takakura, K., Saito, I., Shinohara, Y., Asano, T., and Yasuhara, H., 1998, Ebselen in acute ischemic stroke: a placebo-controlled, double-blind clinical trial. Ebselen Study Group. Stroke 29:12–17. Zhu, L., Gunn, C., and Beckman, J.S., 1992, Bactericidal activity of peroxynitrite. Arch. Biochem. Biophys. 298:452–457.

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COOPERATIVE EFFECTS OF ZINC / SELENIUM AND THIOLS IN THE PROTECTION AGAINST UV-INDUCED GENOMIC DNA DAMAGE

Marie-Jeanne Richard, Nathalie Emonet-Piccardi, Christine Didier, Eric Jourdan, Marie-Thérèse Leccia, Marie-Odile Parat, Jean Cadet, Jean Claude Béani, and Alain Favier

The skin is continuously exposed to environmental insults including solar radiation (SR): ultraviolet B and A (UVB, 290–320nm and UVA, 320–400nm). Due to recreational habits and the use of UVB absorbing sunscreens, human exposure to SR is increasing. As a consequence, the risks of skin damage have increased. While the precise mechanisms remain elusive, reactive oxygen species (ROS) are thought to play a major role in skin damage. Many studies in cell cultures, animals and more recently in humans have demonstrated that antioxidants (scavengers and metalloenzymes) can prevent UV-induced cell damage. Among the many possible antioxidant approaches, the cooperation between zinc (Zn), selenium (Se) and thiol compounds will be considered here.

1. PHOTO-OXIDATIVE STRESS AND GENOTOXIC EFFECTS OF SOLAR RADIATION Sunlight exposure causes many deleterious effects: acute effects such as erythema and acute sunburn, but also in the long term it causes more serious lesions such as premature skin aging and the development of skin cancers (Taylor et al., 1990). Indeed, UV radiation is the major environmental factor that affects the DNA of skin cells. UVB is mainly genotoxic through the direct absorption of UV radiation by DNA. The carcinogen effectiveness of UVA seems to be related to an oxidative stress in which DNA bases are oxidized (Cadet et al., 1997). An addition, UVB and UVA are capable of inducing mutations of p53, a transcription factor which is involved in DNA repair (Fig. 1). The respective and synergistic roles of both UVB and UVA in carcinogenesis

Address all correspondence to: Dr M. J. Richard; LBSO Laboratoire de Biologie du Stress Oxydant, Hôpital A. Michallon, BP 217-38043 Grenoble; France. Telephone: 476765147; Fax: 476765664; email: [email protected] Trace Elements in Man and Animals 10, edited by Roussel et al.,

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are now established and lead to the development of a new strategy in photoprotection based not only on sunscreens but also on an antioxidant strategy. Indeed high doses of UV alter antioxidants (Fuchs et al., 1989). The destruction is much more important within the epidermis than in the dermis suggesting that maintaining antioxidant capacity is a relevant question in the limitation and prevention of the photodamage (Fuchs et al., 1998).

2. EFFICACY OF THE PAIR ZINC/THIOLS AS ANTIOXIDANT AND GENOPROTECTOR 2.1. Zinc as Antioxidant The antioxidant properties of Zn (Favier, 1995; Bray et al., 1990) can be related to multiple actions, the most commonly mentioned being Zn interference with the absorption of transition metals, which catalyze the Fenton reaction and amplify ROS production. Zn is also ascribed to stabilization of Cu-Zn superoxide dismutase (SOD). Nevertheless, in Zn-depleted cells, no modification in SOD activity was observed (Parat et al., 1997). In contrast the zinc-chelator, N,N,N' N'-tetrakis (2-pyridylmethyl)ethylene diamine (TPEN), which induced intracellular Zn deprivation, triggers a glutathione (GSH) depletion accompanied by an efflux of oxidized glutathione (GSSG). Several studies have dealt with the relationship between Zn and GSH and suggest that GSH binds Zn lowering intracellular GSH concentration and signaling a need for GSH synthesis. The binding of Zn to cysteinyl residues with a stronger affinity than iron (Fe) is one mechanism of Zn protective effect against protein oxidation. Zn is also implicated in DNA protection. In previous studies we have shown that Zn addition significantly increases the survival rate of normal skin fibroblasts exposed to UVA radiation and decreases lipid peroxidation of these cells (Leccia et al., 1993). Zn depletion sensitizes cells to DNA stand breaks whereas Zn supplementation lowers them (Parat et al., 1996; Leccia et al., in press). Using the single-cell gel electrophoresis we confirmed that Zn protects cells from DNA damage induced by UVA (Emonet et al., 1998). One explanation for DNA protection involves competition between Zn and prooxidant metals found to nucleic acids. Another protective mechanism is represented by metallothioneins (MT). Indeed, as well as having a role in metal homeostasis, MT have scavenging properties through their cystein residues. Hanada et al. (1991) demonstrated that increasing MT

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synthesis suppressed UVB-mediated sunburn cell formation in mouse skin suggesting a protective effect of MT. Moreover, MT-null mouse provides direct evidence of the photoprotective effect of cellular MT in the skin (Hanada et al., 1998). MT are expressed constitutively within human skin only in epidermal cells, whereas MT induction is reported both in epidermis and dermis upon UVB irradiation (Anstey et al., 1996). Nuclear MT are thought to protect DNA against oxidative damage caused by UVR through their antioxidant capacity or their ability to promote the synthesis of nucleic acids and proteins by supplying Zn to the enzymes involved in their synthesis (Jacob et al., 1998).

2.2. Zinc and Apoptosis In TPEN treated cells, intracellular Zn chelation induced apoptosis (Parat et al., 1997). The number of apoptotic cells was dose and time dependent and cells exposed to TPEN plus Zn were not apoptotic. In contrast, Zn treatment decreased the mitochondrial dysfunction observed in dying cells as well as prevented the UVA-induced apoptosis (Leccia et al., in press). Different mechanisms could explained such results. Zn protects cystein from oxidation maintaining protein structure and activity and it regulates the activation of 2 enzymes involved in apoptosis: endonucleases (Marini et al., 1998) and caspase-3 (Perry et al., 1997).

2.3. Zinc and DNA Repair P53 contributes to prevent the replication of cells exposed to genotoxic factors, thereby limiting the risk of propagation of genetic abnormalities leading to cancer. DNA damage allows p53 to accumulate and to interact with other cellular factors. These factors mediate conformational modifications of p53 and activate the capacity to bind to defined DNA sequences. Induction of p53 results in permanent or transient cell-cycle arrest and this may help the completion of extensive DNA repair. In other cells, induction of p53 results in apoptosis and facilitates the elimination of cells that contain genes damaged beyond repair (Levine, 1997). Zn plays an important role in maintaining the tertiary structure of the DNAbinding domain of p53. Cells exposed to the metal chelator, TPEN, p53 accumulated in a form unable to bind DNA which correlates with a change in p53 protein conformation (Verhaegh et al., 1998). Removal of the chelator allowed p53 to fold back in the native conformation and to recover DNA-binding activity. These data have important potential implications. Zn might play a role as signaling molecule in the control of unaltered genetic message. Disruption of this control contributes to carcinogenesis by impairing p53 functions. Oxidative stress such as UV irradiation that oxidizes thiols groups might disturb p53 metalloregulation.

3. PLACE OF THIOL COMPOUNDS IN THE ANTIOXIDANT PHOTOPROTECTION MEDIATED BY SELENIUM Selenium can attenuate the mutagenic effects of DNA adducts. Two antioxidant systems: glutathione peroxidase (GSH-Px) / glutathione (GSH) and thioredoxin reductase (TR) / thioredoxin (Trx) have to be considered (Fig. 2).

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3.1. Are Se and GSH Synergistic Against UV Radiation? GSH-Px is an important enzyme whose role is not restricted to the catalysis of H2O2 but concerns also the reduction of lipid peroxides. As previously reported, Se prevented UVA-induced cell death and UVA-induced lipid peroxidation (Leccia et al., 1993). Previous works from Leist et al., (1996) using the comet assay showed that Se improves genetic stability of cultured cells. Interestingly, we demonstrated that an adequate Se supply afforded a significant protection against UVA-induced DNA damage (EmonetPiccardi et al., 1998). In order to study the respective efficiency of GSH-Px and GSH, cells were treated with N-acetyl-cystein (NAC), a GSH precursor, 4 hours before irradiation. NAC treatment maintained GSH levels even in irradiated cells. A significant protection against UVA lethal effects and DNA damage were demonstrated. Interestingly, no additive effects of Se and NAC on genomic DNA damage were observed whereas an additive effect concerning UV- induced cell death was found (Emonet et al., 1997). Thus, the major antioxidant selenodependent system present in the nucleus should be TR/Trx rather than GSH-Px/GSH.

3.2. What Are the Roles of the Couple Thioredoxin / Thioredoxin Reductase System in the Skin? The TR/Trx couple is an ubiquitous redox system. TR is a FAD containing flavoenzyme that uses NADPH to reduce Trx. TR activity is controlled by the amount of available Se (Powis et al., 1997). Trx is one of the major redox regulatory molecules which determines the oxidation state of the other protein thiols (Powis et al., 1997b). Trx has been presumed to function as a key component in DNA synthesis through its ability to reduce ribonucleotide reductase (RR). Trx also exerts specific redox control over some transcription factors regulating gene transcription. Trx has also a radical scavenging activity (Powis et al., 1997b). The epidermis represents the first line of defense against UV light, therefore the surface of the skin must be able to counteract its penetration and to neutralize ROS. In this pathway, Trx and TR are very important (Schallreuter, 1989). Indeed, by activating tyrosinase, oxidized Trx controls the biosynthesis of melanin, the main pigment that

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absorbs UV radiation. Trx can also participate in melanocyte growth leading to cutaneous hyperpigmentation (Funasaka et al., 1997). UVB has been shown to induce the production and release of Trx in keratinocytes which upregulates the DNA synthesis of melanocytes. The possible consequences of a decrease in TR activity due to selenium deficiency are not known. If low Se leads to a decreased ability of cells to undergo apoptosis this might lead to an increased risk of developing cancer, thus explaining the association between low dietary Se and increased incidence of human cancer. In summary, Se and Zn, in close conjunction with thiols, prevent both cell death and DNA induced damage. The need of antioxidant combination therapies has to be considered as an integral part of the multi-faceted approach in photoprotection.

REFERENCES Anstey, A., Marks, R., Long, C., Navabi, H., Perase, A., Wynford-Thomas, D., and Jasani, B., 1996, In vivo photoinduction of metallothionein in human skin by ultraviolet irradiation. J. Pathol. 178:84–88. Bray, T.M. and Bettger, W.J., 1990, The physiological role of zinc as an antioxidant. Free Rad. Biol. Med. 8:281–291. Cadet, J., Berger, M., Douki, X, and Ravanat, J.L., 1997, Oxidative damge to DNA. Formation, measurement and biological significance. Rev. Physiol. Biochem. Pharmacol. 131:2003–2007. Emonet, N., Leccia, M.T., Favier, A., Béani, J.C., and Richard, M.J., 1997, Thiols and selenium: protective effect on human skin fibroblasts exposed to UVA radiation. J. Photochem. Photobiol. 40:84–90. Emonet-Piccardi, N., Richard, M.J., Ravanat, J.L., Signorini, N., Cadet, J., and Béani, J.C., 1998, Protective effects of antioxidants against UVA-induced DNA damage in human skin fibroblasts in culture. Free Rad. Biol. Med. 29:307–313. Favier, A., 1995, Zinc-ligand interactions and oxygen free radiacl formation, in Handbook of metal-ligand interactions in biological fluids, (G. Berthon ed), pp. 876–887, M. Dekker, New York. Fuchs, J., 1998, Potentials and limitations of the natural antioxidants RRR-alpha-tocopherol, L-ascorbic and b-carotene in cutaneous photoprotection, Free Rad. Biol. Med. 25:848–873. Fuchs, J., Hufletjt, M.E., Rothfuss, L., Wilson, D.P., Gerardo, C., and Packer, L., 1989, Impairment of enzymic and non enzymic antioxidants in skin by photooxidative stress. J. Invest. Dermatol. 93:769–773. Funasaka, Y. and Ichihashi, M., 1997, The effect of UVB induced adult T cell leukemia-derived factor/ thioredoxin (ADF/TRX) on survival and growth of human melanocytes. Pigment Cell Res. 10:68–73. Hanada, K., Gange, R.W., Siebert, E., and Hassan, T., 1991, Protective effects of cadmium chloride against UVB injury in mouse skin and in cultured human cells: a possible role of cadmium induced metallothionein. Photodermatol. Photoimmunol. Photomed. 8:111–115. Hanada, K., Sawamura, D., Tamai, K., Baba, T., Hashimoto, I., Muramatsu, T., Miura, N., and Naganuma, A., 1998, Novel function of metallothionein in photoprotection: metallothionein-null mouse exhibits reduced tolerance against ultraviolet B injury in the skin. J. Invest. Dermatol. 111:582–585. Jacob, C., Maret, W., and Vallee, B., 1998, Control of zinc transfer between thionein, metallothionein, and zinc proteins. Proc. Natl. Acad. Sci. 95:3489–3494. Leccia, M.X, Richard, M.J., Beani, J.C., Faure, H., Monjo, A.M., Cadet, J., Amblard, P., and Favier, A., 1993, Protective effect of selenium and zinc on UVA-damage in human skin fibroblasts. Photochem. Photobiol. 58:548–553. Leccia, M.T., Richard, M.J., Beani, J.C., and Favier, A., (in press) Zinc protects against UVA1 induced DNA damage and apoptosis in cultures human fibroblasts. Biol. Trace Elem. Res. Leist, M., Raab, B., Maurer, S., Rosick, U., and Brigelius-Flohe, R., 1996, Conventional cell culture media do not adequatly supply cells with antioxidants and thus facilitat peroxide-induced genotoxicity. Free Rad. Biol. Med. 3:297–306. Levine, A., 1997, P53: The cellular gatekeeper for growth and division. Cell 89:323–331. Marini, M. and Musiani, D., 1998, Micromolar zinc affects endonucleolytic activity in hydrogen peroxidemediated apoptosis. Exper. Cell Res. 239:393–398. Parat, M.O., Richard, M.J., Béani, J.C., and Favier, A., 1997, Involvement of zinc in intracellular oxidant/antioxidant balance. Biol. Trace Elem. Res. 60:187–204.

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Parat, M.O., Richard, M.J., Pollet, S., Hadjur, C., Favier, A., and Béani, J.C., 1996, Zinc and DNA fragmentation in keratinocyte apoptosis: its inhibitory effect in UVB irradiated cells, J. Photochem. Photobiol. B, 7:101–106. Perry, D., Smyth, M., Stennicke, H., Salvesen, G., Duriez, P., Poirier, G., and Hannun,AY., 1997, Zinc is a potent inhibitor of the apoptotic protease, caspase-3. J. Biol. Chem. 25:18530–18533. Powis, G., Gasdaka, J., and Baker, A., 1997, Redox signaling and the control of cell growth and cell death. Adv. in Pharmacol. 38:329–359. Powis, G., Gasdaka, J., Gasdaska, P., Berggren, M., Kirkpatrick, D.L., Engman, L., Cotgreave, I., Angulo, M., and Baker, A., 1997, Selenium and the thioredoxin redox system: effects on cell growth and death. Oncology Res. 9:303–312. Schallreuter, K., Lemke, K.R., Hill, H., and Wood, J., 1994, Thioredoxine reductase induction coincides with melanin biosynthesis in brown and black guinea pigs and in murine melanoma cells. J. Invest. Dermatol. 103:820–824. Schallreuter, K. and Wood, J., 1989, Free radical reduction in the human epidermis. Free Rad. Biol. Med. 6:519–532. Taylor, C.R., Stern, R.S., Leyden, J.J., and Gilchrest, B.A., 1990, Photoaging/photodamage and photoprotection. J. Am. Acad. Dermatol. 22:1–15. Verhaegh, G.W., Parat, M.O., Richard, M.J., and Hainaut, P., 1998, Modulation of p53 protein conformation and DNA-binding activity by intracellular chelation of zinc. Mol. Carcinog. 21:205–214.

17

TRACE ELEMENTS AND ENZYME REDOX CENTERS

Marc Fontecave Laboratoire de Chimie et Biochimie des Centres Rédox Biologiques DBMS-CB CEA, Université Joseph Fourier, CNRS 17 Avenue des Martyrs 38054 Grenoble Cedex 9 France

The elements that must be incorporated into biological systems for any organic chemistry to take place are H, C, N and O. Unfortunately all the compounds which these elements form together are not stable relative to and in the presence of in air. All biological systems should burn but they do not so over reasonable periods of time because of kinetic barriers. Biology must start from and activation of these small “primitive” molecules absolutely depend on transition metals. It is therefore likely that life had to start from the inorganic catalysis of reactions. Most of biological organic reactions are not kinetically competent for cell survival. They need to be catalyzed by enzymes. It is now well established that a large proportion of these enzyme activities depend on transition metal ions: this is the case for a number of electron transfer reactions, oxidations, reductions, hydrolysis reactions and so on. Also life in air is only possible because of the availability of metalloproteins which are able to bind molecular oxygen and transport it (hemoglobin). Among the most important biological metals are zinc, iron and copper. Why?

1. RELATIVE ABUNDANCE The first explanation resides in the relative abundance of the various elements on the earth. There is a relatively good correlation between the amount of a given metal in the earth crust and the amount of the same metal in biological tissues. As a consequence, iron is the most abundant metal followed by zinc. Copper concentration is about half as

Adress all correpondence to: Professor M. Fontecave—Tel: 33476889103—Fax:33476889124—mail:[email protected] Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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high as iron. Molybdenum, as molybdate, is also important and is present in biological systems (nitrogenase, nitrate reductase). Aluminium the first transition metal on the earth is absent from biological systems because of its insolubility and its inaccessibilty. The data thus suggest that availabilty is certainly one of the important factors determining which elements have been selected for utilization by organisms (Ochiai, 1978).

2. CHEMICAL REACTIVITY A second factor is related to the fact that certain elements are inherently best suited to certain biological functions and that organisms tend to adopt those elements that best fulfill the required task (Ochiai, 1978). For example Zn(II) is, among a number of divalent ions, one of the most potent Lewis acid, due to one of the largest “ionic potential” value, Zeff/r, where Zeff is the effective electrical charge of the cation and r is the effective radius. It has thus the potential to polarize a bond that is to undergo reaction and can catalyze making and breaking bonds in organic molecules. It can bind water molecules and induce a strong polarization of the HO-H bond thus converting water into a strong nucleophile. This explains why Zn(II) is used in a huge number of peptidases, proteases, phosphatases, ... It has a number of advantages with respect to divalent cations possessing similar polarizing ability, such as Cu(II) or Co(II). One is the abundance (see above). Another resides in the flexibility of Zn(II) with respect to coordination structure. Because Zn(II) has a electronic configuration, its polarizing effect is isotropic, leading to flexible coordination. The requirements for binding substrates are less rigid than those for other cations. Finally as Zn(II) is not a redox cation, its catalytic activity cannot be affected by variations of the redox potential of the medium. Iron and copper are the most widely utilized metals with redox properties in living organisms. Redox potentials provide a relevant parameter which make it clear why nature have selected these metals. First the whole range of redox potentials obtained with iron and copper systems lie inside of the decomposition limit of water. Co(III)/Co(II) or Cr(III)/Cr(II) cannot be candidates for redox catalysts as they would oxidize or reduce water, respectively. Second, the reason why iron is so widely used in organisms is twofold. One is chemical: iron can accomodate a very wide range of reduction potentials from— 0.5 to 0.6V, which can be finely tuned by slight variations of the coordination sphere. It can thus catalyze a large number of redox reactions useful to living organisms. The other is historical: in the oceans of the primitive earth, where life is believed to have started, iron, in the form of the soluble ferrous Fe(II) iron, was plentiful and readily available to organisms. Only later when oxygen appeared at the surface of the earth iron precipitated as ferric oxides and hydroxides. Copper allows a more limited range of redox potentials and is suggested to appear in living organisms later during evolution when air-dependent oxidation of insoluble Cu(0) and Cu(I) species resulted in soluble Cu(II). However, it is important to note that similar reactions can be catalyzed by both copper and iron centers (one classical example is found in superoxide dismutases).

3. IRON, COBALT AND DNA SYNTHESIS A key process for living organisms is DNA synthesis and repair on which depends cell proliferation. DNA synthesis depends on a balanced supply of the four

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deoxyribonucleotides (Reichard et al., 1998; Fontecave, 1998). In all living organisms, with no exception so far, this is achieved by reduction of the corresponding ribonucleoside diphosphates, NDPs, or triphosphates, NTPs. This reaction is catalyzed by a fascinating family of allosterically regulated metalloenzymes, named ribonucleotide reductases (RNRs). Thus DNA synthesis is not possible in the absence of transition metals, iron and cobalt more specifically (Mulliez et al., 1997). It is now generally accepted that life was first based on RNA and that the emergence of a ribonucleotide reductase was the key event that allowed the transition from the RNA to the DNA world (Reichard, 1997; Riera et al., 1997). According to that concept, we would expect to find only one type of enzyme with the same general structure in all organisms. Instead, we find, in contemporary metabolism, at least three distinct classes of RNR, which probably are the products of divergent evolution from a common ancestor. The evolutionnary relationship between these three classes has been recently discussed in a number of excellent review articles by Peter Reichard (Reichard, 1997; Reichard, 1993). Class I RNRs are strictly aerobic a2b2 enzymes and their substrates are ribonucleoside diphosphates. They are divided into two subclasses Ia and Ib. Class Ia RNRs are found in all types of eukaryotes, several viruses, few prokaryotes and bacteriophages. Both proteins Rl (a2) and R2 (b2) from Escherichia coli have been crystallized and their three-dimensional structure determined at high resolution (Nordlund et al., 1993; Uhlin et al., 1994). Protein Rl contains the binding sites for both substrates and allosteric effectors. Complexes of protein Rl with substrates and effectors have been structurally characterized (Eriksson et al., 1997) showing that the substrate site contains the three conserved redox active cysteines which were previously suggested to participate in ribonucleotide reduction. Protein R2 contains a tyrosyl radical essential for enzyme catalysis and a non heme diiron center, in which the ferric ions are linked by an oxo and a bidentate glutamate bridge, on each polypeptide chain. The role of the iron center is, in its reduced diferrous form, to react with oxygen in order to make an oxidizing high-valent iron species which is responsible for the oxidation of a specific tyrosine to a tyrosyl radical (Fontecave, 1998).

The importance of the link between iron metabolism and DNA synthesis through ribonucleotide reductase in eukaryotes has been little appreciated so far. It is now well established that iron chelators have a drastic effect on ribonucleotide reduction in human cells, leading to depletion of deoxyribonucleotide pools and inhibition of DNA synthesis (Cooper et al., 1996). This is something to consider when patients are treated with some chelators such as desferrioxamine, under circumstances of iron overload or oxidative stress pathologies. Class Ib RNRs are also a2b2 enzymes found in bacteria. They are closely related to class Ia enzymes, with similar Fe-radical center and amino acid sequences except for the lack of the first 50 N-terminal amino acid residues in the large Rl protein. As the N-terminus provides residues for binding the allosteric effectors, ATP and dATP, this difference results in differences in the allosteric regulation of ribonucleotide reduction (Reichard et al., 1998). Class II RNRs are found in bacteria and archaea (Reichard, 1997; Riera et al., 1997). They are active both aerobically and anaerobically. What characterizes a class II RNR is the requirement for adenosylcobalamin (AdoCbl). The enzyme facilitates

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homolysis of the Co-C bond of Adobl for generating an essential cysteinyl radical (Licht et al., 1996). A three-dimensional structure is not available so far, but elegant studies from JoAnne Stubbe have demonstrated that ribonucleotide reduction depends on the presence of three essential redox-active cysteines in the active site and proceeds much as in class I RNRs (Booker et al., 1994).

Class III RNRs are oxygen-sensitive enzymes found in some facultative anaerobes and bacteriophages. On the basis of sequence comparisons, it seems likely that methanogens also use a class III enzyme for deoxyribonucleotide synthesis (Reichard et al., 1998). The prototype is the enzyme that we discovered in 1989 in anaerobically growing E. coli cells (Fontecave et al., 1989). Biochemical and spectroscopic studies have shown that it is an a2b2 enzyme (Ollagnier et al., 1996). The large component a2 contains the substrate and the allosteric effector binding sites and, in its active form, a glycyl radical (Gly681 in E. coli) absolutely required for catalysis (Sun et al., 1996). Its three dimensional structure has been recently determined (Logan et al., 1999) The small component b2 contains an iron sulfur center which catalyzes the reduction of Sadenosylmethionine by flavodoxin to generate a putative 5'-deoxyadenosyl radical. The latter is supposed to be a precursor of the glycyl radical on protein a2 (Mulliez et al., 1993; Ollagnier et al., 1997).

As can be seen from equations 1,2 and 3, in all cases the metal centers are absolutely required for the generation of an essential protein radical. These examples (activation of molecular oxygen or sulfonium compounds by iron complexes or activation of Co-C bonds) represent fascinating reactions and illustrate the richness of the redox chemistry of trace elements in living systems.

REFERENCES Booker, S., Licht, S., Broderick, J., and Stubbe, J. 1994, Coenzyme B12-dependent ribonucleotide reductase: evidence for the participation of five cysteine residues in ribonucleotide reduction, Biochemistry 33:12676. Cooper, C.E., Lynagh, G.R., Hoyes, K.P., Hider, R.C., Cammack, R., and Porter, J.B. 1996, The relationship of intracellular iron chelation to te inhibition and regeneration of human ribonucleotide reductase, J. Biol. Chem. 271:20291. Eriksson, M., Uhlin, U., Ramaswamy, S., Ekberg, M., Regnström, K., Sjöberg, B.-M., and Eklund, H. 1997, binding of allosteric effectors to ribonucleotide reductase protein Rl, Structure 5:1077. Fontecave, M., Eliasson, R., and Reichard P. 1989, Oxygen-sensitive ribonucleoside triphosphate reductase is present in anaerobic E. coli, Proc. Natl. Acad. Sci. USA 86:2147. Fontecave, M. 1998, Ribonucleotide reductase and radical reactions, Cell. Mol. Life Sci. 54:684. Licht, S., Gerfen, G.J., and Stubbe, J. 1996, thiyl radicals in ribonucleotide reductases, Science 271:477. Logan, D.T., Andersson, J., Sjöberg, B.-M., and Nordlund, P. 1999, A glycyl radical site in the crystal structure of a class III ribonucleotide reductase, Science, 283:1499. Mulliez, E., Fontecave, M., Gaillard, J., and Reichard, P. 1993, an iron-sulfur center and a free radical in the active anaerobic ribonucleotide reductase from E. coli, J. Biol. Chem. 268:2296. Mulliez, E. and Fontecave, M. 1997, Structure and reactivity of the metal centers of ribonucleotide reductases, Chem. Ber. 130:317. Ochiai, E. 1978, Principles in bioinorganic chemistry, J. Chem. Educ. 55:631

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Ollagnier, S., Mulliez, E., Gaillard, J., Eliasson, R., Fontecave, M., and Reichard, P. 1996, The anaerobic E. coli ribonucleotide reductase, J. Biol. Chem. 271:9410. Ollagnier, S., Mulliez, E., Schmidt, P.P., Eliasson, R., Gaillard, J., Deronzier, C., Bergman, T, Gräslund, A., Reichard, P., and Fontecave, M. 1997, Activation of the anaerobic ribonucleotide reductase from E. coli, J. Biol. Chem. 272:24216. Nordlund, P. and Eklund, H. 1993, Structure and function of the E. coli ribonucleotide reductase protein R2, J. Mol. Biol. 232:123. Reichard, P. 1993, From RNA to DNA, why so many ribonucleotide reductases ?, Science 260:1773. Reichard, P. 1997, The evolution of ribonucleotide reduction, Trends in Biochem. Sci. 22:81. Reichard, P. and Jordan, A. 1998, Ribonucleotide reductases, Ann. Rev. Biochem. 67:71. Riera, J., Robb, F.T., Weiss, R., and Fontecave, M. 1997, Ribonucleotide reductase in the archaeon Pyrococcus furiosus: a critical enzyme in the evolution of DNA genomes ?, Proc. Natl. Acad. Sci. USA 94:475. Sun, X., Ollagnier, X., Schmidt, P.P., Atta, M., Mulliez, E., Lepape, L., Eliasson, R., Gräslund, A., Fontecave, M., Reichard, P., and Sjöberg, B-M. 1996, The free radical of the anaerobic ribonucleotide reductase from E. coli, J. Biol. Chem. 271:6827. Uhlin, U. and Eklund, H. 1994, Structure of ribonucleotide reductase protein Rl, Nature 370:533.

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IMPORTANCE OF TRACE ELEMENTS IN TRANSCRIPTION FACTOR NF-κB ACTIVATION

Jacques Piette Laboratory of Virology & Immunology Institute of Pathology B23 University of Liège B-4000 Liège, Belgium

and the other members of the Rel family of transcriptional activator proteins are a focal point for understanding how extracellular signals induce the expression of specific sets of genes in higher eukaryotes (Baeuerle and Henkel, 1994; May and Ghosh, 1998). Unlike most transcriptional factors, proteins of this family reside in the cytoplasm and must therefore translocate into the nucleus in order to function. The nuclear translocation of Rel proteins is induced by an extraordinarily large number of agents such as bacterial and viral pathogens, immune and inflammatory cytokines, or a variety of agents that damage cells such as oxidizing agents and radiation. Remarkably, is the main transcription factor activated by oxidative stress and then a potential target for modulation by trace elements. The Rel protein family has been divided into two groups based upon differences in their structures, functions, and modes of synthesis. The first group consists of p50 and p52 which are synthesized from precursor proteins of 105 and 100 kDa, respectively (Fig. 1). The mature proteins, which are generated by proteolytic processing, have a so-called Rel homology domain that includes motives for DNA-binding and dimerization with a nuclear localization signal (Fig. 1). The mature proteins form functional Rel dimers with themselves or other members of the family, and dimers containing the unprocessed proteins remain sequestered in the cytoplasm. The second group of Rel proteins, including RelA, c-Rel, RelB, v-Rel (Fig. 1) are not synthesized as precursors. Two types of Rel protein complexes are found in the cytoplasm prior to induction. The first type consists of Rel homo- or heterodimers (e.g. p50 and RelA) bound to a member of the family of inhibitory proteins Bcl-3, Fig. 2). Members of this family share a characteristic ankyrin repeat motif that is required for Telephone: 32-4-366.24.42, fax: 32-4-366.24.33; E-mail: [email protected] Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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their interactions with Rel proteins, and a C-terminal PEST sequence thought to be involved in protein degradation (Beg and Baldwin, 1993) (Fig. 2). The second type of complex consists of a heterodimer formed between a mature Rel protein (e.g. RelA) and an unprocessed Rel protein precursor (e.g., p105). Activating signals lead to the phosphorylation of both the Based on many observations, a model was proposed in which phosphorylation of results in its degradation, thereby allowing to translocate to the nucleus and bind sites (Israel, 1995). The potential key phospho-acceptor residues are Ser-32 and Ser-36 (Fig. 2). When mutated individually or in combination, they prevent phosphorylation following stimulation (Brown et al., 1995; Traenckner et al., 1995). Since the S32/S36A mutant is resistant to phosphorylation induced by various stimuli, different signaling pathways seem to result in the phosphorylation of these amino acids. Recently, an alternative mechanism of activation was demonstrated in Jurkat T cells treated with pervanadate, a protein tyrosine phosphatase inhibitor and T cell activator (Imbert et al., 1996). This mechanism involves tyrosine residue 42 phosphorylation but not degradation of While the carboxy terminal PEST sequence is not required for signal-induced degradation, its removal stabilizes free in unstimulated cells, indicating its requirement for constitutive turn-over of free (Van Antwerpen et al., 1996). This induced degradation is rapid and, in some cell types, can be completed within 10 minutes. It is also extensive and produces no obvious intermediates. Thus, the induced degradation is a remarkably efficient process which is an obligatory step in the activation of The phosphorylation event on Ser-32 and -36, which is carried out by a multisubunit kinase of high molecular weight (>700kDa), is required for an additional modification of at Lys-21 and -22, namely, multiubiquitination (DiDonato et al., 1997; Mercurio et al., 1997). Thus, it became clear that (i) phosphorylation of preceedes ubiquitination and (ii) ubiquitination is a signal for degradation. Recently, two groups have systematically fractionated activity that phosphorylated at Ser-32 and Ser-36 (DiDonato et al., 1997; Mercurio et al., 1997). The fraction with the highest activity was enriched with polypeptides of 85, 87, and 64kDa. Microsequence analysis of the 85kDa polypeptide followed by a partial cloning of the cDNA revealed that it was a previously identified ser/threo kinase of unknown function called CHUK. The CHUK kinase has been renamed or IKK-1 (745 amino acids) while another related kinase found in the complex has been designated or IKK-2 (756 amino acids). It has been found that can phosphorylate the Ser-32 and -36 of efficiently. Two candidate IKK kinases are NIK and MEKK1 (Nakano et al., 1998), but their physiological roles are not clear (Karin and Delhasse, 1998).

HOW TRACE ELEMENTS COULD INFLUENCE NF-κB ACTIVATION Sulphur- and Selenium-Containing Componds Among the water-soluble antioxidants known to inhibit NF-κB activation, sulphur derivatives are the most efficient (Schreck et al., 1991, 1992a, 1992b). The mode of action of these compounds is mainly through a replenishing of the intracellular GSH pool or by directly furnishing a pool of sulphur to several redox proteins such as thioredoxin (see above). Dietary selenium has been shown to enhance GSH peroxidase (GPX), an

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essential enzyme in the detoxification of both lipo- or hydroperoxides (Sappey et al., 1994). Enhanced GPX activity significantly contributes to a decrease in inducibility by proinflammatory cytokines, phorbol esters, or ROS (Sappey et al., 1994; Krebs-Remy et al., 1996; Renard et al., 1997). Another mode of action by which selenium could decrease cellular sensitivity to oxidative stress would be to increase the activity of thioredoxin reductase, a flavoenzyme responsible for the reduction of thioredoxin (Powis et al., 1997). On the other hand, it has recently been shown that sodium selenate can prolong the MAPK and S6 kinase activation in rat adipocytes treated with insulin, indicating the involvement of these kinases in the insulin-mimetic actions of selenium (Hei et al., 1998). Since MEKK1, a MAPK3 family member, has been shown to be part of the transduction pathway, one can suspect that selenium, in some cell types, could in some way directly affect one or several members of the signalosomes leading to phosphorylation.

ZINC Zinc is an essential element for cell growth, and especially for the immune system (Wellinghausen and Rink, 1998). Zinc deficiency, as well as zinc above normal level due to high-dose treatment, leads to an impaired immune function, because it has been shown tobe involved in specific interactions with immunologically important serum proteins, signal transduction components, and membrane functions. Zinc is involved in the catalytic center of many enzymes with various activities but has also been shown to be crucial for DNA-binding proteins. Gene expression cannot occur without zinc. Concerning the activation transduction machinery, zinc is putatively important for several members of the transduction machinery such as IKKs, which are zinc-finger proteins (Regnier et al., 1997). In T lymphocytes, several transduction pathways controlling immune responses (involving contain zinc-finger proteins, and it is possible that the in vivo functioning of these proteins could be affected by dietary zinc. Recently, it has been shown that the expression of T lymphocyte is elevated by dietary zinc deficiency and diet restriction in mice. This might contribute to altered thymocyte maturation, apoptosis and lymphopenia (Lepage et al., 1999). Knowing that is important for the transduction pathways leading to Y42 phosphorylation of by various stresses, these results demonstrate once again the importance of the Zn status in activation. On the other hand, both secretory (S-) and lysosomal (L-) sphingomyelinases (SMases), which contain several highly conserved zinc-binding motifs, are directly activated by zinc (Schissel et al., 1998). L-SMase is exposed to cellular Zn during trafficking to lysosomes and this exposition is required for its activity. In contrast, the pathway targeting S-SMase to secretion appears to be relatively sequestred from cellular pools of Zn; thus, S-SMase requires exogenous Zn for full activity. Because L-SMase has been shown to be activated upon the internalization of proinflammatory cytokine receptors, Zn deficiency could directly affect this enzymatic activity and be responsible for impairment of activation.

NICKEL AND COBALT Cobalt and nickel, as well as hyperoxia, stimulate the production of erythropoetin (EPO) through a putative interaction with oxygen sensors by changing the redox status of the central iron atom of heme proteins (Porwol et al., 1998). Then, Co and Ni could

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well be important for activation by influencing the cell redox status, which has been shown to be a crucial determinant for phosphorylation.

IRON The involvement of iron in many cellular processes such as gene regulation has been widely demonstrated (see Kuhn, 1998; Fleming and Andrews, 1998 for review). The discovery of iron regulatory proteins (IRPs) has provided a molecular framework in which the coordinate regulation of iron metabolism can be fully understood. IRPs bind to ironresponsive elements in specific mRNAs and regulate their utilization. The target of IRP action now appears to extend well beyond proteins that function in the storage (ferritin) or cellular uptake (transferrin receptor) of iron to include those involved in other aspects of iron metabolism as well as in the tricarboxylic cycle, and in signaling pathways. Multiple factors modulate the RNA-binding activity of IRPs, including iron, nitric oxide, phosphorylation by protein kinase C, oxidative stress, and hypoxia/reoxygenation (Eisenstein and Blemings, 1998). All these factors also influence activation, demonstrating an important overlap between cellular conditions influencing these important factors. Because IRPs are key modulators for the uptake and metabolic fate of iron in cells, they are the focal points for the modulation of cellular iron homeostasis in response to a variety of agents and circumstances. Beyond the well-defined chemical functions of iron in Fenton reactions and in the generation of highly reactive oxygen species (hydroxyl radicals, perferryl radicals, etc.) (Valentine et al., 1998), iron is an important co-factor for many proteins. In transduction pathways involved in activation, iron is important for lipoxygenase (Fig. 3). In mammals, lipoxygenases catalyze the formation of hydroperoxides as the first step in the biosynthesis of several inflammatory mediators

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(see Prigge et al., 1997). The substrate of this reaction, arachidonic acid, is the key precursor of two families of potent physiological effectors. It is the branch point between two central pathways: one, involving the enzyme cyclooxygenase, leads to the synthesis of prostaglandins and thromboxanes; the other, involving lipoxygenases, leads to the synthesis of leukotrienes and lipoxins, compounds that regulate important cellular responses in inflammation and immunity. Lipoxygenases are large non-heme, iron-containing enzymes that use molecular oxygen for the dioxygenation of arachidonic acid to form hydroperoxides, the first step in the biosynthetic pathways leading to leukotrienes and lipoxins. Because of the importance of these compounds, lipoxygenases have been the subject of extensive study ranging from detailed kinetic measurements to cloning, expression, and site-directed mutagenesis. The structure reveals that the 839 amino acids in the protein are organized in two domains: a N-terminal domain and a large, mostly helical C-terminal domain. The iron is present in the C-terminal domain facing two internal cavities that are probably the conduits through which the fatty acid and molecular oxygen gain access to the metal (Kuban et al., 1998). Numerous iron chelators (desferrioxamine, pyrrolidine dithiocarbamate, etc.) have been shown to significantly reduce activation by oxidative stress or by pro inflammatory cytokines (Schreck et al., 1992a, Sappey et al., 1995, Lee et al., 1997). Concommitantly to the inhibition of activation, phosphorylation of has also been shown to be abrogated (Lee et al., 1997), showing that the iron-catalyzed reaction in the transduction pathway is an early event corresponding either to the reaction leading to ROS release (lipoxygenase) or to the phosphorylation step of (Fig. 3). While iron is definitively an important element in the transduction pathways leading to activation, many incertainties still exist as to the nature of the reaction where iron is involved.

MANGANESE Manganese is an important co-factor of one of the primary antioxidant enzymes, manganese-containing superoxide dismutase (MnSOD). It has been shown that the transcriptional and DNA-binding ability of AP-1 and but not of SP-1, is inhibited (by 50%) in the MCF-7 cell line overexpressing MnSOD (Li et al., 1998). When transiently expressed, MnSOD inhibited AP-1 but increased transactivation, which can be abolished by sodium pyruvate, a hydrogen peroxide scavenger. It has also been suggested that tumor suppression by overexpressing MnSOD is related to a modulation of AP-1 and which causes a down-regulation of genes responsible for tumor malignant phenotype (Li et al., 1998).

ACKNOWLEDGMENTS JP is Research Director at the Belgian National Fund for Scientific Research (NFSR, Brussels, Belgium).

REFERENCES Baeuerle, P.A. and Henkel, T., 1994, Function and activation of NF-κB in the immune system, Annu. Rev. Immunol. 12:141–179.

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Beg, A.A. and Baldwin, A.S., 1993, The proteins: multifunctional regulators of transcription factors, Genes. Dev. 7:2064–2070. Brown, K., Gersterberger, S., Carlson, L., Franzoso, G., and Siebenliest, U., 1995, Control of proteolysis by site-specific, signal-induced phosphorylation, Science 267:1485–1488. DiDonato, J.A., Hayakawa, M., Rothwarf, D.M., Zandi, E., and Karin, M., 1997, A cytokine-responsive kinase that activates the transcription factor Nature 388:548–554. Eisenstein, R.S. and Blemmings, K.P., 1998, Iron regulatory proteins, iron responsive elements and iron homeostasis, J. Nutr. 128:2295–2298. Fleming, M.D. and Andrews, N.C., 1998, Mammalian iron transport: an unexpected link between metal homeostasis and host defense, J. Lab. Clin. Med. 132:464–468. Hei, Y.J., Farahbakbshian, S., Chen, X., Battell, M.L., and McNeill, J.H., 1998, Stimulation of MAP kinase and S6 kinase by vanadium and selenium in rat adipocytes, Mol. Cell. Biochem. 178:367–375. Imbert, V., Rupec, R.A., Livolsi, A., Pahl, H.L.,Traenckner, E.B.-M., Mueller-Dieckmann, C., Farahifar, D., Rossi, B., Auberger, P., Baeuerle, P.A., and Peyron, J.F., 1996, Tyrosine phosphorylation of activates without proteolytic degradation of Cell. 86:787–798. Israel, A., 1995, A role for phosphorylation and degradation in the control of activity, Trends Genet. 11:203–205. Karin, M., and Delhasse, M., 1998, JNK or IKK, AP-1 or which are the targets for MEK kinase 1 action?, Proc. Natl. Acad. Sci. U S A. 95:9067–9069. Kretz-Remy, C., Mehle, P., Mirault, M-E., and Arrigo, A.P., 1996, Inhibition of phosphorylation and degradation and subsequent activation by glutathion peroxidase overexpression, J. Cell. Biol., 133:1083–1093. Kuban, R.J., Weisner, R., Rathman, J., Veldink, G., Nolting, H., Sole, V.A., and Kuhn, H., 1998, The iron ligand sphere geometry of mammalian 15-lipoxygenases, Biochem. J. 332:237–242. Kuhn, L.C., 1998, Iron and gene expression: molecular mechanisms regulating cellular iron homeostasis, Nutr. Rev. 56:54–75. Lee, R., Beauparlant, P., Elford, H., Ponka, P., and Hicott, J., 1997, Selective inhibition of phosphorylation and HIV-1 LTR-directed gene expression by novel antioxidant compounds, Virology 234:277–290. Lepage, L.M., Giesbrecht, J.A., and Taylor, C.G., 1999, Expression of T lymphocyte p56(lck), a zinc-finger signal transduction protein, is elevated by dietary zinc deficiency and diet restriction in mice, J. Nutr, 129:620–607. Li, J.J., Oberley, F.W., Fan, M., and Colburn, N.H., 1998, Inhibition of AP-1 and by manganesecontaining superoxide dismutase in human breast cancer cells, FASEB J. 12:1713–1723. Mercurio, F., Zhu, H., Murray, B.W., Shevchenko, A., Bennett, B.L., Li, J.W., Young, D.B., Barbosa, M.L., and Mann, M., 1997, IKK-1 and IKK-2: cytokine-activated kinases essential for activation, Science 278:860–866. Nakano, H., Shindo, M., Sakon, S., Nishinaka, S., Mihara, M., Yagita, H., and Okumura, K., 1998, Differential regulation of kinase and by two upstream kinases, kinase and mitogenactivated protein kinase/ERK kinase kinase-1, Proc. Natl. Acad. Sci. USA. 95:3537–3542. Porwol, T., Ehleben, W., Zierold, K., Fandrey, J., and Acker, H., 1998, The influence of nickel and cobalt on putative members of the oxygen-sensing pathway of erythropoietin-producing HepG2 cells, Eur. J. Biochem. 256:16–23. Powis, G., Gasdaska, J.R., Gasdaska, P.Y., Berggren, M., Kirkpatrick, D.L., Engman, L., Cotgreave, I.A., Angulo, M., and Baker, A., 1997, Selenium and the thioredoxin redox system: effects on cell growth and death, Oncol. Res. 9:303–312. Prigge, S.T., Boyington, J.C., Faig, M., Doctor, K.S., Gaffney, B.J., and Amzel, L.M., 1997, Structure and mechanism of lipoxygenases, Biochimie 79:629–36. Regnier, C.H., Song, H.Y., Gao, X., Goeddel, D.V., Cao, Z., and Rothe, M., 1997, Identification and characterization of an kinase, Cell 90:373–383. Renard, P., Zachari, M.-D., Bougelet, C., Mirault, M.-E., Haegeman, G., Remacle, J., and Raes, M., Effects of antioxidant enzyme modulations on interleukin-1-induced activation, Biochem. Pharmacol. 53:149–160. Sappey, C., Legrand-Poels, S., Best-Belpomme, M., Favier, A., Rentier, B., and Piette, J., 1994, Stimulation of glutathione peroxidase activity decreases HIV type 1 activation after oxidative stress, AIDS Res. Hum. Retroviruses. 10:1451–1461. Sappey, C., Boelaert, J.R., Legrand-Poels, S., Forceille, C., Favier, A., and Piette, J., 1995, Iron chelation decreases and HIV type 1 activation due to oxidative stress, AIDS Res. Hum. Retroviruses 11:1049–1061.

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Schissel, S.L., Keesler, G.A., Schuchman, E.H., Williams, K.J., and Tabas, I., 1998, The cellular trafficking and zinc dependenece of secretory and lysosomal sphingomyelinase, two products of the acid sphingomyelinase gene, J. Biol. Chem. 273:18250–18259. Schreck, R., Rieber, P., and Baeuerle, P.A., 1991, Reactive oxygen intermediates as apparently widely used messengers in the activation of the transcription factor and HIV-1, EMBO J. 10:2247–2258. Schreck, R., Meier, B., Mannel, D.N., Dröge, W., and Baeuerle, P.A. 1992a, Dithiocarbamates as potent inhibitors of nuclear factor activation in intact cells, J. Exp. Med. 175:1181–1194. Schreck, R., Albermann, V., and Baeuerle, P.A., 1992b, Nuclear factor an oxidative stress-responsive transcription factor of eukaryotic cells (a review), Free Rad. Commun., 17:221–227. Traenckner, E.B.M., Pahl, H.L., Henkel, T., Schmidt, S., Wilk, S., and Baeuerle, P.A., 1995, Phosphorylation of human on serines 32 and 36 controls proteolysis and activation in response to diverse stimuli, EMBO J. 14:5433–5441. Valentine, J.S., Wertz, D.L., Lyons, T.J., Liou, L.L., Goto, J.J., and Gralla, E.B., 1998, The dark side of dioxygen biochemistry, Curr. Opin. Chem. Biol. 2:253–262. Van Antwerp, D.J. and Verma, I.M., 1996, Signal-induced degradation of association with and the PEST sequence in are not required, Mol. Cell. Biol. 16:6037–6045. Wellinghausen, N. and Rink, L., 1998, The significance of zinc for leukocyte biology, J. Leukoc. Biol. 64:571–577.

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TRACE ELEMENTS Metabolism and Oxidative Modifications of Lipoproteins

Y. Rayssiguier and A. Mazur Centre de Recherches en Nutrition Humaine Unité Maladies Métaboliques et Micronutriments INRA, Theix 63122 St-Genès-Champanelle France

Plasma lipoproteins are the source of the cholesterol that accumulates in the arterial wall and several lines of evidence suggest that oxidation of lipoproteins plays a role in atherogenesis (Ross, 1999). Oxidized LDL is taken up by the macrophages through the scavenger receptor pathway resulting in unregulated cholesterol accumulation and foam cell formation. Moreover, atherosclerosis is clearly an inflammatory disease and growth factors, cytokines and vasoactive substances influence the progression of atherosclerosis (Ross, 1999). Thus, dietary effects on atherosclerosis are not limited to the effect of dietary fats and cholesterol and other dietary factors such as micronutrients, including trace elements, contribute to altering the risk. The trace elements Cu, Zn, Se are well known essential components of antioxidant defenses while iron acts as an important mediator in cell injury accompanying oxidative stress. This review is focused on recent data suggesting that the potential role of essential trace elements in atherogenesis including dyslipidemia and vascular response is related to oxidative stress and antioxidant defenses.

1. TRACE ELEMENTS AND LIPOPROTEINS Inadequate dietary intake of trace elements in experimental models affects lipoprotein metabolism and results in dyslipidemia. It is clear that there are specific effects of different trace elements on lipoprotein metabolism (Rayssiguier and Mazur, 1995; Strain, 1998; Fields, 1999). The dyslipidemia may have various origins related to disturbances in lipid absorption, lipoprotein synthesis and secretion, modification in activities and gene expression of key proteins (enzymes, receptors, apolipoproteins) involved in lipid metabolism. In these experimental models, despite the association of oxidative stress with hyperlipidemia, a possible relationship between diet induced oxidative stress and diet Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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induced hyperlipidemia has not been closely examined. However, this possibility exists. For instance, vitamin E decreases plasma cholesterol concentration in hyperlipidemic hamster and Watanabe rabbit (Kubow et al., 1996; Willingham et al., 1993). Moreover, there is substantial evidence that free radicals induce a cellular response by changing gene expression (Jackson et al., 1998). Thus, we suggest that oxidative stress could play a causal role in hyperlipidemia associated with inadequate intake of trace elements. Copper

Cu deficiency in rats induces hypercholesterolemia and changes in lipoprotein concentration and composition (Lei, 1990; Mazur et al., 1992; Nassir et al., 1993; Allen and Klevay, 1994; Nassir et al., 1994). This hypercholesterolemia is mainly the result of an increase in HDL1 and LDL. An increase in plasma apo B levels results mostly from an increase in apoB 100 in LDL. The increase in plasma apo E is related to the increase in HDL1 fraction. Several studies were designed to examine apoprotein synthesis and gene expression to examine the mechanisms responsible for hyperlipemia. We have reported that hepatic apo B synthesis is enhanced and is associated with increased plasma apo B levels. The increase in apo B 100 synthesis reflects post-translational alterations in gene expression. In contrast, the increase in plasma apo E level is related to impaired metabolism of apo E rich HDL because we have shown that apo E synthesis is not affected. In our experimental model, Cu deficiency does not affect plasma apo A1 levels and no changes in liver synthesis and liver apo A1 mRNA levels were observed. In other studies, Cu deficiency increases the plasma apo A1 level as well as hepatic apo A1 production and secretion (Hoogeveen et al., 1995). The reasons for these different results are unclear. HMG CoA reductase activity is elevated in Cu deficient rats indicating that cholesterol synthesis is increased. It was proposed that excessive production of glutathione in the liver, probably resulting from increased oxidant stress, may activate HMG CoA reductase (Kim et al., 1992). Recent studies suggest that hepatic iron overload in Cudeficient rats may contribute to hypertriglyceridemia and hypercholesterolemia (Fields and Lewis, 1997). In contrast, reduction in iron intake was shown to exert a beneficial effect on plasma lipids in rats fed a Cu-deficient diet. Hepatic lipid peroxidation was also decreased. Cholesterolemia is higher in Cu-deficient rats fed fructose than starch, and a fructose diet increases lipid susceptibility to peroxidation (Rayssiguier et al., 1996). Thus, in these experimental models the possibility exists that hypercholesterolemia may be the result of increased peroxidation in the liver.

Zinc Zinc deficiency in rats results in a dyslipidemic lipoprotein profile (Nassir et al., 1996). The decrease in plasma total cholesterol is due to reduction in the HDL fraction containing apo A1. Zn deficiency specifically decreases hepatic apo A1 gene expression that may be responsible for the reduction in plasma apo A1 levels (Wu et al., 1998). However, there are several in evaluating the specificity of Zn deprivation on lipid metabolism because Zn-deficient animals have decreased food intake. When experiments are conducted with gastric tube feeding, Zn deficiency does not decrease plasma lipids and furthermore the effect of Zn deficiency depends on the type of fat (Eder and Kirchgessner, 1997). Hyperlipemia is observed in Zn-deficient rats fed linseed oil rich in PUFA. When Zn-deficient rats are fed olive oil, hyperlipemia is not observed. Zn deficiency causes increased oxidative stress, which is more pronounced in rats fed linseed

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oil than in rats fed olive oil. In this model, hyperlipemia observed in Zn-deficient rats could be associated with marked oxidative stress.

Selenium Selenium deficiency in the rat leads to dyslipidemia, the main effect being hypercholesterolemia. Increase in plasma cholesterol is associated with changes in plasma lipoprotein and apolipoprotein concentrations. In particular, there is a marked increase in apo E and HDL1 concentrations (Stone et al., 1994; Mazur et al., 1996; Nassir et al., 1997). Hepatic lipoprotein receptor levels (LDL receptor and HDL binding proteins) and apoprotein synthesis are not affected. Hypercholesterolemia is related to increased HMG CoA reductase activity in liver microsomes. Metabolic interrelationships between Se and vitamin E are well known. Oxidative damage in many tissues is more severe during combined Se and vitamin E deficiency than with deficiency of either antioxidant alone. We showed that vitamin E/Se-deficient rats had different pattern of plasma lipoproteins, compared to control and Se-dencient animals. An increased concentration of plasma LDL which corresponds to increased apo B levels occurs in double deficiency suggesting again that oxidative stress may influence lipoprotein distribution (Mazur et al., 1996).

Iron Iron overload has been shown to cause hypercholesterolemia in rats (Dabbagh, 1994). In some experiments, an increase of body Fe stores alone does not induce hypercholesterolemia but hypercholesterolemia occurs when high Fe diet is combined with decreased antioxidant defenses (Fields, 1999).

2. OXIDATIVE MODIFICATIONS OF LIPOPROTEINS AND VASCULAR RESPONSE Studies suggest that adequate trace element intake acts primarily by reducing oxidative modification of lipoproteins and vascular injury caused by free radical reaction rather than by altering plasma lipid levels.

Copper We have shown that copper deficiency in rats increases the susceptibility of lipoproteins to in vitro peroxidation (Rayssiguier et al., 1993; Motta et al., 1996; Mazur et al., 1998). At present, we have no precise explanation for such increased susceptibility to oxidation of lipoproteins from Cu deficient rats. Possible mechanisms include reduced antioxidant defenses and alteration in lipid composition of lipoproteins. As a generalized increase in lipid peroxidation occurs in dietary Cu deficiency (Nelson et al., 1992; Rayssiguier et al., 1993), Cu deficiency may facilitate cell mediated LDL oxidative modification in vivo. Moreover, the increased inflammatory response in Cu deficiency has probably a profound influence on pathogenesis (Schuschke et al., 1994). Cu deficiency in rats is associated with endothelial damage. NO, thromboxane and prostacyclin metabolisms are affected adversely by impaired oxidative defenses leading to greater vascular tone and thombogenesis (Allen and Klevay, 1994). Moreover, decreased lysyl oxidase activity may result in the elaboration of abnormal extracellular matrix protein that may

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cause destabilization of advanced atherosclerotic plaques (Allen and Klevay, 1994). While dietary Cu is clearly an antioxidant nutrient essential for cardiovascular health in animal models, copper ions are very effective in catalyzing the oxidative modification of LDL in vitro and some epidemiological evidences support the paradox that high levels of Cu may promote atherosclerosis. In fact, whether free copper is involved in lipoprotein modification in vivo is still unproven and epidemiological data are inconsistent (Ferns et al., 1997). For instance, serum Cu and ceruloplasmin levels have been suggested to be independent risk factors for CHD operating through oxidative modifications of LDL. In fact, atherosclerosis is an inflammatory disease and ceruloplasmin is increased by inflammation. Thus, the association between serum copper or ceruloplasmin and CHD may be attributed to the inflammation process rather than to the pro-oxidant effect of Cu (Klipstein-Grobusch et al., 1999). In two recent studies, Cu supplementation was found not to affect plasma lipoprotein and not contribute to lipoprotein oxidation. However, decreased susceptibility of red blood cells (RBC) to peroxidation (Rock et al., 1999) and increased lipoprotein oxidation lag time in subjects with suboptimal copper status (Jones et al., 1997) justify further inquiry into the concept that some individuals would benefit from increased copper intake.

Zinc This metal inhibits lipoprotein oxidation in vitro (Wilkins and Leake, 1994) and moderate zinc deficiency in rats produces VLDL and LDL with abnormally high sensitivity to in vitro oxidation (Faure et al., 1991; DiSilvestro and Blostein-Fujii, 1997). The amount of Zn in the lipoproteins does not appear to contribute to these results. Possible mechanisms for the changes in lipoprotein oxidation include modification in lipid composition and decreased antioxidant reserve of lipoproteins. Moreover, zinc affects the inflammatory process of atherosclerosis. Furthermore, zinc is a protective and critical nutrient for maintenance of endothelial integrity (Connell et al., 1997). plays a central role in regulating the cytokine network and hence its activation may be a major factor contributing to pathogenesis of atherosclerosis. Zinc supplementation markedly attenuates TNF mediated activation of These studies suggest that zinc may protect against transcription factors sensitive to oxidative stress and upregulation of inflammatory cytokines (Connell et al., 1997). Thus, zinc may exhibit anti-atherogenic properties by being able to function as an antioxidant and also by preventing metabolic and physiologic derangement of the vascular wall. However, the effect of Zn supplementation to alleviate the development of atherosclerotic lesions in experimental models or in atherosclerotic patients has not been clearly established.

Selenium Se deficiency in rats produces VLDL and LDL with abnormaly high sensitivity to in vitro oxidation. This result may be explained in part by a high unsaturated to saturated fatty acid ratio in lipoproteins from Se-deficient rats. Moreover, decreased antioxidant reserve is a possible mechanism for changes in lipoprotein oxidation (Mazur et al., 1996). Se supplementation can inhibit progression of atherosclerosis in animal models. Se supplementation was found to inhibit atherosclerosis in hypercholesterolemic rabbits more effectively than vitamin E alone, but with equal potency as probucol and this was independent of the effects on plasma cholesterol concentrations (Schwenke and Behr, 1998). Apo E-deficient transgenic mice are an attractive model for atherosclerosis studies.

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In the absence of apo E, lipoprotein remnants are not carried to the liver where they are normally metabolized and the mice become hypercholesterolemic. Atherosclerotic lesions develop that are similar to those in humans. Se supplementation results in a 30% reduction in lesions. Se supplementation increases GSH content and GPX activity in mice peritoneal macrophages and reduces LDL oxidation by macrophages (Rosenblat and Aviram, 1998). Thus, the increase in antioxidant defenses following Se supplementation contributes to the attenuation of the atherosclerotic process in this experimental model. However, there is still no unequivocal evidence for a role for Se deficiency as a causative factor in CVD in humans.

Iron Excessive iron has been proposed to be a potent risk factor for CHD. Iron overload increases the development of atherosclerotic lesions in hypercholesterolemic rabbits (Araujo et al., 1995). Oxidative modification of LDL in vitro is absolutely dependent on low concentration of iron ions (or copper). The possibility exists that deposits of iron in the arterial wall following iron loading stimulate in vivo LDL oxidation. Moreover, iron overload results in a pro-oxidative liver environment that would influence lipoprotein synthesis. Under these circumstances LDL would have a decreased antioxidant reserve and would be more susceptible to oxidation in the arterial wall when exposed to oxidative stress. Furthermore, iron overload increases myocardial damage caused by anoxia reperfusion. Thus, iron overload may amplify the damaging effect of superoxide overproduction in a very broad spectrum of inflammatory or ischemia-related conditions. Although excessive iron has been proposed as a risk factor for coronary heart disease in man, findings have been controversial (Tuomainen et al., 1998). The assessment of whether iron (and copper) status is related to the cardiovascular risk in man is complicated by the fact that biochemical indicators of status are modified by the inflammatory process. Other minerals and trace elements may also contribute to vascular risk and involve similar mechanisms. For instance, Mg deficiency leads to the activation of cellular systems such as monocytes/macrophages inducing the secretion of mediators (free radicals, cytokines) which are involved in inflammatory events. The inflammatory response contributes to the development of hyperlipemia, oxidative modifications of lipoproteins, and endothelial injury. In addition, animal studies have provided evidence for Mg modulation of atherogenesis (Rayssiguier et al., 1996; Malpuech-Brugère et al., 1999). To conclude, there is substantial evidence that micronutrients when consumed in appropriate amounts may help to protect against atherosclerosis. Most of the data however are from animal studies. The results warrant additional clinical and epidemiological studies examining the role of trace element status and nutritional interactions in CVD.

REFERENCES Allen, K.G.D. and Klevay, L.M., 1994, Copper: an antioxidant nutrient for cardiovascular health, Current Opinion of Lipidology 5:22–28. Araujo, J.A., Romano, E.L., Brito, B.E., Parthé, V., Romano, M., Bracho, M., Montano, R.F., and Cardier, J., 1995, Iron overload augments the development of atherosclerotic lesions in rabbits, Arteriosder. Thromb. Vase. Biol. 15:1172–1180. Connell, P., Young, V.M., Toborek, M., Cohen, D.A., Barve, S., McClain, C.J., and Hennig, B., 1997, Zinc attenuates tumor necrosis factor-mediated activation of transcription factors in endothelial cells, J. Am. Col. Nutr. 16:411–417.

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Dabbagh, A.J., Mannion, T., Lynch, S.M., and Frei, B., 1994, The effect of iron overload on rat plasma and liver oxidant status in vivo, Biochem. J. 300:799–803. DiSilvestro, R.A. and Blostein-Fujii, A., 1997, Moderate zinc deficiency in rats enhances lipoprotein oxidation in vitro, Free Radical Biology & Medicine 22:739–742. Eder, K. and Kirchgessner M., 1997, Concentrations of lipids in plasma and lipoproteins and oxidative susceptibility of low-density lipoproteins in zinc-deficient rats fed linseed oil or olive oil, J. Nutr. Biochem. 8:461–468. Faure, P., Roussel, A.M., Richard, M.J., Foulon, T., Groslambert, P., Hadjian, A., and Favier, A., 1991, Effect of an acute zinc depletion on rat lipoprotein distribution and peroxidation, Biol. Trace Elem. Res. 28:135–146. Ferns, G.A.A., Lamb, D.J., and Taylor, A., 1997, The possible role of copper ions in atherogenesis: the Blue Janus, Atherosclerosis 133:139–152. Fields, M., 1999, Role of trace elements in coronary heart disease, Br. J. Nutr. 81:85–86. Fields, M. and Lewis, C.G., 1997, Hepatic iron overload may contribute to hypertriglyceridemia and hypercholesterolemia in copper-deficient rats, 46:377–381. Hoogeveen, R.C.A.J.M., Reaves, S.K., and Lei, K.Y., 1995, Copper deficiency increases hepatic apolipoprotein A-I synthesis and secretion but does not alter hepatic total cellular apolipoprotein A-I mRNA abundance in rats, J. Nutr. 125:2935–2944. Jackson, M.J., McArdle, A., and McArdle, F., 1998, Antioxidant micronutrients and gene expression, Proc. Nutr. Soc. 57:301–305. Jones, A.A., DiSilvestro, R.A., Coleman, M., and Wagner, T.L., 1997, Copper supplementation of adult men: effects on blood copper enzyme activities and indicators of cardiovascular disease risk, Metabolism 46:1380–1383. Kim, S., Pi Yu Chao, and Allen, K.G.D., 1992, Inhibition of elevated hepatic glutathione ablishes copper deficiency cholesterolemia, FASEB J. 6:2467–2471. Klipstein-Grobusch, K., Grobbee, D.E., Koster, J.F., Lindemans, J., Boeing, H., Hofman, A., and Witteman, J.C.M., 1999, Serum caeruloplasmin as a coronary risk factor in the elderly: the Rotterdam Study, Br. J. Nutr. 81:139–144. Kubow, S., Goyette, N., Kermasha, S., Stewert-Phillip, J., and Koski, K.G., 1996, Vitamin E inhibits fish oil-induced hyperlipemia and tissue lipid peroxidation in Hamsters, Lipids 31:839–847. Lei, K.Y., 1990, Role of copper in lipid metabolism, CRC Press, Boca Raton, Fl. Malpuech-Brugère, C., Nowacki, W., Rock, E., Gueux, E., Mazur, A., and Rayssiguier, Y., 1998, Enhance tumor necrosis factor α production following endotoxin challenge in rats is an early event during magnesium deficiency. Biochem. Biophys. Acta 1453:35–40. Mazur, A., Gueux, E., Bureau, I., Feillet-Coudray, C., Rock, E., and Rayssiguier, Y., 1998, Copper deficiency and lipoprotein oxidation, Atherosclerosis 137:443–445. Mazur, A., Moundras, C., Gueux, E., Rock, A.M., Grolier, P., Chardigny, J.M., Rock, E., and Rayssiguier, Y., 1996, Selenium and vitamin E deficiencies affect plasma lipoprotein concentrations and their susceptibility to peroxidation in the rat, 9th Int. Symp. on Trace Elements inMan and Animals (TEMA9), Banff, Alberta, Canada, May 19–24, 1996. Mazur, A., Nassir, F., Gueux, E., Cardot, P., Bellanger, J., Lamand, M., and Rayssiguier, Y., 1992, The effect of dietary copper on rat plasma apolipoprotein B, E plasma levels, and apolipoprotein gene expression in liver and intestine, in: Biological Trace Element Research, Volume 34, pp. 107–113, The Humana Press, Inc. Mazur, A., Nassir, F., Gueux, E., Moundras, C., Bellanger, J., Grolier, P., Rock, E., and Rayssiguier, Y., 1996, Diets deficient in selenium and vitamin E affect plasma lipoprotein and apolipoprotein concentrations in the rat, Br. J. Nutr. 76:899–907. Motta, C., Gueux, E., Mazur, A., and Rayssiguier, Y., 1996, Lipid fluidity of triacylglycerol-rich lipoproteins isolated from copper-deficient rats, Br. J. Nutr. 75:767–773. Nassir, F., Blanchard, R.K., Mazur, A., Cousins, R.J., and Davidson, N.O., 1996, Apolipoprotein B mRNA editing is preserved in the intestine and liver of Zinc-deficient rats, J. Nutr. 126:860–864. Nassir, F., Mazur, A., Gueux, E., Sérougne, C., and Rayssiguier, Y., 1994, Apolipoprotein A-I, A-IV and E synthesis in the liver of copper-deficient rats, Lipids 29:727-729. Nassir, F., Mazur, A., Sérougne, C., Gueux, E., and Rayssiguier, Y., 1993, Hepatic apolipoprotein B synthesis in copper-deficient rats, FEBS 322:33–36. Nassir, F., Moundras, C., Bayle, D., Sérougne, C., Gueux, E., Rock, E., Rayssiguier, Y. and Mazur, A., 1997, Effect of selenium deficiency on hepatic lipid and lipoprotein metabolism in the rat, Br. J. Nutr. 78:493–500.

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Nelson, S.K., Ching-Jang Huang, Mathias, M.M., and Allen, K.G.D., 1992, Copper-marginal and copperdependent superoxide dismutasse activity, and increase aortic lipid peroxidation in rats, J. Nutr. 122:2101–2108. Rayssiguier, Y., and Mazur, A., 1995, Metal ions and lipid metabolism, in: Handbook of Metal Ligand Interactions in Biological Fluids, Volume 2, pp. 858–870 (G. Berthon, ed.), Marcel Dekker, New York. Rayssiguier, Y., Mazur, A., Gueux, E., and Rock, E., 1996, Magnesium deficiency affects lipid metabolism and atherosclerotic process by a mechanism involving inflammation and oxidative stress, pp. 251–255, In: Current Research in Magnesium (M.J. Halpern and J. Durlach, eds), John Libbey & Company Ltd., London. Rayssiguier, Y., Gueux, E., Bussière, L., and Mazur, A., 1993, Copper deficiency increases the susceptibility of lipoproteins and tissues to peroxidation in rats, J. Nutr. 123:1343–1348. Rayssiguier, Y., Gueux, E., Rock, E., and Mazur, A., 1996, Sucrose diet increases the cardiovascular defects of copper deficiency in rats by enhancing oxidative damage, 9th Int. Symp. on Trace Elements in Man and Animals (TEMA-9), Banff, Alberta, Canada, May 19–24, 1996. Rock, E., Mazur, A., Rayssiguier, Y., Kehoe, C., O’Connor, J.M., Bonham, M.P., and Strain, J.J., 1999, Effect of copper supplementation in middle aged people on plasma antiooxidants and red blood cell oxidizability: Foodcue study, TEMA 10. Rosenblat, M. and Aviram, M., 1998, Macrophage glutathione content and glutathione peroxidase activity are inversely related to cell-mediated oxidation of LDL: in vitro and in vivo studies, Free Rad. Biol. & Med., 24:305–317. Ross R., 1999, Atherosclerosis—An inflammatory disease, in: Mechanisms of Disease, Volume 340 (F.H. Epstein, ed.), pp. 115–126, Massachusetts Medical Society. Schuschke, D.A., Saari, J.T., Miller, F.N., 1994, The role of the mast cell in acute inflammatory responses of copper-deficient rats, Agents Actions 42:19–24. Schwenke, D.C. and Behr, S.R., 1998, Vitamin E combined with selenium inhibits atherosclerosis in hypercholesterolemic rabbits independently of effects on plasma cholesterol concentrations, Circ Res. 83:366–377. Stone, W.L., Scott, R.L., Stewart, E.M., and Kheshti, A., 1994, Lipoprotein alterations in the spontaneously hypertensive rat fed diets deficient in selenium and vitamin E, P.S.E.B.M. 206:130–137. Strain, J.J., 1998, Trace elements and cardiovascular disease, in: Role of Trace Elements for Health Promotion and Disease Prevention, Volume 29 (B. Sandströme, P. Walter eds.), pp. 127–140, Bibl Nutr Dieta, Basel, Karger. Tuomainen, T.P., Punnonen, K., Nyyssönen, K., and Salonen, J.T., 1998, Association between body iron stores and the risk of acute myocardial infarction in men, Circulation 97:1461–1466. Wilkins, G.M. and Leake, D.S., 1994, The oxidation of low density lipoprotein by cells or iron is inhibited by zinc, FEBS Letters 341:259–262. Willingham, A.K., Bolanos, C., Bohannan, E., and Cenedella, R.J., 1993, The effects of high levels of vitamin E on the progression of atherosclerosis in the Watanabe heritable hyperlipidemic rabbit, J. Nutr. Biochem. 4:651–658. Wu, J.Y.J., Reaves, S.K., Yi Ran Wang, Yan Wu, Lei, P.P., and Lei, K.Y., 1998, Zinc deficiency decreases plasam level and hepatic mRNA abundance of apolipoprotein A-I in rats and hamsters, Am. J. Physiol. 275:C1516–C1525.

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CO-LOCALIZATION OF Cu/Zn-SUPEROXIDE DISMUTASE (SOD-1), NITRIC OXIDE SYNTHASE (NOS), AND ZN/CU-METALLOTHIONEIN (MT) IN RAT BRAIN 1

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Masashi Okabe , Toshiyuki Hosokawa , Shigeru Saito , Takeshi Saito , 3 1 Masaaki Kurasaki , and Hidesuke Shimizu 1

Department of public health and environmental medicine The Jikei University School of Medicine Tokyo 105-8461 Japan 2 Center for Research and Development in Higher Education Hokkaido University Sapporo 060-0809 Japan 3 Department of Environmental Medicine and Informatics Graduate School of Environmental Earth Science Hokkaido University Sapporo 060-0810 Japan 4 Department of Hygiene and preventive Medicine Hokkaido University School of Medicine Sapporo 060-8638 Japan

SUMMARY Co-localization of NOS, MT and SOD-1 in rat brain was visualized by tissueblotting technique. Co-localization of the proteins was observed in the granular cells of the cerebellum. The results suggest the possibility of the synthesis of nitroso-thionein (NO-MT) from NO and MT under the endogenous oxygen radical free environment in the region. Moreover, the MT immuno-reactivity and NO releasing ability were observed in the same fractions from the chromatography. NO and Zn were considered to be

Address all correspondence to: Dr M. J. Richard; LBSO Laboratoire de Biologie du Stress Oxydant, Hôpital A. Michallon, BP 217-38043 Grenoble; France. Telephone: 476765147; Fax: 476765664; email: [email protected] Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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released from cerebellar NO-MT by replacement with Hg and Cu. From these results, MT was expected to be a NO acceptor and/or donor, the different roles of MT between acceptor and/or donor of NO could be regulated by the concentration of Cu ions in the tissue cells.

1. INTRODUCTION NO is recognized as a bi-functional (beneficial/deleterious) molecule, and its balance may be modulated by redox potential and/or thiol groups in the tissue (Lipton et al., 1993). These histo-chemical factors are, however, still unclear. In this study, to understand the function of NO in the brain, the histo-chemical environment for the preservation of NO, distribution of oxygen radical scavenging activity and a thiol rich protein MT in rat brain were revealed.

2. MATERIALS AND METHODS Male Wistar rats (180–200 g body weight) were used. The animals were received a trans-cardiovascular perfusion with 40mM Tris-33mM HCl containing 154mM NaCl (500 ml/kg) after an overdose of pentobarbital anesthesia (60mg/kg IP). The procedures of histochemistry and chromatography were carried out according to our previous reports (Okabe et al., 1993; Okabe et al., 1996; Okabe et al., 1998). The contents of NO in the fractions were measured with fluorometric method (Cook et al., 1996). The immunoreactivity against anti-MT antibody E9 of each fraction was measured by competitive ELISA.

3. RESULTS Localization of SOD, NOS and MT in the Brain The distributions of the proteins showed as a striking pattern in the parasagittal section blots (Fig. 1, A; SOD activity, B; NOS and C; MT). In the blots, high contents of proteins were detected in the granular layer of the cerebellum (Fig. 1, A, B and C; arrows). On the contrary, the proteins scarcely observed in corpus callosum and in corpus medullare of the cerebellum (arrow heads in Fig. 1).

Chromatographic Behavior of MT and NO The elution profile of MT on Superdex75 FPLC column was shown in Fig. 2, A, as an immuno-reactivity against MT in each fractions. The major peak of the reactivity was identical to that showed the profiles of NO releasing by addition of Hg ions (final concentration was 1 mM) as shown in Fig. 2, B. Moreover, it was revealed the major metal component of MT was zinc (Fig. 1, C).

NO Releasing from Nitroso-Thionein (NO-MT) by Addition of Metal Ions The amounts of released NO from cerebellar NO-MT and commercially available MT (Sigma) by treatment with metal ions were shown in Fig. 3A and B. In both case, Hg and Cu ions showed NO releasing ability, whereas Zn and Cd failed to release NO

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from the proteins. The releasing potential of Hg ions was about two folds larger than that of Cu ions. The commercial Zn-MT released NO up to 25% in comparison with cerebellar MT under the same conditions.

4. DISCUSSION Crosstalk of SOD-1, NO and MT The region showed high SOD-1 activity was also well known as a forming region of NO, a rapid diffusible retrograde signal transducer, which synthesized by NOsynthase; NOS, EC 1.14.23 (Bredt et al., 1990). With excessive release of both NO and reactive oxygen radical, the balance between NO and oxygen radical might be overwhelmed, leading to cell death. As suggested by Lei et al. NO is necessary but not sufficient for neuronal injury and is toxic in the presence of oxygen radical (Lei et al., 1992). The results in the study, the localizations of both SOD-1 and Zn/Cu-MT were identical to that of NOS. This observation may lead the further insight that NO generating regions require oxygen radical free environment to maintenance to express a beneficial NO function and to prevent the formation of toxic peroxynitrite. Moreover, excess NO might be temporally captured by thiol-rich proteins such as Zn/Cu-MT in the tissue. The formed NO-MT indicated NO releasing potentials respond to Cu which an essential trace element. These findings permit us to speculate that the micro environment at the brain was also arranged by SOD-1 and Zn/Cu-MT for expression of beneficial NO function.

Non-Enzymatic Releasing of NO from Nitroso-Thiol In this study we observed the releasing of NO from NO-MT by addition of Hg and/or Cu ion. The releasing of NO was not only from NO-MT, but also Cook et al.

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reported from other S-nitroso thiols such as S-nitrosoglutathione (GSNO) and/or Snitrosylated bovine serum albumin (Cook et al., 1996). The potential upon releasing of NO from NO-MT by Hg and Cu ion was not equivalent, Hg ion was approx. 4 times higher than that of Cu ion. Similarity was reported also by Cook et al., they described that the releasing potential of Hg ion was about 4 times higher than that of Cu ion against GSNO. On the other hand, the sequence of binding affinity of metal ions against thiols is Hg > Cu > Cd > Zn, from these observations on the releasing of NO from nitrosoproteins, we postulated that the binding affinity of NO to cysteine residues of peptides and/or proteins may be equivalent to the value between the binding affinity of Cd and that of Cu, or rather near to that of Cu. Up to now, the toxicity of heavy metals such as Hg has been explained by denaturing of the enzymes due to bind the heavy metal ions to cysteine residues of active sites. Based on the results, we would like to propose the unregulated NO releasing from NO pooled proteins as an additional mechanism of Hg toxicity. On the other hand, The releasing of NO caused by Cu ion was not so rapid, rather continuously (in our experiment, 15 ~ 30min, data not shown) than the case of Hg (within 3min, not shown). The moderate potentials of NO releasing from S-NO of Cu ion may be a novel bio-function of Cu as a essential trace element. REFERENCES Bredt D., Hwang P., and Snyder S., 1990, Localization of nitric oxide synthase indicating a neural role for nitric oxide, Nature 347:768–770. Cook J., Kim S., Teague D., Krishna M., Roberto P., Mitchell J., Vodovotz Y., Nims R., Christodoulou D., Miles A., Grisham M., and Wink D., 1996, Convenient colorimetric and fluorometric assays for S-nitrosothiols, Anal. Biochem., 238:150–158. Lei S., Pan Z., Aggarwal S., Chen H., Hartman J., Sucher N., and Lipton S., 1992, Effect of nitric oxide production on the redox modulatory site of the NMDA receptor-channel complex, Neuron 8:1087– 1099. Lipton S., Choi Y., Pan Z., Lei S., Chen H., Sucher N., Loscalzo J., Singel D., and Stamler J., 1993, A redoxbased mechanism for the neuro protective and neuro destructive effects of nitric oxide and related nitroso-compounds, Nature. 364:626–632. Okabe M., Nyakas C., Buwalda B., and Luiten P., 1993, A Novel Method for Direct Transfer of Native Proteins from Sectioned Tissue to Blotting Membrane: Procedure and Some Applications, J. Histochem. Cytochem. 41:972–934. Okabe M., Nakayama K., Kurasaki M., Yamasaki F., Aoyagi K., Yamanoshita O., Sato S., Okui T., Ohyama T., and Kasai., 1996, Direct visualization of copper-metallothionein in kidney of LEC rat: Application of auto-fluorescence signal of copper-thiolate cluster, J. Histochem. Cytochem. 44:865–873. Okabe M., Saito S., Saito T., Ito K., Kimura S., Niioka T., and Kurasaki M., 1998, Histochemical localization of superoxide dismutase activity in rat brain, Free Rad, Biol. Med., 24:1470–1476.

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EFFECT OF DOLOMITE AND THIAMINE SUPPLEMENTATION ON SERUM TOTAL ANTIOXIDANT STATUS AND BIOELEMENTS CONCENTRATION IN LEAD-INTOXICATED RATS

Z. Krejpcio and R. Wojciak Department of Human Nutrition and Hygiene Agricultural University Wojska Polskiego str. 31, 60-624 Poznan Poland

1. INTRODUCTION The results of recent studies [Sandhir et al., 1994; Naarala et al., 1995; Dabrowska et al., 1996; Ercal et al., 1996] have shown that lead depletes glutathione and protein bound sulfhydryl groups leading to the production of reactive oxygene species, lipid peroxidation, DNA damage, and altered calcium homeostasis. Since nutritional factors such as calcium, magnesium, thiamine, and other nutrients can influence lead absorption and toxicity, the objective of this study was to investigate the effect of dolomite and thiamine supplementation on serum total antioxidant status and some bioelements concentration in rats exposed to lead acetate.

2. MATERIALS AND METHODS The study was carried out on 6-week-old male Wistar rats fed for 8 week period semipurified diets (20% casein, 10% soy oil, 10% sucrose, 50.5% wheat starch, 1–5% potato starch, 3.5% mineral mixture and 1% vitamin mixture) supplemented with a combination of two levels of dolomite (20 and 40g/kg diet), and two levels of thiamine (hydrochloride, 25 and 50mg/kg diet) and lead acetate (500mgPb/kg diet, approx. 30% in a factorial design Moreover, two reference groups (K0-standard diet without lead and the supplements, and K1-standard diet with 500mgPb/kg diet) were established. Animals had free access to food and distilled water. At the end of the Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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experiment rats were sacrificed (thiopental injection) to draw blood and remove inner organs for biochemical analyses. The total antioxidant status (TAS, mmol/l) was measured by the degree of suppression by serum of the stable radical ABTS using the Randox Laboratories kit. The concentration of Ca, Mg, Zn and Cu in serum was determined by the flame atomic absorption spectrometry method (Zeiss AAS-3, with BC). For statistical evaluation of the results, the ANOVA and linear regression analyses at p < 0.05 were applied.

3. RESULTS The effect of dolomite and thiamine supplementation on the serum parameters is presented in Tables 1 and 2. As shown in Table 1, the mean serum TAS was significantly

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decreased (by 56%) in the rats exposed to lead vs. the control group (K0). Dolomite and thiamine supplementation significantly increased serum TAS (by 155–175%) in all tested groups of animals poisoned with lead acetate. Besides, despite lead ingestion, rats fed diets supplemented with dolomite and thiamine exhibited the increased TAS values even in comparison with the control K0. The level of dolomite and thiamine supplementation did not affect serum TAS in the rats exposed to lead, however, an interaction of these factors was observed (Table 2). The serum Ca concentration did not differ, whereas serum Mg was markedly elevated in the group I and IV. The level of dietary supplements did not affect the serum Ca and Mg concentrations, however, an interaction of the dietary supplements was found for serum Mg. The serum Zn concentration was significantly decreased in the rats receiving the higher level of thiamine. The serum Cu concentration increased in the group I and decreased in the group II, in comparison with the control K1. Also, the interaction of dolomite and thiamine was noticed in the rats exposed to lead. Relationships between pairs of the studied biochemical parameters as well as liver Pb and Fe contents (not shown in this report) in the rats were calculated using a linear regression analysis. It was found that the serum TAS was positively correlated with the serum Mg concentration (r = 0.29, p < 0.05) and inversely correlated with the serum Ca/Mg ratio (r = –0.37, p < 0.05) as well as the liver Pb (r = –0.69, p < 0.001) and Fe content (r = –0.48, p < 0.001) in the rats intoxicated with lead. Additionally, the serum Ca was positively correlated with Mg (r = 0.63, p < 0.001) and Zn (r = 0.42, p < 0.01). The serum Mg was proportional to the serum Zn (r = 0.33, p < 0.05) as well.

4. DISCUSSION In the literature there is an evidence that Pb ions are able to accelerate Fe-dependent lipid peroxidation which may have physiological significance in lead poisoning [Monteiro et al., 1986; Quinlan et al., 1988]. The mechanisms by which lead promotes oxidative stress are not clear. Quinlan et al. (1988) suggested that Pb ions, which can not participate in redox radical reactions, change membrane structure restricting phospholipid movement and facilitating the propagation of peroxidation. According to HermesLima (1991) the participation of free radicals in plumbism may occur at three distinct levels. Firstly, the ALA overload induced by lead inhibition of the hem biosynthesis pathways, could be a source of oxygen formation in vivo. Secondly, the binding of Pb ions to biological membranes could facilitate propagation of Fe-dependent lipid peroxidation. Thirdly, lead could accelerate lipid peroxidation induced by ALA-generated oxygen radicals. The depressed TAS accompanied by increased Pb and Fe contents in the liver, obtained in this study, support the second hypothesis showing that Pb accelerates Fentontype lipid peroxidation induced by Fe. As it is known, lead damages the hem synthesis leading to an increase of free Fe and its accumulation in the liver, which may explain the decrease of reducing power of serum caused by lead ingestion. The dolomite supplementation significantly increased the serum TAS which may be a result of reduction of lead absorption from the gut in the presence of calcium and magnesium as well as interaction of these metals at the metabolic level. The weak positive correlation between serum TAS and Mg, and negative one between serum TAS and Ca/Mg ratio confirms the protective role of Mg against free radical formation and lipid peroxidation reported by Garcia et al. (1998). There was no influence of moderate dolomite supplementation on serum Ca, Mg, Zn and Cu concentration that may result from homeostatic mechanisms responsible for regulation of these ions in the body fluids. The depression of serum Zn

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caused by high doses of thiamine may result from its chelating properties reported by Bratton et al. (1981).

5. CONCLUSIONS Lead induces lipid peroxidation in rats exposed to this metal. Dolomite and thiamine supplementation attenuates lead-induced oxidative stress. Moderate dolomite supplementation does not influence serum Ca, Mg, Zn and Cu concentration while high doses of thiamine decrease serum Zn concentration in rats exposed to lead.

REFERENCES Bratton, G.R., Zmudzki, J., Bell, M.C., and Warnock, L., 1981, Thiamine effects on lead intoxication and deposition in tissues. Therapeutic potential. Toxicol. Appl. Pharmacol., 59:164–172. Dabrowska-Bouta, B., Struzynska, L., and Rafalowska, U., 1996, Does lead provoke the peroxidation process in brain synaptosomas?, Molecular and Chemical Neuropathol.,29:127–139. Ercal, N., Treeratphan, P., Lute, P., Hammond, T.C., and Matthews, R.H., 1996, N-acetylocysteine protects Chinese hamster ovary CHO cells from lead-induced oxidative stress, Toxicology, 108(l–2):57–64. Garcia, L.A., Dejong, S.C., Martin, S.M., Smith, R.S., Buettner, G.R., and Kerber, R.E., 1998, Magnesium reduces free radicals in an in vivo coronary occlusion-reperfusion model, J. Am. Coll. Cardiol., 32(2):536–539. Hermes-Lima, M., Valle, G.R.V., Vercesi, A.E., and Bechara, E.J.H., 1991, Damage to rat liver mitochondria promoted by delta-aminolevulinic acid-generated oxygene species: connection with acute intermittent porphyria and lead poisoning, Biochimica and Biophysica Acta, 1056:57–63. Monteiro, H.P., Abdalla, D.S.P., Faljoni-Alario, A., and Bachara, E.J.H., 1986, Generation of active oxygene species during complex autoxidation of oxyhemoglogin and delta-aminolevulinic acid. Biochimica and Biophysica Acta, 881:100–106. Naarala, J.T., Lovikkanen, J.J., Ruotsalainen, M.H., and Savolainen, K.M., 1995, Lead amplifies glutamateinduced oxidative stress, Free Radic. Biol. Med, 19(5):689–693. Quinlan, G.J., Halliwell, B., Moorhouse, C.P., and Gutteridge, J.M.G., 1988, Action of lead(II) and aluminium (III) ions on iron-stimulated lipid peroxidation in liposomes, erythrocytes and rat liver microsomal fractions, Biochimica and Biophysica Acta, 962:196–200. Sandhir, R., Julka, D., and Gill, K.D., 1994, Lipoperoxidative damage on lead exposure in rat brain and its implication on membrane bound enzymes, Pharmacol. & Toxicol., 74(2):66–71.

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PROTECTIVE EFFECTS OF ANTIOXIDANTS ON IRON-INDUCED FREE RADICAL-MEDIATED DAMAGING PROCESSES IN HUMANS AND ANIMALS

Igor Afanas’ev, Elena Ostrachovich, Irina Deeva, and Ludmila Korkina Russian State Medical University Osato Bio-Medical Institute Moscow, Russia

1. INTRODUCTION Iron is believed to be a major catalyst of the transformation of ubiquitous, physiologically important and slightly toxic superoxide ion into highly reactive hydroxyl (or hydroxyl-like) free radicals through the superoxide-driven Fenton reaction:

Various therapeutic schemes comprising chelators and antioxidants have been recently developed to ameliorate adverse clinical effects associated with iron-driven oxygen radical overproduction in human organism. However, although numerous antioxidants, free radical scavengers, and chelators have been under extensive investigation in thousands of laboratories around the world, only a few of them are feasible for the clinical use. Among them natural non-toxic mixtures containing metal chelators, free radical scavengers and antioxidants seem to be of particular interest. In this work we compared the in vitro and ex vivo effects of Bio-normalizer (BN, the complex nutriceutical product prepared by microbial and yeast fermentation of unripe papaya fruits) with effects of some classical antioxidants on the iron-induced free radical-mediated damage to erythrocytes of thalassemic patients as well as to liver and peritoneal macrophages of iron-overloaded (IOL) rats. Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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2. EXPERIMENTAL Iron overloading in rats was performed by the intraperitoneal injection of ferrous sulfate (1 mL, 100 µM, 3 times a day during 24 hours). After that, BN was given 200mg a day for 7 days. The concentration of TBAR products in liver microsomes and oxygen radical release from peritoneal macrophages (by means of luminol-dependent chemiluminescence, LDCL) were determined twice before and after BN administration. Spontaneous and quinone-induced free radical formation in thalassemic erythrocytes was measured by cytochrome c reduction in the presence or absence of antioxidants. The level of MetHb formed was calculated from the absorbance at 560, 577 and 630 nm.

3. RESULTS AND DISCUSSION 3.1. Effects of Antioxidants on Free Radical Processes in Iron-Overloaded Rats We have earlier shown (Afanas’ev et al., 1995) that bioflavonoid rutin and lipoic acid (LA) decreased lipid peroxidation in liver microsomes and free radical production by phagocytes of IOL rats. Similarly, BN inhibited lipid peroxidation in IOL microsomes and oxygen radical release by peritoneal macrophages. As is seen from Table 1, the efficiency of the antioxidants studied decreased in the range: rutin > BN > LA. Unlikely other antioxidants studied, besides the suppression of iron-induced free radical-mediated processes BN was able to remove excessive iron from microsomal and phagocyte membranes (Table 1).

3.2. Effects of Antioxidants on Free Radical Production by Thalassemic Erythrocytes Oxidative stress associated with abnormal oxidation of hemoglobin and accumulation of intracellular “labile” iron is considered as an origin of short lifespan of thalassemic erythrocytes. We studied the effects of antioxidants on the oxygen radical release and oxidation of oxyhemoglobin in erythrocytes isolated from the patients with thalassemia major. In the preliminary experiments, using normal erythrocytes, we found that BN, rutin, NAC, and ubiquinone suppressed though to different extent spontaneous and menadione (MD)-stimulated oxidation of to MetHb (Table 2). BN and rutin, being the most effective inhibitors, were selected for experiments with thalassemic erythrocytes. BN decreased spontaneous and MD-stimulated

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oxidation by 50–30% and the rate of cytochrome c reduction by 85–20% (Figs. 1 and 2). In contrast, rutin, being an effective inhibitor of MD-induced oxidative processes in thalassemic erythrocytes (the inhibition of 30–60%), significantly enhanced spontaneous free radical production.

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These findings pointed out at the principal difference in the protective mechanisms of BN and rutin. Since difference between BN and rutin was observed only in thalassemic cells, it could be explained in three ways: (1) by BN unique ability to remove free iron from the membrane that was proven in experiments with IOL rats; (2) by BN direct protective effect towards abnormal hemoglobin oxidation; (3) by the BN free radical scavenging effect. On the other hand, rutin was able neither to remove iron from cells nor to affect hemoglobin oxidation, therefore, its antioxidant effect seems depend exclusively on its oxygen radical scavenging activity. It is difficult to explain the stimulating effect of rutin on the intensity of spontaneous free radical formation. We have earlier shown that iron-rutin complexes were usually redox inactive the catalysis of the formation of hydroxyl radical by the Fenton reaction. But, it seems that rutin may activate iron deposits by some unknown yet mechanism in thalassemic erythrocytes increasing free radical release from them. At the same time, rutin remains an effective inhibitor of oxidative processes in thalassemic erythrocytes under pro-oxidant conditions. On the whole, BN is apparently the most perspective inhibitor of iron-induced free radical-mediated pathological processes in a number of human diseases.

REFERENCE Afanas’ev, I.B., Ostrachovitch, E.A., Abramova, N.E., and Korkina, L.G., 1995, Different antioxidant activities of bioflavonoid rutin in normal and iron-overloading rats: Biochem. Pharmacol. 50:627–635.

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COMPLEXES OF FLAVONOIDS WITH IRON AND COPPER AS A NEW WAY OF DECREASING THE OXIDATIVE DAMAGE INDUCED BY TRANSITION METAL SUPPLEMENTATION L. G. Korkina1, E. A. Ostrachovich1, G. A. Ibragimova1, and I. B. Afanas’ev2 1

Russian State Medical University 1 Ostrovityanova, Moscow 117513 Russia 2 Vitamin Research Institute 14A Nauchny Pr., Moscow 117820 Russia

1. INTRODUCTION Ron and copper ions are essential elements in a wide variety of metabolic processes. The acquired or inherited deficiency of these metals causes the impaired cellular metabolism and physiology that leads to the development of a number of human pathologies such as Menkel disease and iron-deficient anemia. An appropriate supplementation with iron and copper is necessary to avoid health complications associated with metal deprivation. Unfortunately, a vast majority of widely used iron- and copper-containing supplements comprises transition metals in the redox active form that apparently increases the risk of reactive oxygen species overproduction with consecutive oxidative damage to biomolecules, cellular membranes, and tissues (Halliwell and Gutteridge, 1984). A main goal of the present study was to develop new non-toxic iron and copper complexes with increased bioavailability and significant antioxidant properties. As a ligand, bioflavonoid rutin known as an antioxidant and metal chelator (Afanas’ev et al., 1989) was used. Being incorporated in the complexes with rutin and inorganic anions, Fe and Cu lost completely their pro-oxidant ability and increased substantially both free radical scavenging and antioxidant activities of rutin. Due to this and the high bioavalability of rutin, Fe- and Cu-rutin complexes protected cellular membranes, DNA, and lung tissue against oxidative damage. Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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2. MATERIALS AND METHODS 2.1. Materials All reagents, solvents, and mediums were purchased from Sigma Co. (St Louis, USA) if not said otherwise.

2.2. Preparation of Metal-Rutin Complexes Metal-rutin complexes, Fe(rut)(oxalate) and were prepared by mixing rutin with iron or copper salts in Tris-HCl or phosphate buffers at pH 7.4. The formation of complexes was characterized by the shift of a long-wave rutin maximum at 366 nm to the right, the maximum shift being achieved at the 1:1 stoichiometry.

2.3. Superoxide production in the xanthine-xanthine oxidase system was measured by the standard cytochrome c reduction method (McCord and Fridovich, 1968).

2.4. Lipid Peroxidation (LPO) and Superoxide Production in Liver Microsomes LPO was determined by the Aust method (1987) with slight modifications. LPO in microsomes (0.5 mg protein/mL) was initiated by and ADP in 0.1 M phosphate buffer, pH 7.4, in the presence or absence of rutin or its complexes. The reaction was started by adding of 0.3mM NADPH. After 30min incubation at 37°C, was added to stop oxidation, and TBA reactive products (TBAR) were measured by standard procedures. In parallel experiments, the rate of cytochrome c reduction (cyt c) and intensity of lucigenin-dependent chemiluminescence (Luc-CL; lucigenin, 40 were registered.

2.5. Asbestos- and bleomycin-induced chromosomal aberrations were determined in culture of human lymphocytes by method described in details previously (Korkina et al., 1992).

2.6. Lung inflammation and fibrosis in rats were induced by intra-tracheal instillation 1 ml) in of either the suspension of asbestos fibers (1mg/ml, 1 ml) or bleomycin ( physiological solution. Inflammatory reaction was assessed by the wet lung weight, neutrophil count and protein content in broncho-alveolar fluid, and the intensity of fibrosis by hydroxyproline level in the lung tissue.

3. RESULTS AND DISCUSSION We hypothesized that the complex formation of rutin with iron and copper may enhance the antioxidant properties of bioflavonoid due to the additional metal-

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containing superoxide-dismuting center. This suggestion was confirmed by the effects of complexes on the rate of superoxide production in both xanthine-xanthine oxidase and microsomal systems (Table 1) All the complexes studied inhibited superoxide formation in a concentrationdependent manner. Noteworthy, values of and Fe(rut)(oxalate) were tenfold smaller than that of rutin itself. Unlike Fe(rut)(oxalate) and the superoxide-dismuting and LPO inhibiting effects of were comparable with those of rutin. Therefore, the complexation of Fe and Cu ions with rutin led to the entire lack of pro-oxidant action of metals. Furthermore, the rutin molecule gained a novel free radical scavenging center. Probably due to the highly elevated antioxidant activity, Fe- and Curutin complexes were more effective than rutin in protecting microsomal membranes

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against the iron-induced LPO (Table 1) and DNA damage induced by both asbestos and bleomycin (Table 2). It has been well documented that the mutagenic and fibrotic effects of asbestos fibers and antibiotic bleomycin mainly depended on iron-initiated oxygen radical formation with subsequent pro-oxidant attack to DNA, proteins, and polysaccharides (Korkina et al., 1992). In present work we found that when given per os to the animals exposed to asbestos or bleomycin, metal-rutin complexes prevented lung inflammation and fibrosis (Table 3).

REFERENCES Afanas’ev, I.B., Dorozhko, A.I., Brodskii, A.V., Kostyuk, V.A., and Potapovitch, A.I., 1989, Chelating and free radical scavenging mechanisms of inhibitory action of rutin and quercetin in lipid peroxidation, Biochem. Pharmacol., 38:1763–1769. McCord, J.M. and Fridovich, I., 1968, The reduction of cytochrome c by milk xanthine oxidase, J. Biol. Chem., 243:5753–5757. Aust, S.D., 1987, Lipid peroxidation, in: Handbook of methods for oxygen radical research (R.A. Greenwald, ed.), pp. 203–207, CRC Press, Boca Raton. Halliwell, B. and Gutteridge, J.M.C., 1984, Oxygen toxicity, oxygen radicals, transition metals and disease, Biochem. J., 219:1–14. Korkina, L.G., Durnev, A.D., Suslova, T.B., Cheremisina, Z.P., Daugel-Dauge, N.O., and Afanas’ev, I.B., 1992, Oxygen radical-mediated mutagenic effect of asbestos on human lymphocytes: suppression by oxygen radical scavengers, Mut. Res., 265:245–253. Korkina, L.G. and Afanas’ev, I.B., 1997, Antioxidant and chelating properties of flavonoids, in: Advances in pharmacology, Volume 38 (H. Sies, ed.), pp. 151–163, Academic Press, Inc., New York.

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ONCOGENE ACTIVATION AND APOPTOSIS AS POSSIBLE MECHANISM OF ANTITUMOUR EFFECT OF FERRIC-SORBITOL-CITRATE

Marija Poljak-Blazi, Marijeta Kralj, and Marijana Popovic-Hadzija Rudjer Boskovi. Institute Bijenicka 54, 41001 Zagreb Croatia

1. INTRODUCTION Iron participates in several biological reactions and is essential to virtually all known forms of life. Iron-containing proteins of the respiratory chain are involved in electron transport to provide the energy for cellular functional activities. Iron is also required for DNA synthesis, cell growth and multiplication. Iron, and iron-containing compounds, could act as carcinogens, but they could also inhibit proliferation of normal or malignant cells. For example iron-containing, anti-anaemic drug, ferric-sorbitol-citrate complex (FSC- or Jectofer, Astra, Linz), inhibited proliferation of cultured mouse melanoma GHC, KB and HeLa cells and caused tumour regression in vivo, but did not affect the proliferation of the non-malignant fibroblast line L929, HBS and VERO cells (Poljak-Blazi et al., 1985, 1998). An increased number of cells in and early S phase suggested that iron excess blocked the cell cycle before the onset of DNA synthesis (Flajsig and Poljak-Blazi, 1990). For a better understanding of the mechanism of drug action, influence of FSC on DNA fragmentation and expression of c-Myc, Bcl-2, and p53 (wild and mutant) protein in CaCo2 cells was examined.

2. MATERIALS AND METHODS Ferric-sorbitol-citrate complex—FSC was used in the form of the anti-anaemic drug Jectofer (Astra, Linz, Austria). The final concentration of the drug was to Fe. Cell cultures were incubated under standard conditions for 72 hours. The cell growth (percentage of control) was estimated using MTT assay. c-Myc, p53 (wild, wp53 and mutated type, mp53), and Bcl-2 proteins were detected by modified immunocytochemical method described by Kranz and co-workers (1989). The number of positive Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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(brown) or negative (blue) cells was evaluated under the light microscope (x502). Each sample was done in triplicate and a three hundred cells per sample was counted. Apoptotic DNA fragments were isolated according to the method described by Herrmann et al. (1994), and separated by electrophoresis in 1.5% agarose gels. The results were analysed using the ANOVA test.

3. RESULTS AND DISCUSSION After treatment, for 72 hours, with FSC we observed impaired viability of four carcinoma (Ca) cells line: CaCo2 (colon Ca), MiaPaCa2 (pancreatic Ca), Hep2 (laryngeal Ca) and HeLa (cervix Ca) measured by MTT assay. The most pronounced growth inhibitory effect was observed on HeLa and CaCo2 cells. In spite of that, all concentrations of FSC stimulated the proliferation of non-malignant HEF522 (human embryonic fibroblasts) cells (Fig. 1). Thus, Jectofer killed cancer cells preferentially because the activities of cellular antioxidants that protect normal cells against oxidative damage may be decreased in cancer cells. The specific antitumour activity of Jectofer could be explain with possibility that cancer cells are less able than normal cells to inactivate the free radicals and are therefore less able to survive oxidative damage. In order to answer the question if the biological activity of FSC include oncogenes activation and apoptosis the degree of DNA fragmentation (apoptosis) which followed the morphological changes and growth inhibition of the FSC-treated CaCo2 cells were determined after 72 hours of culturing. In CaCo2 cells, FSC-induced DNA fragmentation resulted in numerous irregular oligonucleotide fragments and looked on electrophoresis like a smear line. During the apoptotic process, a concomitant regulation of several genes could occur. In our case

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c-Myc protein expressed 71% control cells and 62.8% of treated cells. Protein of wild p53 gene was detected in 36% control and in 34% treated cells, while mp53 gene was present in high incidence (57%) in control cells and was further increased to 70% in treated cells. Bcl-2 protein was present in 68% control CaCO2 cells, but after treatment with FSC the percent of Bcl-2 positive cells significantly decreased to 52% (Fig. 2). Thus, growth modification of CaCo2 cells caused by FSC involves interference with the diminished expression of Bcl-2, and an over expression of mp53 oncogene. Expression of c-Myc and wp53 protein was not significantly altered.

4. CONCLUSION The specific antitumour effect of FSC involves DNA damage; apoptosis and oncogene alteration. Apparently p53 could induce apoptosis by at least two distinct pathways. Namely, opposite to wp53 mutant of p53 is a potent inducer of apoptosis. In considering drug therapy, the question as to whether a given apoptotic stimulus might trigger more than one apoptotic pathways is of great importance. Selective inhibition of growth and viability seems possible that FSC-iron could act through the mechanism of the cellular growth regulation that differ between normal and malignant cells. It is well know that malignant cells are less able to inactivate the oxygen radicals, because the activities of cellular antioxidants that protect normal cells against oxidative damage may be decreased in cancer cells. Thus FSC-iron (iron from Jectofer) might work through the toxicity of reactive oxygen species (ROS) or through perturbation of cellular iron metabolism. It could be important in strategy in the treatment of cancer and thus warrants continued investigation.

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REFERENCES Flajsig, I. and Poljak-Blazi, M., 1990, Influence of iron on proliferation and cell cycle kinetics on cultured malignant and non-malignant cells. Oncology 47:443–446. Herrmann, M., Lorenz, H.M., Voll, R., et al., 1994, Rapid and simple method for the isolation of apoptotic DNA fragments. Nuc. Acid Res. 22:5506-5507. Kranz, B.R., Thiel, E., and Thierfelder, S., 1989, Immunocytochemical identification of meningeal leukaemia and lymphoma: Polly-L-lysine-coated slides permit multimarker analysis even with minute cerebrospinal fluid cell specimens. Blood 73:194–213. Poljak-Blazi, M., Stancic-Rokotov, D., and Ferle-Vidovic, A., 1985, Inhibitory effect of iron on melanoma B16 growth. Period. Biol. 87:17–21. Poljak-Blazi, M., ••arkovic, N., and Schaur, R.J., 1998, Impaired proliferation and DNA synthesis of a human tumour cell line (HeLa) caused by a short treatment with FSC and the lipid peroxidation product 4-hydroxinonenal. Cancer Biother. Radioph. 13:395–402.

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CHANGES IN OXIDANT AND ANTIOXIDANT STATUS IN RATS FED DIFFERENT AMOUNTS OF SELENIUM AND EXPOSED TO NO X J. Gromadzinska1, W. Wasowicz1, K. Rydzynski1, and J. Neve2 1

The Nofer Institute of Occupational Medicine 8 Teresy St. 90-950 Lodz, Poland 2 Free University of Brussels B-1050 Brussels, Belgium

Nitrogen oxides are oxidizing gases found in air pollutants originating from combustion of coal and liquid fuels. The mechanism by which affects respiratory epithelium and the whole body is unknown. It is postulated that induces oxidation of macromolecules in epithelial lining fluid via free radical reactions and/or release of free radicals during phagocytosis. In order to protect the organism from oxidative stress, it is necessary to maintain appropriate antioxidative potential. The aim of the study was to examine whether appropriate concentration of antioxidants in the diet may modify toxic effects of environmental oxidants on the lungs. Three groups of rats were fed a diet with different Se content: low-Se normal and high-Se Erythrocyte glutathione peroxidase (GSH-Px) activity was monitored weekly during the study. After 12 weeks of feeding, 25% of the animals from each of the groups were whole-body exposed in dynamic inhalation chambers to 0.5, 1.5 or 5.0ppm for 4 week (6 hours/day, 5 days/week). The control group consisted of animals which were exposed to atmospheric air in the above mentioned conditions and consumed appropriate amounts of Se. Plasma Se levels in rats fed rich Se diet did not differ in animals exposed to 0.5 ppm .., but were lowered in other exposed groups of animals as compared with controls p < 0.001, and for controls). In animals on normal Se diet, plasma Se concentration was lower in animals exposed to 5 ppm p < 0.001), only in the low Se rats, plasma Se concentration was on the same level in both the exposed and the control group Erythrocyte GSH-Px activity in low-Se rats was inversly proportional to the extent of exposure: 15.4 ± 3.7u/g

This work was supported in part by GRANT KBN 995/P05/97/13

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Hb (p < 0.0001) after exposure to 0.5ppm 24.5 ± 6.6 u/gHb (p < 0.005) after exposure to 1.5ppm and 26.6 ± 6.4 u/gHb (p < 0.005) after exposure to 5.0ppm vs. 38.9 ± 9.6 u/gHb in controls. Brochoalveolar lavage fluid (BAL) GSH-Px activity in rats fed high Se-diet was higher in all exposed groups as compared to controls. In low-Se and normal-Se rats exposed to 0.5 and 1.5ppm BAL GSH-Px activity was significantly higher than in the corresponding control groups. In rats fed low Se diet and exposed to 5.0 ppm GSH-Px BAL was by 35% lower (p < 0.001) as compared with the corresponding control group. In all exposed groups of animals, the concentration of blood plasma low molecular antioxidants, measured as total reactive antioxidative potential (TRAP), was increased proportionally to the intensity of exposure. Statistically significant increase of thiobarbituric acid reactive substances concentration in BAL was observed in animals exposed to 0.5ppm but not in other groups. Results of our study indicated that different Se levels in the diet may influence the antioxidant potential of animals exposed to

26

ZINC PROTECTS GENOMIC DNA FROM SOLAR LIGHT INJURY A Possible Role for Zinc-Induced Metallothionein?

E. Jourdan, Emonet-Piccardi N., Favier A., Beani J. C., and Richard M. J. LBSO, UJF Faculte de Pharmacie Domaine de La Merci 38700 La Tronche France

The essential role for Zn(II) in the apoptosis, survival, growth and metabolism of cells is partly explained by the requirement of metalloenzymes. It has been proposed that metallothionein (MT) may be an excellent candidate in the regulation of Zn metabolism. Recently some studies showed that Zn-MT can act as a zinc donors for Zn-apoenzymes, or a zinc chelator for Zn-metalloenzymes, suggesting that metallothionein are most than a zinc “storage protein”. Furthermore MT is a promising intracellular acute phase reactant protein in the pathogenesis of stress and shock. In this work we investigated the role of Zn-induced MT in the protection of genomic DNA from solar light. Cells were irradiated with a solar simulator Dermolum UM-W. The doses correspond to those used to induce an erythema. We induced MT in HaCat, Human keratinocytes cell line, by Zn treatment (zinc chlorure, for 72h). MT induction and expression were studied by northern blotting and western blotting. DNA strand-breaks and alkali-labile sites were quantified by the comet assay. The cellular localisation of MT was investigated by immunostaining. Solar irradiation induces serious DNA damage. Zn significantly decreased the yeild of DNA damage. This protection increased gradually as a function of Zn treatment. The maximal protection was obtained for 72h of incubation. We observed that maximal levels of MT were also reached for 72h. The immunostaining of MT in HaCat Keratinocytes after Zn treatment showed a new distribution for MT in cells: MT level increased in the cytoplasm whereas a nuclear pool of MT can be

This work has received the international prize SFERETE 1997

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visualised. Solar light at physiological doses also induced MT protein synthesis and a new distribution of MT. We can conclude that MT accumulate in the nucleus of Zn-treated HaCaT cells increasing intracellular antioxidant capacity and more specifically protecting DNA from the oxidative attack. We demonstrated that MT induction was correlated with a significant decrease of DNA damage induced by a single solar-simulated irradiation. Induction of MT in the cytoplasm and the nucleus constitutes another line of defence for the cells as MT have scavenger properties and could change the overall redox potential. Nevertheless a highest apometallothionein level in Zn deficiencies conditions could be stressful by changing Zn cellular homeostasis and Zn protein transfer as we previously described using Zn chelator.

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THE EFFECT OF CARBAMYLATION ON GLUTATHIONE PEROXIDASE AND COPPER-ZINC SUPEROXIDE DISMUTASE

Heather E. Roxborough, Cheryl Reid, Jane McEneny, Caroline Mercer, Dorothy McMaster, Maireard O’Hare, and Ian S. Young School of Clinical Medicine The Queen’s University of Belfast Belfast, Northern Ireland

Carbamylation occurs when groups and thiol groups on amino acids react with ureaderived cyanate. Protein carbamylation is increased in renal failure, and the activity of several proteins is known to be altered as a result of carbamylation. We have previously reported that the ferroxidase activity of caeruloplasmin is inhibited as a result of carbamylation (Roxborough et al., 1995) The aim of this study was to determine whether carbamylation can inhibit the actions of sereval enzymes whose activity is reduced in renal failure, including lipoprotein lipase (LPL) and the antioxidant enzymes glutathione peroxidase (GPx) and Cu-Zn Superoxide dismutase (Cu-ZnSOD). Enzymes were carbamylated in vitro by incubation with potassium cyanate in borate buffer for various periods of time (0, 5, 30, 60, 120, 180min) followed by dialysis to remove excess cyanate. Carbamylation had little effect on the activity of Cu-ZnSOD (0min, activity 22.1 ± 0.3U/ml; 5min, 21.9 ± 0.3; 30min, 21.4 ± 0.3; 60min, 21.7 ± 0.3; 120min, 21.6 ± 0.5; 180min 20.9 ± 0.3). GPx activity was markedly inhibited by carbamylation (0min, 100%; 5min, 103 ± 9%; 30min, 80 ± 4%; 60min, 73 ± 2%; 120 min, 36 ± 17%; 180min 34 ± 5%). There was a strong correlation between the degree of carbamylation and the loss of GPx activity. LPL activity was also substantially reduced by carbamylation (0min protein; 180min p<0.01). Carbamylation may contribute to the reduced activity of a number of enzymes (including GPx and LPL) observed in chronic renal failure. 131

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REFERENCES Roxborough H.E., Millar C.A., McEneny J., and Young I.S., 1995, Carbamylation inhibits the ferroxidase activity of caeruloplasmin, Biochem. Biophys. Res. Comm. 214:1073–1078. Address all correspondence to: Dr. Heather Roxborough; Cardiovascular Research, Rayne Institute, St Thomas’ Hospital, London, SE1 7SH; email: [email protected]

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ANTHRACYCLIN RESISTANCE OF GLC4 TUMORAL CELLS AND INTRACELLULAR SELENIUM METABOLISM

M. Andriollo, P. Guiraud, M. J. Richard, and A. Favier Laboratoire de Biologie du Stress Oxydant (LBSO) Faculté de Pharmacie Domaine de la Merci 38700 La Tronche France

The development of acquired resistance to antineoplastic agents like anthracyclins (adriamycin: ADR) represents a significant problem in the treatment of patients with cancer. Growing evidences show that anthracyclin resistance seems to result at least partly from the development of mechanisms preventing reactive oxygen species generation and an increased enzymatic capacity to detoxify free radicals in cells. A lot of studies have demonstrated that elevated levels of glutathione are associated with resistance phenomenon, but the mechanism is not yet known. We have investigated the effect of a selenium treatment on the antioxidant status in human small cell lung carcinoma cell line (GLC4). Different selenium concentrations were added to parental GLC4 cells (GLC4) and resistant GLC4 cells (GLC4/ADR). Levels of total glutathione (GSHt), reduced glutathione (GSH), intracellular selenium and activity of selenium-dependent glutathione peroxidase (Se-GPx) were determined in these two cell lines. An important difference in the selenium metabolism was observed between GLC4 and GLC4/ADR. Selenium level increased in a dose-dependent manner in the two cell lines, however selenium was more incorporated in the resistant cell line. Total glutathione level was higher in GLC4/ADR cells about 3 fold more than in control cells). Nevertheless, the addition of selenium had no significative effect on GSHt levels in the two cell lines. Basal Se-GPx activity was considerably higher in the parental cell line. Selenium treatment had no effect on the Se-GPx activity in the resistant cells, whereas activity raised by 3-fold in the GLC4 cells. Several reports have demonstrated that elevated levels of GSH, together with detoxifying enzymes using GSH like GPx might contribute to resistance in chemotherapy. In 133

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this work we have shown that GSH level was increased in resitant GLC4 cells, while SeGPx activity was considerably reduced and not responsive to selenium. Selenium metabolism seems abnormal in GLC4/ADR cells, further works are conducted to try to elucidate the mechanisms involved.

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ANTIPROLIFERATIVE ABILITY OF FERRICSORBITOL-CITRATE AND FERROCENES FOR MALIGNANT CELL LINE, HEP2 AND F10 M. Poljak-Blazi1, A. Ferle Vidovic1, V. Rapic2, and D. Škare1 1

Rudjer Boškovic Institute Bijenicka 54, 10001 Zagreb, Croatia 2 Faculty of Food Technology and Biotechnology University of Zagreb, Croatia

BACKGROUND The complex mechanism of iron influence on cell proliferation is not entirely understood. This transition metal plays a critical role in several essential oxidationreduction systems within the cell, e.g., catalase, cytochromes and ribonucleotide reductase. Being an efficient oxidant, iron is also of crucial importance not only in physiology of cell and tissue oxidation but in oxidative stress as well. Some iron compounds could have antitumour effect. The most likely mechanisms by which iron could affect carcinogenesis involve its functions in tumour cell growth, proliferation, respiration and oxidative metabolism. We have shown earlier that the non-toxic, ironcontaining, anti-anaemic drug ferric-sorbitol-citrate complex (FSC—Jectofer, Astra, Linz), inhibited proliferation of cultured mouse melanoma cells and caused tumour regression in vivo, but did not affect the proliferation of the nonmalignant fibroblast line L929. The same drug also inhibited the proliferation of malignant KB, HeLa and GHC cell lines in vitro, but did not appreciably alter proliferation of normal HBS and Vero cells. It was found that the ferrocenes have also antitumour effect on the Erlich ascites in mice.

AIM AND PROCEDURE In this paper antiproliferative activity of FSC and two ferrocenes against human malignant Hep2 (laryngeal carcinoma), and mouse melanoma F10 was evaluated. Ferrocenes F168 and F169 was synthetized in our laboratory. As control cells human (HEF) and mice (L929) fibroblasts were used. Every day, during 6 days of culturing 135

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proliferative ability of cells was determined by trypan blue exclusion assay. Viability of cells, after treatment for 72 hours, was measured by MTT assay.

RESULTS The tested ferric-sorbitol citrate and ferrocenes F168, F169 were very potent in inhibiting the growth of malignant cell lines (Hep2, F10), whereas they had no inhibitory effect on HEF or L929 fibroblasts. The generation time was longer for treated malignant cells. For example; of nontreated Hep2 cells was 13 hours but after treatment with Jectofer or ferrocenes this value was 15 h. Stronger effect was find out for F10 cells where the for control cells was 19 h but after treatment with iron compounds it was 26 h. The most pronounced growth inhibitory effect was observed on F10 cells. The most potent was ferrocene 169. The concentration of of used iron compounds, after incubation for 6 days, inhibited the proliferation of F10 cells by 80% (F168), 70% (F169) and 50% (Jectofer). The difference of the drug action was discovered after treatment for 72 hours. In that case treatment with ferrocenes to M) impaired the viability of malignant and the nonmalignant cells also. And there were about 50% of viable cells left. In spite of that Jectofer (in all concentrations) did not diminished the viability of non malignant cells under 80 percent.

CONCLUSION Control of tumour cell growth through perturbation of cellular iron metabolism is a potentially important strategy in the treatment of cancer and thus warrants continued investigation. The Ferric-sorbitol-citrate (Jectofer) and tested ferrocenes are very interesting in that fields because of their specific antitumour effect and they could be further tested and submitted as a new antitumour substances.

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MODULATION OF SP1 AND BINDING ACTIVITY BY ALUMINUM IN HELA CELLS

C. Garrel, M. Osman, and A. Favier Laboratoire de Biologie du Stress Oxydant (LBSO) UFR Pharmacie Medecine Grenoble, France

Aluminum is considered as a potentially toxic metal which has been linked to various neurological diseases such as amyotrophic lateral sclerosis, the Parkinsonism dementia complex of Guam and Alzheimer disease. Although aluminum is not an redox metal, different studies have show that aluminum’s cytotoxicity in neurological diseases would be link to an oxydative stress. However, the relationship between aluminum and free radicals still remains unclear. On the other hand, it is now well established that the DNA binding activity of two transcription factors: and SP1 is regulated by redox control mechanisms. The aim of our study was to investigate if aluminum is able to modulate the DNA binding activity of these two transcription factors. By electrophoretic mobility shifft assay we have shown that an 3 hours and 24 hours incubation of HeLa cells with various concentrations of aluminum sulfate led to an activation fo the DNA binding activity of in a dose dependant way, while DNA SP1 binding activity decrease. We have also shown, by the same method, that aluminum is able to decrease DNA SP1 binding activity in an cellular system. So, concerning SP1, the date support the hypothesis that the effect of aluminum on this transcription factor may be due to a direct interation of aluminum with the protein SP1. Further experiments are needed to determine if these effect of aluminum on and SP1 transcription factors result or not from an involment of this metal in an intracellular oxidative process.

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ANTIOXIDANT MICRONUTRIMENTS INSULIN-SENSITIVITY

P. Faure, F. Couzy, D. Barclay, E. Rossini, M. J. Richard, J. Arnaud, A. Favier, and S. Halimi *LBSO, Université Joseph Fourier 38700 La Tronche, France **Nestlé Research Centre Vers-chez-les-Blancs, 1000 Lausanne 26 Switzerland

We recently demonstrated insulin resistance and oxidative stress in high fructose fed-rats, and that vitamin E improves their insulin sensitivity. To clarify the role of oxidative stress on insulin action, we evaluated the effects of high fructose diets containing antioxidant micronutriments (Zn, Se, Cu, Vitamin E, carotene). Control groups received standard or high fructose diets (fructose: 55% of carbohydrates). Indicators of glucose metabolism (Insulin sensitivity by glucose clamp technique; plasma glycemia; triglycerides and fructosamine; liver glycogen) and antioxidant status (enzymes, micronutriments) were measured. The association of Zn (50mg/kg of diet), Se vitamin E (500mg/kg) and carotene (50mg/kg diet) led to a 30% improvement of insulin sensitivity, lower plasma triglyceride (p < 0.001) and liver glycogen levels (p < 0.001). Furthermore, the ratio of reduced glutathione to oxidized glutathione was higher when insulin sensitivity was improved. Erythrocyte Se-GPX and Cu-Zn-SOD activities were also increased in rats receiving antioxidant micronutriments. This study confirms the link between insulin activity and antioxidant protection, opening new therapeutic perspectives for management of diabetes.

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EXACERBATED IMMUNE STRESS RESPONSE IN EARLY MAGNESIUM DEFICIENCY IN THE RAT

A. Mazur, C. Malpuech-Brugère, W. Nowacki, E. Rock, and Y. Rayssiguier Centre de Recherches en Nutrition Humaine Unité Maladies Métaboliques et Micronutriments INRA, Theix, 63122 St Genès Champanelle France

Magnesium (Mg) plays an essential role in a wide range of fundamental cellular functions and many clinical signs and disease states are attributed to the altered Mg homeostasis. In a variety of animal models, Mg deficiency was shown to affect the immune system. In the rat Mg deficiency leads within a few days to the characteristic inflammatory syndrome. Since it appears that this deficiency affects the maintenance of host regulatory mechanisms in inflammation we have evaluated the immune stress response in Mg-deficient rats. As previously reported we have observed inflammatory symptoms associated to this deficiency, with the maximum after about one week on the deficient diet. These symptoms were concomitant with increased leukocyte count, plasma IL6, acute phase proteins and the presence of a large number of activated peritoneal macrophages. After one week on the Mg-deficient diet LPS challenge resulted in 70% mortality within 3 h induced, while there was no lethal effect of LPS in control rats. The vulnerability of Mg-deficient rats to LPS was associated with higher TNF alpha plasma values. Mg-deficient animals that received Mg supplementation before endotoxin challenge had significantly higher survival. At day 2 of Mg deficiency there was also a significant increase in plasma TNF after LPS challenge in deficient rats as compared to controls. Other results showing the increased sensitivity to the immune stress of Mgdeficient animals were also obtained by using PAF and live bacteria. Peritoneal cells (mainly macrophages) from deficient rats showed enhanced superoxide anion production and calcium mobilizing potency following in vitro stimulation, as well as after 2 and 8 days of deficiency. These studies indicate that an abnormal calcium handling induced by low extracellular Mg in vivo may be at the origin of exacerbated inflammatory response. These results also suggest that activated or primed state of immune cells is an early event occurring in Mg deficiency. Whether inflammatory response is also sensitive to Mg status in man merits further examination. 139

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ZINC PROTECTS HUMAN ENDOTHELIAL VASCULAR CELLS AGAINST THE GLUCOSE INDUCED CYTOTOXICITY 1

K. Lalanne1, S. Bouvard1, N. Wiernsperger2, P. Faure1, A. Favier , and S. Halimi1 LBSO-Grenoble, France LIPHA, Lyon, France

Morphological and functional abnormalities of vascular endothelium may represent a pathway to the accelerated atherosclerotic complications of diabetic patients. The mechanism underlying the effects of high glucose on endothelium are not clear even if it is almost certain that oxidative stress may play a significant role in these pathological events. It known that glucose may induce apoptosis of these cells and that antioxidant may protect them against the deleterious effect of glucose. To investigate the cell damage induced by glucose-dependent oxidative stress, we have employed an in vitro system of human endothelial veins cultured in elevated glucose concentrations. The effects of an essential micronutriment, zinc, was experimented in this model. Cytotoxicity of glucose was determined by the blue trypan technique, 7 or 14 days after glucose incubation (25 and 50mM) or Zinc (5, 25 and We found indeed that, the number of survival endothelial cells, incubated with glucose significantly decreased at J7 as well as J14 in a dose dependent manner. Moreover, endothelial cells preincubated with Zinc 25 and were protected from glucose induced cytoxicity. Complementary studies will be performed in order to measure the effect of zinc on endothelial cell redox state including reduced glutathione and antioxidant enzymes. Moreover the effect of metformin alone or associated with zinc will be experimented in this system as we recently demonstrated that this antidiabetic agent leads to an antioxidant effects (1) in vivo. Coculture technique will be used associating endothelial and vascular smooth cell in order to evaluate the effect of zinc on vascular smooth cells as their proliferation is linked to the endothelial cell functions.

REFERENCE P. Faure, E. Rossini, N. Wiernsperger, M.J. Richard, A. Favier, and S. Halimi. Diabetes, In press, 1998.

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EFFECTS OF ELEVATED DIETARY CU AND CD CONCENTRATIONS ON OXIDATIVE STRESS, CELL PROLIFERATION, AND APOPTOSIS IN ATLANTIC SALMON (SALMON SALAR L.) PARR Marc H. G. Berntssen1, Sjoerd E. Wendelaar Bonga2, and Amund Maage1 1

Institute of Nutrition Directorate of Fisheies P.O. Box 185, N-5002 Bergen Norway 2 Department of Animal Physiology Facutly of Science University of Nijmegen Toernooiveld 6525 ED Nijmegen The Netherlands

Elevated dietary Cu and Cd can cause peroxidation of lipids. Toxic consequences of membrane lipid oxidation may result in loss of membrane integrity and subsequent apoptosis and/or necrosis. Salmonid tissues, which are characterised by high concentrations of polyunsaturated fatty acids, are especially susceptible to pro-oxidants. Both Cu and Cd can occur in elevated concentrations in commercial fish feed. This can be due to either contamination (Cd) or supplementation of trace elements to the feed (Cu). The present study (one month exposure) was conducted to asses the effects of dietary Cu (control, 35 or 700mg/kg) and Cd (control, 0.5 or 250mg/kg) on tissue oxidative stress and intestinal cell turn over. Both Cu and Cd accumulated in intestine > kidney > liver of exposed fish. Dietary Cd had an inhibiting effect on selenium dependent glutathione peroxidase activity in the intestine. The intestine showed highest lipid peroxidation, measured as TBARS. Rats of intestinal and cell proliferation increased following exposure to dietary Cu and Cd, no necrosis was observed. Results from this study indicate oxidative stress in elevated dietary Cd and Cu exposed Atlantic salmon, resulting in regulated celldeath.

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MODULATION OF COPPER DEFICIENCY INDUCED OXIDATIVE STRESS BY DIETARY POLYPHENOL IN THE RAT

I. Bureau, C. Feillet-Coudray, E. Gueux, E. Rock, A. Mazur, and Y. Rayssiguier Centre de Recherche en Nutrition Humaine Unité Maladies Métaboliques et Micronutriments INRA, Theix 63122 St-Genès-Champanelle

France

The mechanism of injury during Cu deficiency may result from a reduction in antioxidant defenses. Since polyphenol compounds possess antioxidant properties, the present study was undertaken to determine whether rutin supplementation to Cudeficient rats protects the animals against the severity of Cu deficiency. Weanling male Wistar rats were fed a purified diet for 6 weeks, containing sucrose as the sole dietary carbohydrate and were assigned to one of 3 groups: Cu-adequate, Cu-deficient, and Cudeficient, rutin supplemented (1%). The Cu concentrations of the diets were 0.6mg/kg (deficient diets) and 6mg/kg (control diet). Cu-deficient rats had a lower body weight and higher relative liver and heart weight than control rats, but there was no mortality in any group. Cu-deficient rats had lower liver Cu concentrations and hematocrit values. Rutin supplementation of Cu-deficient rats does not protect against these consequences of Cu deficiency. Cu-deficient rats had decreased plasma antioxidant capacity (measured as trolox equivalent) compared to control rats. After exposure of tissue homogenate to iron induced lipid peroxidation, thiobarbituric acid reactive substances (TBARS) were significantly higher in heart from deficient rats compared to control rats. Rutin supplementation of Cu-deficient rats ameliorated the antioxidant status as indicated by a significant increase in plasma antioxidant capacity and a significant decrease in susceptibility of heart to in vitro peroxidation. In conclusion, Cu-deficient rats are more susceptible to peroxidative damage. Rutin administration ameliorates the antioxidant status of Cu-deficient rats but is unable to provide a complete protection against the pathological consequences of Cu deficiency. The results suggest either the effects of Cu deficiency are not limited to reduced antioxidant defenses, or the effects of decreased Cu, Zn, SOD activity cannot be completely counteracted by antioxidant supplementation. 142

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THIOREDOXIN/THIOREDOXIN REDUCTASE SYSTEM Their Roles in DNA Stability and Cell Adaptation to Ultraviolet Radiation

Didier C.*, Richard M.-J.*, Beani J. C., and Favier A.* *LBSO/LCR7 n°8 Université J. Fourier F-38043 Grenoble Cedex 03 France

Growing evidence has indicated that cellular redox status regulates various aspects of cellular function, modulates cell cycle, and takes part in carcinogenesis. Thioredoxin (Trx), in combination with the selenoprotein thioredoxin reductase (TR), is one of the component which has been shown to participate in the maintenance of this intracellular redox status. Previously we demonstrated that UVA irradiation is an oxidative stress which induces a modification of the redox status by glutathione consumption. Until today no information was available concerning Trx in UVA irradiated cells. In this work we investigated the consequences of UVA irradiation on Trx/TR system in cutaneous cells. We first reported that overexpression of human Trx (transfection experiments) as well as exogenous human recombinante (Trx addition into the culture medium) protected UVA irradiated cells from cell death. Using comet assay we oberved that this redox protein decreased DNA damage in irradiated cells. Interestingly we demonstrated that this intracellular signalling protein is induced by ultraviolet A radiation. Trx increased from 6h to 12h and returned to basal levels after 24h. In addition, TR activity was determined in human cutaneous fibroblastes and modulated by selenium supply. The link between the modification of the redox status define by Trx/TR system and DNA damage in UVA and UVB irradiated cells will be studied. In this work attempts were made to delineate the implication of selenium in cellular response to DNA damage, and in the regulation of redox status.

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TOXIC EFFECT OF CIPROFLOXACIN MAY BE THE RESULT OF A FREE RADICAL PATHWAY A. Gürbay1,2, B. Gonthier2, D. Daveloose3, F. Hincal1, and A. Favier2 1

Department of Toxicology Faculty of Pharmacy University of Hacettepe Ankara, 06100, Turkey 2 laboratory of Biology of Oxidative Stress (LBSO/LCR 7 No 817) Universite Joseph Fourier F-38043 Grenoble Cedex 09, France 3 Department of Biophysique Centre de Recherche du Service de Santé des Armées La Tranche, France

Fluoroquinolones (FQs), such as ciprofloxacin (CPFX), represent an important class of antimicrobial agents used in the treatment of a wide range of infectious diseases. However, these drugs are also associated with a low incidence of adverse effects related to gastrointestinal and central nervous system (CNS) function. In previous studies, we showed that CPFX induced oxidative stress in cerebral and hepatic tissues of rats in vivo, and on cultured human fibroblast cells in vitro. The protective effect of vitamin E on these systems was also shown. In this study, the aim was first, to investigate the possible cytotoxic effects of CPFX on primary culture of astrocytes, and then to determine the mechanism of its action on hepatic microsomal system; free radical pathway was evidenced by Electron Spin Resonance (ESR) spectroscopy using spin-trapping technique. The cultured cells were incubated with CPFX at various concentrations and cytotoxicity was determined by neutral red and MTT methods. A decrease in cell viability was showed with the higher concentrations of drug after 48, 72 and 96 hours of incubation. In the microsomal system, CPFX induced free radical production in a dose and time-dependent manner. Free radical production was completely inhibited by iron chelators such as desferroxamine and DTPA. Furthermore, treatment of microsomes with vitamin E provided significant protection. Actually, similar experiments are carried out using ESR spectroscopy in order to demonstrate the same radical formation in cerebral microsomes. In addition, mass spectroscopy experiments will allow us to identify the trapped radical. 144

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TRACE ELEMENT SPECIATION IN HUMAN BODY FLUIDS

Peter Brätter, Andrea Raab, and Andrea N. Richarz Hahn-Meitner-Institut Berlin Department Trace Elements in Health and Disease Glienicker Str. 100, D-14109 Berlin Germany

In order to maintain vital processes trace elements must be present in the organism within certain concentration ranges. They fulfill special biological functions e.g. as catalysts in the synthesis of proteins and enzymes, as active centres of proteins and as structure-forming parts of molecules. In body fluids many metal containing proteins are present that are involved in transport processes, acute phase reactions or in the protection of cells against damage by radicals. Basically, for clinical investigations almost any body fluid can be used to obtain information about metabolic and catabolic behaviour and the interaction of trace elements, with the choice of the fluid being dependent on the medical indication. Usually those body fluids are preferred that can be easily taken from the human body including urine breast milk and blood. Blood serum and plasma are the body fluids that are usually investigated with respect to trace elements. How much of a trace element enters into the blood circulation after absorption in the gastrointestinal tract depends on its chemical binding form in the foodstuff. It is transported via the serum to sites where metabolic transformation takes place (e.g. the liver) followed by the transport of the resulting metabolites to the sites of action or to the body pools for deposition (Fig. 1). Products of catabolism are also transported via the serum to be excreted or reutilized. The total concentration of a trace element in serum is the result at least all of these processes and it is nearly impossible to determine the contribution of the different compartments to the final concentration by serum analysis. Therefore the common clinical practise of using the trace element level in serum as a marker in the diagnosis of deficiency states must be regarded with a critical eye. Taking homeostatic regulation of the serum level into account even subclinical states cannot be detected by measuring the total element concentration. Deficiency states become evident when the body pools are depleted, at a point too late for preventive care. Address all correspondence to: Dr. Peter Brätter, email: [email protected] Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York. 2000.

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About 75% of the water in the body is located within the cells. Despite the problems caused by the invasive sampling procedure the cytosol of tissues may provide more information on the functions of trace elements than the blood compartments. According to their biological tasks the cells are differentiated with respect to their morphology and structure, the number and arrangement of the organelles and the production of specific chemical species which are dissolved in the cytoplasma. However by measuring only the total amount of the various trace elements in serum or cytosol no information can be obtained about the chemical form of the individual elements. To increase our knowledge of the biological role of trace elements and our understanding of their essentiality, toxicity and bioavailibility as well as the mechanisms of their absorption, transport and storage, identification and quantification of the trace element-binding chemical species present in the clinical sample is necessary. To perform speciation analysis the trace element-binding molecules must be separated and then the elements detected in the fraction. In recent years it has been shown that trace element speciation in biological fluids can be performed online by combining high performance liquid chromatography (HPLC) with mass spectrometry (MS) or atomic emission spectrometry (AES) using an inductively coupled plasma (ICP) or offline using neutron activation analysis (NAA) or atomic absorption spectrometry (AAS) for element detection. The main analytical challenge in the speciation of clinical samples is maintaining the in-vivo situation unchanged. This

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means that the integrity of the metal ligand interaction must be maintained both during the preanalytical steps and during the chromatographic separation itself. Structural alterations can occur when the analytical environment of the species differs from the physiological environment the species was drawn from. Because of the underlying separation mechanisms the reliability of reversed-phase, affinity or ion-exchange chromatography must be questioned. To minimise interactions between labile metal-ligand-protein complexes and the column material size exclusion chromatography by means of gel filtration seems to be an appropriate separation technique. In order to obtain reliable results various parameters of the SEC must be optimised. This includes the choice of the column material, the mobile phase composition, the buffer concentration, pH-value, sample volume and flow rate (Raab et al., 1998). The identification of the trace element binding species in body fluids is still an analytical problem: Commercially available marker substances of human origin representing in-vivo conditions are lacking. In the course of separation and purification of marker substances elements that are weakly bound under in-vivo conditions can go lost and/or be replaced by contaminants. Metalloproteins of human and animal origin can differ in their metal-binding characteristics and differences in the geometric shape of the molecules, as well, influence the retention behaviour during size exclusion chromatography. To demonstrate the benefits of investigating the chemical species of trace elements in body fluids the combination SEC/ICP-MS was used to study the binding pattern in the serum of patients who had undergone a septic reaction and in cytosol obtained from various tissues of the human body.

1. MATERIALS Pooled deep frozen autopsy specimens from human tissues (brain, kidney, liver, myocardium, skeletal muscle) were available for speciation analysis. A 3 g portion was minced and homogenised and aliquots of the supernatant were stored deep-frozen until analysis.

Experimental For the chromatographic separation of cytosol the HiLoad 16/60 column Superdex 75 pg (range: 10–300 kDa, Pharmacia LKB Biotechnology, Upsala, Sweden) was selected. Based on the results of optimisation of the separation with respect to resolution and recovery the following parameters were chosen: mobile phase TRIS, pH = 7.4 adjusted with (65%), flow rate sample volume The serum samples were chromatographed using two connected Asahipak GS-520 columns (0.75 × 25 cm, Asahi Chemical, Tokyo, Japan) with the parameters: eluens TRIS, pH = 7.4 with (65%), flow rate sample volume The starting TRIS-solution was passed through a Chelex 100 column (Bio-Rad Lab., Germany) in order remove any copper, iron and zinc contamination Multielementanalysis was carried out by means of the ICP-MS instrument ELAN 6000 (Perkin Elmer) equipped with a cross-flow nebulizer (Ar flow rate: Ge-72, Rh-103 and Ir-193 were used as internal standards. Details are given elsewhere (Brätter et al., 1998).

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2. RESULTS AND DISCUSSION Speciation of trace elements in cytosols. Figure 2 shows a comparison of the Cu and Cd elution profiles in the cytosol obtained from different tissues. The myocardium cytosol shows a distinctive pattern for Cd. Whereas in the upper molecular mass range no Cd was eluted the still unidentified peak at RT ~ 46 min was only found in the myocard. A coincidence of Cd and Cu can

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be seen at about 42min for all the tissues studied, whereas at a retention time of 35 min the coincidence seems to be specific only for brain tissue. We suggest the presence of isoforms of the metal-binding metallothioneins (MT) because it has been demonstrated that MT-I and MT-II are expressed in nearly all tissues, whereas MT-III is mainly localised in the brain (Uchida et al., 1991). The element pattern obtained from normal brain specimens (Fig. 3) shows the coelution of Cu, Cd and Zn at 35 min suggesting the brain specific isoform MT III which is identical with the so called growth inhibitory factor (GIF). Unfortunately MT III is not available as a marker substance for identification purposes. We therefore used a testing procedure which based on the heat-stability of the MT’s and their high binding affinity for Cd. Denaturation of the cytosols was carried out for 5 min at 95°C followed by a Cd-loading procedure (Bartsch et al., 1990; Eaton and Cherian, 1991). It was found that two Cd-, Cu- and Zn-containing proteins remained in solution after denaturation and that these proteins are able to bind the added cadmium. Thus it is quite probable that the peak at RT = 3 5 min in the chromatogram of Figs. 3 and 4 is MT-III and the second peak at RT ~ 42 min is created by MT-I and MT-II because these tw o isoforms cannot be resolved by SEC. The Cu/Zn-coincidence in the elution pattern at about 31 min was assumed to belong to the antioxidant superoxide dismutase (SOD). Measurement of the SODactivity in the collected fraction (Marklund et al., 1974) showed a very good coincidence between the enzyme and the Cu/Zn-peak (Fig. 4). As regards the manganese peak together with the SOD-activity at RT ~ 27 min in Fig. 4 it can be concluded that, besides the Cu/Zn-SOD, the Mn dependent SOD as well is expressed in brain cells. The speciation of the metalloproteins in brain tissue has gained in interest since the discovery of their relationship to human pathologies including Alzheimer’s disease.

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Speciation analysis may help to increase our knowledge of the physiological role and regulation of these proteins in the brain.

Speciation of Trace Elements in Serum Sepsis is a widespread complication after surgery with a high mortality rate. We studied the changes in the concentration of trace element-binding proteins in the serum of patients from periods of sepsis. In the table below clinical data for two patients are given. Since interleukin IL-6 plays an important role in inflammatory processes its determination in serum is obviously a good indicator for endoseptic events. On measuring the total concentration of essential trace elements it was found that the total concentration of zinc and iron decreases significantly below the reference range when IL-6 increases. Speciation analysis showed a significant correlationbetween the sepsis period and the appearance of a Zn-containing protein in the lower molecular mass range of the elution profiles (Fig. 5a,b). From animal experiments (Sato et al., 1996; Hernán-dez et al., 1998) and studies using cell lines (Schroeder et al., 1990; Snyers et al., 1994) it is known that cytokines such as IL-6 increases the expression of acute-phase proteins including the MT’s. Therefore, the testing procedure for metallothioneins mentioned above was applied. The results suggested that Zn-binding MT eluted at RT = 42 min. It presumably had been released into the serum from damaged cells or even due to apoptosis in the course of sepsis. Despite the appearance of new Zn-binding proteins in the serum its total Znconcentration has decreased. In the table and from the elution profiles of Zn (Fig. 5a,b) it can be seen that simultaneously with the main Zn-binding protein (RT = 37–39min) albumin decreases drastically. The significant decrease of Fe in the serum during sepsis is correlated with the decrease of its binding protein transferrin (Table) which acts, like albumin, as a negative acute-phase protein. Further study is necessary to understand the participation and role of trace elements in processes that are as complex as sepsis. It is to be hoped that trace element speciation in body fluids will prove to be a valuable analytical aid in understanding the mechanisms of the septic reaction and the development of a treatment against it.

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REFERENCES Bartsch, R., 1990, The Cd-Chelex assay: anew sensitive method to determine metallothionein containing zinc and cadmium, Arch of Toxicol. 64:177–180. Bratter, P., Navarro Blasco, I., Negretti de Brätter, Y, and Raab, A., 1998, Speciation as an analytical aid in trace element research in infant nutrition. Analyst 123:821–826. Eaton, D.L. and Cherian, M.G., 1991, Determination of metallothionein in tissues by cadmium-hemoglobin affinity assay, in Methods in Enzymology, Vol 205 (J.F. Riordan and B.L. Vallee eds) pp. 83–88. Academic Press Inc. Hernández, J. and Hidalgo, J., 1998, Endotoxin and intracerebroventricular injection of IL-1 and IL-6 induce rat brain metallothionein-I and -II, Neurochem. Int. 32:369–373. Marklund, S. and Marklund, G., 1974, Involvement of the superoxide anion radical in the autooxidation of pyrogallol and a convenient assay for superoxide dismutase, Eur. J. Biochem. 47:469–474. Raab, A. and Brätter, P., 1998, Separation of metalloprotein complexes in serum by size exclusion chromatography, Optimisation of the separation parameters retention behaviour and recovery employing radiotracers, Journal of Chromatography B 707:17–24. Sato, M., Yamaki, J., Hasmaya, M., and Hojo, H., 1996, Synergistic induction of metallothionein synthesis by interleukin-6, dexamethasone and zinc in the rat, Int. J. Immunopharmac. 18:167–172. Schroeder, J.J. and Cousins, R.J., 1990, Interleukin 6 regulates metallothionein gene expression and zinc metabolism in hepatocyte monolayer cultures, Proc. Natl. Acad. Sci. USA 87:3137–3141. Snyers, E. and Content, J., 1994, Induction of metallothionein and stotnatin by interleukin-6 and glucocorticoids in a human amniotic cell line, J. Biochem. 223:411–418. Uchida, Y, Takio, K., Titani, K., Ihara, Y., and Tomonaga, M., 1991, The growth inhibitory factor that is deficient in the Alzheimer’s disease brain is a 68 amino acid metallothionein-like protein, Neuron 7:337–347.

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GELFILTRATION AND ANION-EXCHANGE CHROMATOGRAPHY FOR THE SEPARATION OF VANADIUM BINDING PROTEINS IN PLASMA OF RATS, RABBITS, AND HUMANS

K. De Cremer and K. R. Cornelis Institute for Nuclear Sciences Laboratory for Analytical Chemistry University of Gent Proeftuinstraat 86, B-9000 Gent Belgium

1. INTRODUCTION Vanadium is a transition element that is known to be essential for mammals but so far not for humans (Nielsen and Uthus, 1990). In serum of healthy persons, the concentration is very low (0.031 ng/mL) and kept between narrow margins (Cornelis et al., 1981). Vanadium can inhibit enzymes but also activate enzymes (e.g. NADH diaphorase) in the body and induce hypertension (Carmignani et al., 1998). In contrast, vanadium has also some therapeutic effects: it has an insulinelike action (Sakurai and Tsuji, 1998) and it is also suggested to be a potential cancer chemopreventive agent (Chatterjee and Bishayee, 1998). In the human body vanadium is mostly present as vanadate(V) in blood and as vanadyl(IV) in tissues. In serum vanadium principally binds to transferrrin and in tissues to ferritin. Previous reports of other researchers (Sabbioni and Marafante, 1978; Chasteen et al., 1986; Pietra et al., 1991) and our experiments shows that there is an interaction of low molecular vanadium with certain stationary phases (gels) of gel filtration columns, so the recovery of vanadium from the column is not 100% and consequently there is a risk of missing peaks or creating ghost peaks. Therefore we compared the behaviour of a vanadate(V) tracer 15.98d) on different columns with a variety of stationary phases. From these results we selected the column with the most acceptable recovery and resolution to do our gel filtration experiments on serum of rats, rabbits and humans. In extension, we tested also an anion-exchange column with the same stationary phase as the selected gel filtration column. Only a few reports (Sabbioni et al., 1979) of anion-exchange chromatography with vanadium complexes are published. On this self-made anion-exchange column we Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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brought serum of rats, rabbits and humans in order to separate transferrin and albumin which is not possible on a gel filtration column. Previous results (De Cremer et al., 1999) shows that other chromatographic techniques (e.g. cation-exchange chromatography, hydrophobic interaction chromatography, . . .) are not feasible for vanadium.

2. RESULTS Comparison of Different Stationary Phases of a 48-vanadium(V) tracer was brought on different columns and then eluted with a few column volumes of 10mM Tris buffer + 0.15 M NaCl, pH = 7.5. Each experiment was repeated 3 times. Radioactivity was counted with a well-type NaI-detector. The results indicate that vanadium shows a different behaviour on the examined columns. A Sephadex G-25 column with a dextran matrix retains the vanadium(V) tracer quantitatively on the gel. In contrast, a Biogel P6 column (acrylamide), a Hitrap Sepharose butyl or phenyl column (agarose) and a Superose 12 column (agarose) do not retain any vanadium(V) tracer on the gel. Because of the greater resolution of Superose 12 in comparison with other columns, this column was chosen to do gel filtration experiments with serum of rats, rabbits and humans.

Gel Filtration Experiments of 1:5 diluted serum is brought on the Superose 12 column and eluted with approximately 2 column volumes of 10mM Hepes + 0.15 M NaCl, pH = 7.5. Each experiment was repeated 3 times. In the chromatogram of rabbit serum, two major vanadium

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peaks are seen. The first peak is located at an elution volume of 11–15ml, the second at 28–34 ml. The first peak corresponds with a molecular weight of 60,000–80,000 Da and is identified as transferrin, the second peak consists of low molecular vanadium species. This second peak is retained when using a Sephadex column as is seen in previous reports.

Anion-Exchange Experiments of pooled serum fractions from a gel filtration step is brought on a self made Sepharose DEAE Fast Flow column and eluted in a 10mM Hepes buffer, pH = 7.5 with a salt gradient ranging from 0–0.3 M NaCl over 10 column volumes. In the chromatogram of pooled rabbit serum, it can be seen that there is one vanadium peak with a maximum at the elution time of transferrin (transferrin UV-peak not visible). This was tested with a separate elution of a transferrin solution. So, vanadium in serum is bound to transferrin and not to albumin (major UV-peak at 170ml).

3. CONCLUSION In contrast with previous reports and columns we have a recovery of 100% vanadium from the Superose 12 column and a better resolution for proteins. So, we can already conclude that for gel filtration experiments with vanadium complexes this column is a good choice. For anion-exchange Chromatography, a Sepharose DEAE Fast Flow column gives also a recovery of 100% for vanadium, but the resolution for separating albumin and transferrin is not so good. Adapting column dimensions or gradient shape can

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therefore provide a solution. Out of the chromatographic results, we can conclude that vanadium in serum of rats, rabbits and humans principally is bound to transferrin.

REFERENCES Carmignani, M., Volpe, A.R., Sabbioni, E., Felaco, M., and Boscolo, P., 1998, Vanadium and the cardiovascular system; regulatory effects and toxicity, in: Vanadium in the Environment, Volume 2 (J.O. Nriagu, ed.), pp. 181–218, John Wiley and Sons, New York. Chasteen, N.D., Lord, E.M., Thompson, H.J., and Grady, J.K., 1986, Vanadium complexes of transferrin and ferritin in the rat, Biochim. Biophys. Acta, 884:84–92. Chatterjee, M. and Bishayee, A., 1998, Vanadium—A new tool for cancer prevention, in: Vanadium in the Environment, Volume 2 (J.O. Nriagu, ed.), pp. 347–390, John Wiley and sons, New York. Cornelis, R., Versieck, J., Mees, L., Hoste, J., and Barbier, F., 1981, The ultratrace element vanadium in human serum, Biol. Trace Elem. Res., 3:257–263. De Cremer, K., De Kimpe, J., and Cornelis, R., 1999, Stability of vanadium(V)-protein complexes during chromatography, Fresenius J. Anal. Chem., 363:519–522. Nielsen, F.H. and Uthus, E.O., 1990, The essentiality and metabolism of vanadium, in: Vanadium in Biological Systems, (N.D. Chasteen, ed.), pp. 51–62, Kluwer Academic Publishers, Dordrecht. Pietra, R., Alimonti, A., Gallorini, M., Tanet, G., Caroli, S., and Sabbioni, E., 1991, Recent developments of pre-separation procedures for trace elements analysis of biological specimens, Acta Chim. Hung., 128:725–734. Sabbioni, E. and Marafante, E., 1978, Metabolic patterns of vanadium in the rat, Bioinorg. Chem., 9:389–407. Sabbioni, E., Marafante, E., Pietra, R., Goetz, L., Girardi, F., and Orvini, E., 1979, The association of vanadium with the iron transport system in human blood as determined by gel filtration and neutron activation analysis, in: Nuclear Activation Techniques in the Life Sciences 1978, proceedings of a symposium, (IEAE, ed.), pp. 179–192., IEAE, Vienna. Sakurai, H. and Tsuji, A., 1998, Antidiabetic action of vanadium complexes in animals: blood glucose normalizing effect, organ distribution of vanadium, and mechanism for insulin-mimetic action, in: Vanadium in the Environment, Volume 2, (J.O. Nriagu, ed.), pp. 297–315, John Wiley and Sons, New York.

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METABOLISM OF SELENATE ADMINISTERED INTO RATS Speciation of Selenium by HPLC-ICP MS

Kazuo T. Suzuki and Yamato Shiobara Faculty of Pharmaceutical Sciences Chiba University Inage, Chiba 263-8522, Japan

1. SUMMARY

The metabolic fate of selenium (Se) administered intravenously in the form of selenate (natural abundance) to rats (0.3 mgSe/kg body weight) was studied by HPLC-mass spectrometry with ionization by inductively coupled argon plasma (ICP MS) in comparison with that of selenite. Selenate was either excreted directly into the urine within 6 hrs (14% of the dose) or was taken up by the liver without being metabolized in the bloodstream in both cases. The Se of selenate origin taken up by the liver was utilized for the synthesis of selenoprotein P (Sel P) and an increase in the Sel P peak started to be observed within 1 hr after the injection. Selenate taken up by the liver was also methylated for excretion into the urine. However, the production of methylated products was not significant in the urine before 6 hrs after the injection in the selenate group. Although reduction of selenite to selenide was readily observed in vitro in the presence of glutathione (GSH) or on incubation in a liver supernatant fraction, selenate was not reduced in vitro in the presence of GSH or dithiothreitol (DTT). However, selenate was reduced slowly on incubation in a liver homogenate or supernatant fraction, suggesting that the reduction of selenate to selenite takes place during the uptake or immediately after the uptake. The chemical forms of Se in the blood plasma, liver supernatant and urine were determined by the HPLC-ICP MS method, which demonstrated the importance of speciation of each Se metabolite.

E-mail: [email protected] Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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2. INTRODUCTION Both inorganic and organic forms of Se can be utilized as sources of the Se nutrient, and selenite and selenate are the inorganic forms. Selenite was shown to be taken up rapidly and selectively by red blood cells (RBCs) (Suzuki and Itoh, 1997a; Suzuki et al., 1998), and reduced to selenide in RBCs, and then the selenide is exported to the plasma and bound selectively to albumin (Shiobara and Suzuki, 1998). The Se bound to albumin is transferred to the liver and taken up by the liver, and then the Se is utilized for the synthesis of selenoproteins, excreted into the urine or exhaled after being methylated stepwise to monomethylselenol (MMSe), dimethylselene (DMSe), and trimethylselenonium (TMSe) ions (Suzuki and Itoh, 1997b). In our preliminary experiments, the other inorganic form of the Se nutrient, selenate, was suggested to be metabolized through a distinct pathway from that for selenite when injected intravenously into rats. The present study was conducted to elucidate the metabolic pathway for Se administered into bloodstream in the form of selenate by speciating the Se in the bloodstream, liver and urine by the HPLC-ICP MS method (Suzuki, 1996, 1998; Suzuki et al., 1995).

3. MATERIALS AND METHODS Sodium selenate (Se of natural abundance) was injected intravenously into male Wistar rats of 8 weeks of age at the dose of 0.3mgSe/kg body weight, and then blood and urine specimens were obtained. A 20% suspension of RBCs in saline was used for the uptake experiment on selenate and selenite in vitro. The distributions of Se in plasma, liver supernatants and urine were determined on a gel filtration HPLC column of GS520 or GS320 by elution with 10mM Tris-HCl buffer, pH 7.4, at the flow rate of 1.0ml/min (Suzuki, 1996, 1998).

4. RESULTS AND DISCUSSION Although selenite was taken up efficiently by RBCs (Suzuki et al., 1998) within 10min, selenate was not taken up by RBCs in vitro.

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Selenate injected into rats disappeared from the bloodstream much faster than selenite. Selenate was partly excreted directly into the urine without being metabolized in the bloodstream, which was confirmed by speciating Se in the urine by the HPLC-ICP MS method. On the other hand, the major portion of selenate was taken up by the liver, also without being metabolized in the bloodstream. Namely, the Se in the blood plasma was present in the form of selenate until it disappeared from the bloodstream, which is different from the rapid metabolic change of selenite to selenide bound to albumin involving RBCs and albumin in the plasma. Despite the disappearance of Se in the form of selenate, the distribution of Se in the liver supernatant indicated that the Se was similar to that observed after the injection of selenite and was not in the form of selenate. Although selenate was not reduced by GSH or DTT in vitro, it was reduced on incubation with a liver homogenate and supernatant fraction, although the reduction was very slow. These observations suggest that selenate was reduced during the uptake by the liver or in the liver immediately after the uptake. Following the uptake and reduction of the selenate by the liver, the Se was utilized for the synthesis of selenoproteins, the major one being Sel P, and the excessive Se was excreted after being methylated. The same two Se metabolites as those observed in the case of selenite were detected in the liver. One was assumed to be metabolized to the other, the latter being identical to the urinary metabolite that was previously identified as MMSe (but this has to be revised). Our observations can be summarized as shown in the figure.

REFERENCES Shiobara, Y. and Suzuki, K.T., 1998, Binding of selenium (administered as selenite) to albumin after efflux from red blood cells. J. Chromatogr. B, 710, 49–56. Suzuki, K.T., 1996, Simultaneous speciation of endogenous and exogenous elements by HPLC/ICP-MS with enriched stable isotopes. Tohoku J. Exp. Med., 178, 27–35. Suzuki, K.T., 1998, Hyphenated techniques as a tool to speciate biological metals: metallothionein and metalbinding proteins. Analusis, 26, M57–M61. Suzuki, K.T. and Itoh, M., 1997a, Metabolism of selenite labeled with enriched stable isotope in bloodstream. J. Chromatogr. B, 692, 15–22. Suzuki, K.T. and Itoh, M., 1997b, Effects of dose on the methylation of selenium to monomethylselenol and trimethylselenonium ion in rats. Arch. Toxicol., 71, 461–466. Suzuki, K.T., Itoh, M., and Ohmichi, M., 1995, Detection of selenium-containing biological constituents by high-performance liquid chromatography—plasma source mass spectrometry. J. Chromatogr. B, 666, 13–19. Suzuki, K.T., Shiobara, Y., Itoh, M., and Ohmichi, M., 1998, Selective uptake of selenite by red blood cells. Analyst, 123, 63–67.

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FRACTIONATION OF SOLUBLE SELENIUM COMPOUNDS FROM FISH USING SIZEEXCLUSION CHROMATOGRAPHY WITH ONLINE DETECTION BY INDUCTIVELY COUPLED PLASMA MASS SPECTROMETRY Comparison with other Techniques Gunilla Önning1 and Ingvar A. Bergdahl2 1

Biomedical Nutrition Center for Chemistry and Chemical Engineering 2 Department of Occupational and Environmental Medicine Lund University, Lund Sweden

Selenium is an essential element and more than eleven selenoproteins have been identified in mammals (Johnsson et al., 1997). People living in countries with low amounts of selenium in soil may run a risk of getting a too low amount of selenium from the food. It is thus necessary to evaluate the role of different foods for the supply of selenium. Selenium is present in foods mainly as selenomethionine and selenocysteine located in proteins. Fish accumulate significant amounts of selenium and are an important dietary source of this element. Some studies have indicated a low bioavailability of the selenium from fish (Huang et al., 1995; Thorngren and Åkesson, 1987; Meltzer et al., 1993), while in another study (Hagmar et al., 1998) selenium from fish had a marked effect on different markers of selenium status. Since little is known of selenium forms in fish, we have studied soluble selenium compounds in fish species and compared different techniques for detection (ICP-MS, GFAAS) and fractionation (size-exclusion chromatography, ultrafiltration, TCA-precipitation) of the selenocompounds. GFAAS-Zeeman is a commonly available method for the detection of selenium in collected size-exclusion chromatography fractions. However, the detection limit is

Address all correspondence to: Dr Gunilla Önning, Biomedical Nutrition, Chemical Center, PO Box 124, SE221 00 Lund, Sweden; telephone: +46 46 222 82 22; fax: +46 46 222 45 32; email: [email protected] Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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approximately which may not be low enough for some samples. An improvement in the detection of selenium could be achieved by ICP-MS that allows on-line detection of separated selenium compounds at concentrations that occurs in biological samples. Previously ion-exchange chromatography columns have been connected to ICP-MS and the separation and detection of selenate, selenite, Se-methionine and Se-cystine were accomplished (Crews et al., 1996; Pedersen and Larsen, 1997; Bird et al., 1997; Emteborg et al., 1998). The separation (Superdex 75 column, fractionation range 3–70 kDa) and detection of selenium in plaice extract with GFAAS-Zeeman or ICP-MS is shown in Fig. 1. The chromatography was made with a flow rate of 0.5ml/min and 0.5ml extract was injected. For ICP-MS the is presented. One peak was detected with a retention time around 20min, two larger peaks at retention times 34 and 37min, respectively, and finally one peak at a retention time around 48min. The profile correlated well with results obtained using GFAAS-Zeeman analysis. The ICP-MS technique was further evaluated using cod and plaice extracts and a Superdex 200 column (fractionation range 10–600 kDa). The flow-rate was 0.75ml/min and 0.5ml extract was injected onto the column. The limit of detection (calculated as three times the standard deviation of the analytical blank, m/z 82) was The selenium response was linear in the investigated concentration range of (r2 = 0.98). The soluble components from cod and plaice were injected four times each and the variation in the quantitative data for different selenium-containing peaks between runs was small (CV < 10%). Some fish species contain a high amount of low-molecular-weight compounds that are uncharacterised so far (Åkesson and Srikumar, 1994). By using size-exclusion chromatography a size distribution pattern of selenocompounds can be obtained. On the

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other hand, if the interest is mainly to separate the low-molecular-weight compounds from the high-molecular-weight compounds, other techniques, such as ultrafiltration or TCA-precipitation may also be used. We have made a comparison of these three techniques (Fig. 2). The amount of soluble selenium with a molecular weight below 10 kDa, determined with size-exclusion chromatography-ICP-MS, was 27% for cod and much higher, 80%, for plaice. The corresponding figures for ultrafiltration using a membrane with a cut-off at 10 kDa were 20% for cod and 69% for plaice. Using TCA-precipitation gave a similar value for plaice while the obtained value for cod was higher (38%) compared with the other techniques. In summary, the ICP-MS method seems to be able to reproducibly analyse selenocompounds of fish extracts separated on-line with size-exclusion chromatography. Ultrafiltration gave similar values as size-exclusion chromatography, regarding the amount of soluble low-molecular-weight selenocompounds, and is a quick method to isolate these compounds. This study was supported by a grant from the FAIR programme (project CT95-0077).

REFERENCES Åkesson, B. and Srikumar T.S., 1994, Occurrence of low-molecular-weight and high-molecular-weight selenium compounds in fish. Food Chem. 51:45–49. Bird, S.M., Ge, H., Uden, P.C., Tyson, J.F., Block, E., and Denoyer, E., 1997, High-performance liquid chromatography of selenoamino acids and organo selenium compounds. Speciation by inductively coupled plasma mass spectrometry, J. Chrom. A 798:349–359. Crews, H.M., Clarke, P.A., Lewis D.J., Owen, L.M., Strutt, P.R., and Izquierdo, A., 1996, Investigation of selenium speciation in vitro gastrointestinal extracts of cooked cod by high-performance liquid chromatography-inductively coupled plasma mass spectrometry and electrospray mass spectrometry, J. Anal, Atom. Spec. 11:1177–1182. Emteborg, H., Bordin, G., and Rodriguez, A.R., 1998, Speciation of organic and inorganic selenium in a biological certified reference material based on microbore ion-exchange chromatography coupled to inductively coupled plasma atomic emission spectrometry via a direct injection nebulizer or coupled to electrothermal atomic absorption spectrometry, Analyst 123:245–253.

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Hagmar, L., Persson-Moschos, M., Åkesson, B., and Schütz, A., 1998, Plasma levels of selenium, selenoprotein P and glutathione peroxidase and their correlations to fish intake and serum levels of thyrotropin and thyroid hormones: A study on Latvian fish consumers. Eur. J. Clin. Nutr. 52:796–800. Huang, W., Åkesson, B., Svensson, B.G., Schütz, A., Burk, R.F., and Skerfving, S., 1995, Selenoprotein P and glutathione peroxidase (EC 1.11.1.9) in plasma as indices of selenium status in relation to the intake of fish. Br. J.Nutr. 73:455–461. Pedersen, G.A. and Larsen, E.H., 1997, Speciation of four selenium compounds using high performance liquid chromatography with on-line detection by inductively coupled plasma mass spectrometry or flame atomic absorption spectrometry. Fresenius J. Anal. Chem. 358:591–598.

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METABOLISM OF ARSENIC FROM SEAWEED BY MAN AND ANIMALS Speciation in Body Fluids using Liquid Chromatography

Inductively Coupled Plasma Mass Spectrometry

Jörg Feldmann University of Aberdeen Department of Chemistry Meston Walk, Old Aberdeen AB24 3UE, Scotland, UK

1. INTRODUCTION Arsenic and its compounds are widespread in the environment. Recently, there have been real concerns about the arsenic concentration of the inorganic species (arsenite and arsenate) in drinking water. Ingested inorganic arsenic has been associated with increased risk of cancer (e.g., skin cancer) and has been classified by the US EPA as a human carcinogen (cat A) (Chappell et al., 1994). In addition to the inorganic forms of arsenic a wide range of organo-arsenicals have been identified in particular in the marine environment. Especially fish, shellfish and seaweed contains arsenic compounds in relatively high concentrations (up to due to bioaccumulation and biotransformation processes. In order to monitor the daily oral exposure to arsenic it is necessary to distinguish between the different arsenic species due to their different human toxicity. The median lethal dose values in rats for some arsenic compounds found in the environment varied from 14mg arsenite per kg body weight to over 10,600 mg for trimethylarsine oxide (Kaise and Fukui, 1992). Most inorganic as well as the organic arsenic compounds are readily absorbed by the GI tract. In most mammals the inorganic arsenic species (As(III/V)) are going to be methylated to dimethylarsinic acid (DMAA). In particular, humans also excrete

[email protected] Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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substantial amounts of monomethylarsonic acid (MMAA). Arsenobetaine (AsB), the major arsenic species in fish is readily absorbed but not metabolized. Thus, AsB is excreted rapidly into the urine. In order to investigate the exposure of a population to most potent toxic species such as As(III) and As(V), speciation analysis is inevitable. Liquid chromatography coupled to an ICP-MS as an arsenic-specific detector is demonstrated to be a powerful tool to separate the different arsenic species in urine (Le and Ma, 1998). For long term exposure the arsenic content in hair can be used as an indicator, because arsenic is believed to be accumulated in keratin containing materials such as hair and fingernails. The arsenic level in urine is commonly used as a biomarker for a short term exposure. It has been discussed that the concentration of DMAA and MMAA in urine could be used as a biomarker for the short term exposure to inorganic arsenic, whereas the non-toxic AsB from the fish consumption does not increase the level of DMAA and MMAA. Very limited information is available about the metabolism of arsenosugars. The aim of this study is to determine how the arsenosugars are metabolised by man and sheep.

2. MATERIALS AND METHODS The arsenic content of the blood and the wool was determined by ICP-MS (Spectromass 2000 Spectro, Kleve, Germany) after microwave digestion with nitric acid and hydrogen peroxide. The seaweed was freeze dried and subsequently extracted with methanol/water 1:1 (Lai et al., 1997). For speciation analysis three different kind of liquid chromatography (anion exchange (30mM pH6, Hamilton PRP x-100, l.0mL/min), cation exchange 30mM pyridine, pH 3, 35°C, Supelcosil SCX and IP-RP (l0mM TEAM, 4.5mM malonic acid, 0.1% methanol, ODS-2) were employed. The dwell time on each mass (m/z 75, 77) was 200ms. For the validation of the analytical procedure the NIST standard urine SRM2670 with elevated metal level (total 480 +/– was measured by anion exchange and cation exchange (472 and The concentrations of the individual species showed a reasonable good agreement for anion and cation exchange chromatography: AsB: 34; DMAA: 68; MMAA 15; As(V):359; respectively. Metabolism of arsenosugars in humans: 250 g mussel soup was ingested by a 35 year old male volunteer. The urine was sampled and stored at 4 °C until analysis. The mussel soup was extracted and arsenic speciation was determined by ion pairing reverse phase liquid chromatography (IP-RP-HPLC) coupled to ICP-MS. The mussel contained arsenic as AsB and two different dimethyarsinylribosides (sugar X and XI). Metabolism of arsenosugars in sheep: The sheep on the most northern island of the Orkney-Island in Scotland is the only existing flock of the breed North Ronaldsay Sheep (NRS). The entire population of about 2,500 sheep live 3/4 of the year only from seaweed, which they found in the tidal zone or washed on the rocks. The majority of the seaweed are Phaeophyceae in particular Laminara digitata. This seaweed contains about 89mg arsenic per kg dry weight. 85% of the arsenic was found to be the arsenosugars XII, XI and small amounts of X. In January 1999 the sheep were transported from the island in order to be slaughtered. The urine, the blood and the wool of 20 sheep were sampled three days after the deportation. The stomach still contained seaweed. As a control group 5 subjects of Finn Dorset sheep were used. They were kept on normal grass diet, however, the addition of fish containing food additives could not be ruled out.

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3. RESULTS AND DISCUSSIONS The high concentrations of total arsenic in the wool of about hair (n = 20) indicate that the sheep had been exposed to arsenic for a longer time period (Table 1). The concentration were at least two orders of magnitude higher than that of the control group. Although, the blood concentration in the North Ronaldsay sheep (NRS) showed an elevated level, it does not seem to be extremely high. In contrast, the total amounts in the urine sample of NR sheep were extremely high up to a concentration of However, the 20 samples showed a large variability in the arsenic content, which could be minimized by the determination of the creatinine content. But even the average of the arsenic concentration in the urine is found to be at least one to two orders of magnitude higher for the NR sheep than that of the control group, which also have considerably high arsenic levels. The speciation analysis (Fig. 1) revealed that more than 90% of the arsenic in the urine was DMAA. Additionally, considerable amounts of MMAA and tetramethyl-arsonium cation and small amounts of unidentified species were detected in the urine (Table 2). However, no arsenosugars, which have been found to be in the seaweed was detected.

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The mussel experiment: after ingestion AsB was excreted rapidly into the urine. 13.5 hours after ingestion the urine contained besides AsB large quantities of DMAA and four unidentified arsenic species. The retention times of those compounds do not fit to those of the standards available (sugar X, XI, XII, XIII). It can be concluded that the arsenosugars X and XI undergo transformation reactions to DMAA and other organoarsenicals, which could not yet be identified. This transformation reaction is thought to be an enzymatic reaction because the adjustment of the pH on gastric conditions will produce arsenite and unidentified oranoarsenicals but not DMAA.

CONCLUSIONS The arsenosugars in the seaweed were absorbed by humans as well as by the NR sheep. The sheep especially, show tremendously high concentrations of arsenic in the wool and in the urine. Furthermore, the arsenosugars undergo transformation reactions, the metabolites of the arsenosugars XII, XI, and X are the excreted DMAA, MMAA and This raises the question; can the concentration of DMAA be used as a bioindicator for the exposure of humans and sheep to inorganic arsenic if the consumption of arsenosugar containing food increases also the level of DMAA in the urine?

ACKNOWLEDGMENT The author would like to thank Katie John for the preparation of the solutions, Dr. Kevin Woodbridge and Mr. William Stewart for the samples and a special thanks to the Grazing Committee of North Ronaldsay.

REFERENCES Chappell, W.R., Abernathy, C.O., and Cothern, C.R. (eds.), 1994, Arsenic Exposure and health, Science and Technology Letters, Northwood. Kaise, T. and Fukui, S., 1992, The chemical form and acute toxicity of arsenic compounds in marine organisms, Appl. Organomet. Chem. 6:155. Le, X.C. and Ma, M., 1998, Short-column liquid chromatography with hydride generation atomic fluorescence detection for the speciation of arsenic, Anal. Chem. 70:1926. Le, X.C., Li, X.F., Lai, V., Ma, M., Yalcin, S., and Feldmann, I, 1998, Simultaneous speciation of selenium and arsenic using elevated temperature liquid chromatography separation with inductively coupled plasma mass spectrometry detection, Spectrochimica Acta, B 53:899.

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SPECIATION OF SELENIUM IN FOOD AND ANIMAL FEED USING ION CHROMATOGRAPHY ON-LINE MICROWAVE PRETREATMENT HYDRIDE GENERATION ATOMIC ABSORPTION SPECTROMETRY

Magnus Johansson, Guy Bordin, and Adela R. Rodriguez European Commission-Joint Research Centre Institute for Reference Materials and Measurements Retieseweg, B-2440 Geel Belgium

1. INTRODUCTION Selenium is an essential element to most living organisms, it can however also be toxic. The speciation of selenium together with the concentration of the element determines the effect on living organisms. The main source of selenium for mammals is the food. (“Environmental Health Criteria, Selenium,” 1987.) These are reasons for why it is of great importance to develop reliable methods for selenium speciation in foodstuff. The forms of selenium considered in this work are selenite, selenate, selenomethionine and selenocystine. Certainly there are other selenium containing species in organic materials, however the knowledge about selenium speciation is limited. An example of this is the recently identified Se-adenosylhomocysteine in a yeast extract (Casiot et al., 1999). Reliable analytical methods are prerequisites for relevant assessments of selenium species beneficial or toxic effects. Sample pretreatment is crucial in speciation analysis as criteria such as species preservation, representative and high extraction efficiency must be fulfilled. In this report some results will be presented illustrating the problems with sample pretreatment in selenium speciation analysis. The need for certified reference materials (CRMs) in speciation analysis is in this way highlighted.

2. EXPERIMENTAL The carbonate eluent for ion-chromatography was prepared by dissolving ammonium carbonate in water, 15mM, and adding 2% (v/v). Ammonia (supra pure, Merck) Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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was used to adjust the pH to 10. Hydrobromic acid in water 48% (w/w), purity 99.999% with respect to metals (Aldrich, Milwaukee, WI, USA) was used as received. An appropriate amount of potassium bromate (A.C.S. reagent, Aldrich) was dissolved in water to obtain the desired concentration. Solutions of sodium borohydride, 0.5% w/w, were prepared daily in 0.1% w/w of sodium hydroxide used for stabilization. The IC-MWHGAAS system is depicted in Fig. 1. An inert GP-40 gradient pump (Dionex, Sunnyvale, CA, USA) was used to deliver the eluent in the flow-controlled mode to the column PAX100, 50 ×4mm (Dionex). A peristaltic pump (Ismatec, Zurich, Switzerland) was used to deliver the reagents, HBr and used for on-line reduction and oxidation of the separated selenium analyte species. The effluent from the column was merged with the reagents using a mixing manifold. The solution was then passed through a PTFE tubing (0.8mm i.d.) coiled and immersed into the cavity of a focused microwave oven. The gas evolution during microwave heating causes impaired precision due to an irregularly changing flow rate into the hydride generation unit. To prevent this a cooling device and an additional peristaltic pump FIAS 400 (Perkin-Elmer) were incorporated to stabilize the flow rate. The sodium borohydride solution was mixed with the analyte solution using a manifold. The quartz cell for MHS-10 (Perkin-Elmer) was electrically heated to 900°C and installed in a Perkin-Elmer 5,000 atomic absorption spectrometer. Extractions of selenium species from CRM 402, a white clover material was undertaken applying an extraction method described by Emteborg et al. (1998). Approximately 0.5g of CRM 402 was placed in a 100ml conical glass flask, thereafter spikes were added where applicable, 10ml of (1 + 1) with 4% ammonia was added and the flasks set in the ultrasonic bath for 30min. The sample suspension was thereafter centrifuged at 3,500rpm. This extraction procedure was repeated twice. The combined supernatants were filtered using cellulose acetate syringe filters. Further clean-up was achieved by passing the filtered samples twice through a Cl8 cartridge. Sub-samples were taken from the filtered and the fully processed sample solutions for subsequent determination of total selenium concentration by means of ETAAS.

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3. RESULTS AND DISCUSSION All selenium species must be converted into selenite prior to HG as only selenite readily reacts with to form the volatile The initial studies were devoted to studies concerning the conversion rate of the selenium species to selenite and interference’s caused by the pretreatment reagents Seleno-methionine and selenate are the most resistant species to conversion. Selenocystine is relatively easily converted into selenite using these reagents. The concentration of the reagent mixture must be carefully optimised to avoid the formation of an interferent, most probably bromine, causing depression of the analyte response. After optimisation of the system using multivariate techniques all four selenium species were separated and detected within ~5min, detection limits were between 1.6 and based on a injection volume. Similar sensitivity was achieved for all Se species. In Table 1 extraction efficiencies of total selenium from the CRM 402 material after the filtration of the extract and after completed clean up are shown. The extraction efficiency is generally higher for the spiked samples indicating that the intrinsic analytes are more difficult to extract from the sample matrix than the spiked analytes. After the acid extraction procedure approximately 80% of the total selenium content in the non-fortified samples were liberated. However the spiked samples show higher total extraction efficiencies. Comparison between the extraction efficiencies of selenium obtained by determination of total selenium (Table 1) and by speciation, followed by summation of the different species (Table 2), show good agreement for the non-fortified sample. The spiked sample show somewhat lower values using the speciation method. A likely explanation for this difference is the depression of the selenomethionine and selenite response in the speciation mode due to a co-eluting interferent. Furthermore, the retention times are changed for the extracts compared to the standards, this is certainly caused by the matrix present in the extracts. The interferent co-eluting with selenomethionine does most probably origin from ammonia used in the extraction. The changes in retention times or rather the lower resolution causes the peaks of selenomethionine and selenite to partly overlap.

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This induces errors in the measurement of the peak area absorbance of the two peaks. The very low recoveries obtained for selenocystine, see Table 2, suggest that this species is degenerated during the extraction procedure. It is however difficult to say where the product of selenocystine can occur in the chromatogram as no obvious unknown peaks were detected. Selenate is separated from all other peaks and the background absorbance is low at this retention time. From these results it is obvious that the only species reliable to quantify in this sample, using this method, is selenate. The amount of extractable selenate in CRM 402 was determined to be (95% confidence interval) using the standard addition method. This means that the recovery of selenate is assumed to be quantitative, see Table 2. If direct determination using the external calibration curve and correction for the extraction efficiency is used the results is This approach is however very questionable as selenate most likely is not bound into the matrix to the same degree as e.g. selenoaminoacid species and therefore could be expected to have higher extraction efficiency than other species. As the selenium content of CRM 402 is 6.70 ± selenate constitutes approximately 22% of the total selenium if determined by the standard addition method.

REFERENCES Casiot, G., Vacchina, V., Chassaigne, H., Szpunar, J., Potin-Gautier, M., and Lobinski, R., 1999, An approach to the identification of selenium species in yeast extracts using pneumatically-assisted electrospray tandem mass spectrometry, Anal. Commun, 36:77–80. Emteborg, H., Bordin, G., and Rodriguez, A. R., 1998, Speciation of organic and inorganic selenium in a biological certified reference material based on microbore ion-exchange chromatography coupled to inductively coupled plasma atomic emission spectrometry via a direct injection nebulizer or coupled to electrothermal atomic absorption spectrometry, Analyst, 123:245–253. World Health Organisation, 1987, Geneva, Environmental Health Criteria 58, Selenium.

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SELENIUM TRUE ABSORPTION AND TISSUE CONCENTRATION OF RATS AT DIETARY SELENITE, SELENO CYSTEINE, AND SELENO METHIONINE

W. Windisch and M. Kirchgeßner Institute of Nutrition Physiology Technological University of Munich 85350 Freising-Weihenstephan Germany

The objective of this study was to compare the effects of selenite and seleno amino acids on the quantitative Se metabolism and tissue Se retention. For this purpose, 60 growing rats were labeled with and fed for 6 weeks 3 semisynthetic diets which were supplemented with either Na selenite, or seleno cysteine (SeCys), or seleno methionine (SeMet). Total dietary Se content was adjusted uniformly 150ng/g. The Se balance was recorded and finally the animals were killed and dissected. True absorption of dietary Se was 92% of intake in the case of selenite, while with SeCys and SeMet it increased to 98%. The respective endogenous faecal excretions were 10%, 9% and 7% of Se intake. Renal excretions were similar for selenite and SeCys (36% and 33%) but reduced for SeMet (25% of Se intake). The Se retention rose from selenite (47%) over SeCys (56%) to SeMet (67% of Se intake). In comparison to selenite, SeCys did not affect the Se concentrations of organs, blood and bone but increased that of muscle, skin and hair (18%, 6%, 11%). SeMet increased the Se concentrations especially of muscle, hair and skin (78%, 42%, 33%) but also of bone and almost all other organs including brain and testes. Quantitatively, in animals supplied with seleno amino acids the higher Se retention was localised mainly within muscles. The recovery of injected was complete irrespective of the level of Se supply. Se exhalations were therefore not of quantitative importance. The GSH-Px activity in blood plasma was reduced by 5% in the case of dietary SeMet. 173

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The results indicate that SeCys and SeMet are absorbed as intact amino acids which may be included into protein synthesis. The Se bound to this amino acids may thus be withdrawn from Se metabolism and accumulated in tissues without control of Se homeostasis. This effect is especially pronounced in the case of SeMet. The bioavailability may therefore be ordered as follows:

45

EFFECTS OF SELENIUM STATUS ON SELENIUM INCORPORATION INTO PLASMA FRACTIONS AND EXCRETION IN URINE IN MEN INFUSED WITH 74SE SELENITE Y. Xia1, J. Butler2, M. Janghorbani3, P. Ha1, P. Whanger2, J. Olesik4, and L. Daniels4 1

Chinese Academy of Preventive Medicine Beijing, China 2 Oregon State University Corvallis, Oregon, USA 3 Center for Stable Isotope Research Inc., Chicago, Illinois, USA 4 Ohio State University Columbus, Ohio, USA.

The majority of selenium (Se) in plasma is associated with selenoprotein P (SeP) in people consuming deficient or adequate levels of dietary Se, but in contrast the majority of plasma Se is with albumin (Alb) in subjects consuming excess Se in naturally occurring forms. The presence of trimethylselenonium (TMSe) is detectible in the urine only with excess intakes of Se. Chinese men with long term dietary Se intakes over the range of 12 to l, 230g/day were infused for five hours with labeled selenite and the kinetics of daily urine excretion and incorporation into plasma proteins examined for the next seven days. Those subjects with excess selenium (supranutritional) intake (Enshi, China) were infused again after they had consumed food with deficient levels of Se for nine weeks. Subjects with long-term deficient, adequate or supranutritional intakes excreted respectively 4.3 ± 0.4, 12.4 ± 2.2 ans 29.8 ± 3.2% of the infused dose during the seven days of the experiment. The urinary excretion was not affected (28.8 ± 3.4%) in the supranutritional subjects who had consumed the deficient diet for nine weeks. Even though there was a positive correlation of Se intakes with the urinary excretion of this element, this relationship was not linear over the entire rang (deficient, adequate, supranutritional) of Se intake. With increased Se intake the daily urine excretion of this element appears to approach an asymptote, suggesting that the highest Se group eliminated a smaller fraction of infused selenite via the kidneys as compared to the other two groups. This 175

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supranutritional group apparently exhaled greater amounts of the infused Se in their breath. During the depletion period whole plasma Se and SeP, glutathione peroxidase (GPX) and Alb plasma fractions decreased respectively 20, 35, 44 and 41%. The stable isotope of Se was incorporated to the greatest extent into the SeP fraction followed by a small amount in the GPX fraction, but none was incorporated into the Alb fraction. A logarithm plot of the in the SeP fraction against time indicated a half-life of 1.9 to 2.9 days for Se in this selenoprotein. The data suggest a common precursor pool for both plasma SeP and urinary TMSe. Overall, it is concluded that long-term supranutritional intake of Se results in saturation of the selenite-exchangeable metabolic pool which limits continued increases in tissue levels of biologically active Se. Consequently, this may be significant in long-term Se supplementation regimes devised to take advantage of the potential chemopreventive properties of this element. (Supported by NIH grant DK 38341)

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MAGNESIUM EXCRETION AND REDISTRIBUTION IN RAT TISSUES AFTER DISULFIRAM EXPERIMENTAL ADMINISTRATION

L. Kovatsi, M. Tsougas, H. Tsoukali, and D. Psaroulis Laboratory of Forensic Medicine & Toxicology Faculty of Medicine Aristotle University of Thessaloniki 54006 Thessaloniki, Greece

The aim of the present study was to investigate the effect of disulfiram on the excretion and the distribution of magnesium in rat tissues. For this reason, 16 male Wistar rats were used. The animals were divided in two groups (test and control rats) consisting of 8 animals each. The test rats received p.o disulfiram 4mg/kg b.w. for a period of 8 weeks. During the experiment, 24h urine and feces were collected in order to evaluate the effect of disulfiram on the excretion of magnesium. At the end of the experiment, the animals were sacrificed and the target tissues were removed for analysis. Wet digestion with concentrated nitric and perchloric acids (1:1) was used and digests were analysed for the determination of magnesium concentration by flame atomic absorption spectrophotometry in air/acetylene flame. According to our results, disulfiram affects the excretion and the tissue distribution of magnesium in rats. These findings support our previously expressed opinion that drugs or other substances capable to form complexes with elements in vitro, can cause their redistribution into the living organism.

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SELENIUM AND ARSENIC DISTRIBUTION IN SOLUBLE PROTEIN FRACTIONS IN LIVER OF HENS FED WITH ADDED AS 2O3

V. Stibilj, I. Falnoga, D. Cestnik, and R. Jacimovic Department of Environmental Sciences Jozef Stefaný Institute, Ljubljana Slovenia

Selenium has been known for a long time as an essential element for numerous biochemical processes of living organisms. Though the biological role of arsenic is not completely clarified, its antagonistic effects on selenium are well known. A protective effect of As against the toxicity of a variety of forms of Se was demonstrated in several species including rats, dogs, swine, cattle and poultry (Stanley et al., 1994). It was sugested that As acts by enhancing the biliary excretion of Se. The opposite course of events has also been observed, in which selenite can stimulate the excretion of As in the bile of rats. Such experiments have been performed almost exclusively on rats. (Environmental Health Criteria, 1981). In this work we studied how the addition of to the feed with recommended Se content, influences As and Se distribution in the soluble protein fractions in liver of laying hens. We found that a difference between the control and exposed group was found in the Se concentration in the range near the void volume and the metallothionein-like proteins (MT-LP) (position near cytochrome c). Although selenium itself does not trigger the biosynthesis of MT, there are some reports indicating the association of selenium with mammalian MTs under metal induced stress (Takatera et al., 1994). The present study demonstrated the presence and increase of selenium associated with MT-LP in hen’s liver. They were characterised by molecular weight, the absence of UV absorbance, and Cu and Zn contents. 178

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REFERENCES Environmental Health Criteria, 1981. Stanley, T.R., Spann, J.W., Smith, G.J., and Rosscoe, R. Main and interactive effects of arsenic and selenium on mallard reproduction and duckling growth and survival. Archives of Environmental Contamination and Toxicology, 26(1994), s.444–451. Takatera, K., Osaki, N., Yamaguchi, H., and Watanabe, T. HPLC/ICP Mass Spectrometric Study of Selenium Incorporation into Cyanobacterial Metallothionein Induced under Heavy-Metal Stress. Analytical Science. 10(1994), p.567–572

48

METAL DISTRIBUTION IN METALLOTHIONEINS OF CYTOSOLS FROM HUMAN CIRRHOTIC LIVERS

C. Wolf, P. Brätter, and U. Rösick Hahn-Meitner-Institut Berlin Deptartment Trace Elements in Health and Nutrition Glienicker Straße 100, D-14109 Berlin Germany

Fractions containing metallothionein, which were extracted from liver cytosols of humans, were analysed to determine the complete distribution pattern of the metals copper, cadmium and zinc. Samples of cirrhotic livers which had come from organs removed during transplantation were examined for differences in the trace element binding pattern. After the separation of cytosols from the samples, the first step was a membrane ultrafiltration of the solution. This was done in order to separate all high molecular proteins which had molecular weights >100kDa. Using this procedure, the metal content remained in its initial form, contrary to the often used heat treatment of samples, which would have significantly changed the copper distribution. The metallothionein itself was isolated using size exclusion and anion exchange chromatography. Its metal content was determined simultaneously on-line by combination with a ICP-AES as element detector. To enhance the sensitivity of the element detection, an ultrasonic nebulizer was used to produce the aerosols. The calibration of the procedure was done by means a column bypass-injection of elemental standards in the separation system. The reliability of the element determination was confirmed by a quality check of the procedure using commercially available reference proteins. The metallothionein content in the samples was calculated using the determined metal concentrations and the accepted metal/protein ratios for Cu, Cd and Zn. These values were compared with values which were taken from the Comparing various liver samples of different pathogenesis the highest level of Cu-MT was found in primary biliary cirrhosis.

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EFFECT OF RETINOL DEFICIENCY ON LIVER CONCENTRATION OF FE, CR, ZN, MN, CO, CD, AND PB IN RAT

A. C. Anzulovich*, L. B. Oliveros*, L. D. Martínez*, M. Roura**, and M. S. Giménez* *Facultad de Química, Bioquímica y Farmacia Universidad Nacional de San Luis Ejército de los Andes 954 5700 San Luis, Argentina **Serveis Científico-Técnics Universitat de Barcelona Lluis Solé i Sabarís 1-3, 08028-Barcelona Spain

Mammalian liver is the major tissue involved in vitamin A metabolism. Previously we determined in rat that vitamin A deficiency modifies the hepatic enzyme activities related with free radical protection, which need of determined trace elements as cofactors. In the present work we explored the correlations between vitamin A deficiency and concentrations of different cations in the liver, using an in vivo experimental model. Two groups of female Wistar rats were weaned at 21 days of age and immediately assigned randomly to either the experimental diet, devoid of vitamin A (vitamin Adeficient group), or the same diet with 4,000 IU of vitamin A (8mg retinol as retinyl palmitate) per kg of diet (control group). They were fed for 3 months before sacrifice. Diets were prepared according to AIN-93 for laboratory rodents. Body and food intake were registered daily. Plasma and liver samples were taken under reduced light and frozen in the dark at –70 °C until determination of vitamin A concentrations by HPLC. After submitted the tissue samples to acid digestion, the analysis of cations was performed by Inductively Coupled Plasma Mass Spectrometry using a Perkin-Elmer ELAN 6000 ICPMS. Results were expressed as liver. Data were analized by unpaired Student’s test and indicated as mean ± SEM. Statistical significance was accepted at P < 0.05. Vitamin A-deficient rats had lower body weight than control rats (144 ± 6 vs 173 ± 5) and showed subclinical plasma retinol concentration (0.7µmol) and negligible total retinol stores in the liver (<3% of the total liver retinol stores of control rats), which 181

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confirm the vitamin A deficiency. In the vitamin A-deficient rats, the liver content of Fe increased significantly compared with control rats (203 ± 9.5 vs 317 ± 11), while the content of Mn, Zn, and Cr decreased significantly compared with those of control rats (2.8 ± 0.1 vs 1.2 ± 0.1; 27 ± 0.25 vs 15 ± 1 and 0.6 ± 0.02 vs 0.3 ± 0.02, respectively). The other cation concentrations were not affected. The results shown that vitamin A deficiency modifies the depots of several metals in liver which could be an important factor in determining liver susceptibility to oxidative damage. Possible mechanisms for these changes are discussed. Results are also consistent with the hypothesis that vitamin A acts as a physiological antioxidant in vivo.

50

VITAMIN A DEFICIENCY MODIFY THE ANTIOXIDANT DEFENSES AND THE TRACE ELEMENTS LEVEL IN RAT LIVER

A. C. Anzulovich*, L. Oliveros*, D. Martinez*, M. Baucells**, and M. S. Gimenez *Facultad de Química, Bioquímica y Farmacia Universidad Nacional de San Luis

Ejército de los Andes 954 5700 San Luis, Argentina **Serveis Científico-Técnics Universitat de Barcelona Lluis Solé i Sabarís 1-3, 08028-Barcelona Spain

Considering that mammalian liver plays a major role in long chain fatty acids and vitamin A metabolism, we investigated if nutritional vit A deficiency modify in liver the enzymatic defense system components and the concentration of some trace elements which have an important role in free radical protection. Two groups of femal Wistar rats at 21 d age were ramdomly weaned onto either a vit A deficient diet (-A group) or the same diet with vit A 4,000 IU/Kg diet (control group). They were fed for 12 weeks before sacrifice. Serum and liver Vit A concentrations were measured by HPLC. Livers were homogeinized and the spontaneous chemiluminiscence was determined by using a liquid scintillation counter in out-of-coincidence mode. The catalase (CAT), superoxide dismutase (SOD) and glutation peroxidase (GSH-Px) activities weremeasured. The accumulation of end product of lipid peroxidation was determined by measurement of thiobarbituric acid-reactive substances (TBARiS). The Se, Zn and Cu analysis was performed by Inductively Coupled Plasma Mass Spectrometry. Results wzre analized by Studentis test. In relation to the control group, -A group showed: very low levels of serum and liver vit A, which confirms the vit A deficiency; an increase in the liver chemiluminiscence and activities of CAT and GSH-Px (1.8(0.1 vs 2.9 (0.2U/mg protein and 0.09 (0.01 vs 0.15 (0.01 (mol.min/mg protein, respectively), suggesting a high amount of substrates for the enzymes; and a decrease in the (g/g liver of Zn, Cu and Se, indicating that vit A deficiency could interfere with the depots of those trace elements in the liver. In spite of SOD activity and level of TBARiS were not changed, the results show that vit A nutritional deficiency affects the liver antioxidative capacity. 183

51

TRANSPORT OF SILVER AND ITS POTENTIAL INTERACTIONS WITH COPPER METABOLISM IN THE RAT

S. Hanson, S. Donley, H. Rim, and M. C. Linder California State University Fullerton, California 92834-6866 USA

Little is known about the uptake, transport, and metabolism of silver, small quantities of which enter the mammalian organism daily via the diet. However, recent studies by several groups suggest an interaction with copper metabolism, and that while not acutely toxic to the mother, significant doses of ionic silver might interfere with copper transport to the fetus via ceruloplasmin (Shavlovski et al., BioMetals 8: 122–128, 1995). The studies here described were initiated to determine whether the pathway of silver transport to tissues after dietary absorption resembles that for copper, using the rat as a model. Adult female Sprague Dawley rats were injected i.p. with small amounts of silver in the form of (30uCi/rat, equivalent to about 15ug Cu). Blood and tissues were examined for total radioactivity as well as for incorporation into copper binding proteins, from 1 to 52 hours thereafter. As in the case of copper, the liver was the major initial recipient of the radioisotope, and the was rapidly incorporated into liver and some other tissues, like spleen, reaching maximal levels one hour after injection. Over the period examined, about 25% of the radioisotope was in the blood plasma, where it bound primarily to a single component. This component had an apparent molecular weight 750 kDa, as determined by size exclusion chromatography. Only a very small proportion of the radioactivity became incorporated into ceruloplasmin, as determined by immunoprecipitation and native gel electrophoresis. There appeared to be no association of with albumin or transcuprein. The binding of to the 750 kDa protein was of very high affinity, as determined from the off-rate in the presence of pH 7 buffers variously containing NaCl plus phosphate and/or histidine. Addition of 100uM Cu(II), as the 1:1 mol:mol nitrilotriacetate complex, did not release much silver, but there was considerable release upon addition of 2% mercaptoethanol. The major subunits in the partially purified preparation of this silver-binding component were 45 and 145 kDa, as determined by SDS-polyacrylamide gel electrophoresis. These subunits belong to alpha-1-macroglobulin, which has a tetrameric structure and total molecular weight of 760,000. 184

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In lactating rats, the silver tracer did not enter the mammary gland or milk rapidly or in large proportions, which contrasts with what occurs with (see accompanying abstract by Linder et al.). Also in contrast to copper, the silver that entered the milk achieved its highest level at about 24 hours, as compared with one-two hours in the case of copper. We conclude that, at least in the rat, the pathway of distribution of trace amounts of silver ions entering the blood and tissues after absorption from the diet is not the same, involving major differences in the blood plasma protein carriers. The major silver binding protein in plasma appears to be alpha-1-macroglobulin, which is in the same family as transcuprein, one of the principle copper binding proteins involved in the initial phase of copper distribution. This work was supported in part by NSF Grants REU 5369 and DUE 9352396

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DIETARY VANADIUM, P-ATPASE -7A EXPRESSION AND THE INFLUENCE ON LYSYL OXIDASE AND CU ACCUMULATION IN RAT SKIN AND LIVER

Robert B. Rucker, Chang Tai Cui, Eskouhie H. Tchaparian, Alyson E. Mitchell, Michael Clegg, Janet Y. Uriu-Hare, and Carl L. Keen Department of Nutrition University of California Davis, California 95616

Sodium vanadate is a broad spectrum inhibitor of ATPase activity and when ingested is toxic. Vanadium toxicity results in developmental defects (Biol. Trace Elem. Res. 14:193), signs of which include extra cellular matrix (ECM) defects similar to those common to dietary Cu deficiency or intoxication with beta-aminoproprionitrile. As a consequence, we have examined indices of tissue copper metabolism in response to increasing amounts of Na metavanadate in diets fed to weanling rats at 0, 10, 20, 40, or 80 ug V per g of diet. In ECM-enriched tissues, such as tendon or skin, the Cu concentration increases progressively from 0.46 to ~1.0 ug/g tissue in response to dietary V (values plateau at In contrast, no change in liver Cu was observed. Liver Cu values were 3–4 ug/g at all levels of V intake. The V concentration increased in response to dietary V in all of tissues that were examined. Of interest, the activity of the cuproenzyme, plasma ceruloplasm, principally secreted from liver, was not changed in response to dietary V, whereas a cuproprotein secreted from skin and tendon, lysyl oxidase, was inhibited to 10–20% of normal values at intakes of >40 ugV/g diet. These data indicate that though vanadate is an inhibitor of ATPase activity in general, the P-ATPase-7A involved in ECM copper transport and transfer may be particularly sensitive to V. Since Cu and lysyl oxidase are also essential for normal prenatal and neonatal development, we have also the influence of changing Cu status in rat embryos. Cu deficiency results in malformations in rat embryos that are detected by gestation day (GD) 11. Since some of the malformations appear related to abnormal deposition and maturation of extracellular matrix (ECM), we have examined the process further by specifically focusing on lysyl oxidase, which catalyzes the crosslinking of elastin and collagen 186

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in the ECM. Moreover, the relationship between lysyl oxidase expression, i. e. steady state message levels, and the expression of the Cu transporter, such the P- type ATPase-7A associated with Menkes disease was examined. The steady-state lysyl oxidase mRNA levels and the levels of mRNA corresponding to P-ATPase 7A should be at least temporally-related, because of the need to activate lysyl oxidase by Cu transfer processes that involve P-ATPase activity. Lysyl oxidase activity appeared to be constitutively expressed throughout GD 9 to 15. Both lysyl oxidase and P-ATPase-7A mRNAs were easily detected. Importantly, P-ATPase-7A expression was detected during those periods essential to the activation of lysyl oxidase. The expression of both increased relative to gestational age. Supported by a grant from the NIH and USDA-NRI.

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RECOMMENDED DIETARY INTAKES FOR TRACE ELEMENTS New Trends

Janet C. King USDA Western Human Nutrition Research Center University of California Davis, California 956162

Scientific debate has been part of the process for establishing recommended dietary intakes since the first standards were set. This debate, stemming primarily from differences in data interpretation, initially fuels controversy, but then consensus, evolution, and change eventually occurs. In general, the evolution of food habits and general nutrition knowledge drives the controversies and eventual changes in dietary recommendations. 1. HISTORICAL EVOLUTION OF DIETARY STANDARDS Some type of dietary standard has been in place for the past 165 years. The first standard was published in 1835 when the British Merchant Seamanís Act specified a ration of ìlimeî or lemon juice to prevent scurvy among all men in the mercantile service1. About 30 years later, in 1862, the British Privy Council proposed a standard of 3,000 kcal/d and 80 g protein/d to prevent illnesses associated with starvation. This was the first quantitative dietary standard, and it was based on the observed intakes of healthy factory workers. The 1930’s were a time of deprivation caused by the economic depression. Both the British Medical Association and the US Department of Agriculture proposed dietary standards to serve as guidelines for relieving starvation. In 1940, a Committee on Food and Nutrition was set up under the National Research Council (NRC) of the US National Academy of Sciences (NAS) to advise the government on problems concerning national defense. A Committee was established to prepare a set of dietary standards. The report from this Committee was presented to the membership of the American Institute of Nutrition in 1941 and accepted. It also was adopted at the National Nutrition Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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Conference called by President Franklin Roosevelt in May, 1941 and eventually published in 1943. Since then, ten editions of the RDAs have been published with the last published in 1989. In 1997, a draft of a new set of dietary standards formulated by the Food and Nutrition Board of the Institute of Medicine of the NAS, called the Dietary Reference Intakes (DRIs), was published. A complete set of DRIs for all essential nutrients will be completed in 2001. 1.1. Transitions in Dietary Standards from 1835–2000 As one reviews the first 100 years of making dietary recommendations, i.e. from 1835–1935, several key transitions occurred. First, there was a change in the purpose of the standards. They went from being recommendations to relieve starvation and illness stemming from economic and wartime crises to standards for programs to maintain and improve the health of the population, especially women, infants, and children. Second, the approach used to formulate the standards changed. Initially, the recommendations stemmed from observations of the usual pattern of food consumption. Later, they were technical standards based on scientific knowledge of human needs for essential nutrients and energy. Further evolution continued from 1940–2000. The purpose of the standards evolved from less emphasis on prevention of dietary deficiencies to increasing emphasis on the reduction of risk of chronic disease. Furthermore, the uses of the standards increased several-fold from a standard for feeding groups of people to a set of reference values for planning and assessing diets of both groups and individuals. Finally, the food habits of individuals changed. In 1940, individuals obtained all of their nutrients from natural food sources; today nutrients come from a variety of sources—natural foods, fortified foods, and vitamin/mineral supplements. These changes in food habits require a change in the way dietary standards are set. 1.2. Expanding Uses of the Recommended Dietary Allowances The uses of the Recommended Dietary Allowances (RDAs) in nutrition policy and programs in the U.S. in 1989 are contrasted with their use in 1941 ñ to plan and procure food for the military in Table 1. Today, RDAs form the basis of the Federal Food Assistance programs, the food fortification programs, and the development of new nutrient supplements or specially modified foods. They are also used to plan and evaluate the diets of groups and individuals. Although the uses of the RDAs had expanded considerably since the first edition of the RDA was published, the format of the recommendations was essentially unchanged. The next edition of the RDAs needs to reflect the expanded uses of the standards, the concern about reducing the risk of chronic disease, and the multiple sources of nutrients in todayís diet. 2. A NEW PARADIGM FOR DIETARY STANDARDS The Food and Nutrition Board, Institute of Medicine, National Academy of Sciences, initiated a comprehensive effort in 1991 to deal with the growing concern that the RDAs were no longer appropriate standards for the many uses to which they were

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applied. A Standing Committee on the Scientific Evaluation of Dietary Reference Intakes (DRI Committee), composed of nutritional scientists from America and Canada, was appointed to develop a new approach for making dietary recommendations. The changes in the concepts underlying the dietary standards and the proposed responses to each of those changes are summarized in Table 2. The new paradigm calls for the use of functional endpoints of nutrient requirements, consideration of the dietary need for components linked maintaining health, i.e. fiber, choline, carotene, establishment of multiple reference intakes including an upper level, and development of a statistical approach for estimating the inadequacy of a nutrient intake in individuals and groups. This paradigm has been applied to establishing dietary reference intakes (DRIs) for calcium and related nutrients, the B-vitamins.2,3 Although the concepts of this new paradigm are sound, implementation is hindered by the lack of appropriate data. For example, the Panel attempted to set a dietary standard for calcium that reduces the risk for osteoporosis. The association between dietary calcium and osteoporosis is not specific; osteoporosis is not merely due to the lack of calcium, it has many causes. Also, a quantitative or dose-response relationship between dietary calcium and bone mineral density has not been established for all age groups. For this reason, the Committee established an Adequate Intake (AI) instead of a RDA for calcium. An AI is a value based on observed or experimentally determined approximations of nutrient intake by a group (or groups) of healthy people that is used when a RDA cannot be established.2 In other words, the AI for calcium is the intake consumed by individuals appearing to have little risk of low bone mineral density.

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When the data are sufficient, three other dietary standards are proposed:2 Recommended Dietary Allowance (RDA): the average daily dietary intake level that is sufficient to meet the nutrient requirement of nearly all (97 to 98 percent) healthy individuals in a group. Estimated Average Requirement (EAR): a nutrient intake value that is estimated to meet the requirement of half the healthy individuals in a group. Tolerable Upper Intake Level (UL): the highest level of daily nutrient intake that is likely to pose no risk of adverse health effects to almost all individuals in the general population. As intake increases above the UL, the risk of adverse effects increases.

3. A STATISTICAL APPROACH FOR ASSESSING THE ADEQUACY OF NUTRIENT INTAKES OF INDIVIDUALS AND GROUPS If we were able to measure intakes and requirements simultaneously for the same individuals, then we would be able to determine if an individual’s requirements were met or not. Unfortunately, collecting such data is impossible. Therefore, we can only estimate the prevalence of inadequacy in a group or the risk of inadequacy in an individual. The prevalence of inadequacy of a group can be estimated using the following information: The estimated average requirement (EAR) for the nutrient The variance of the distribution of the requirement, and The distribution of usual intakes of the population The proportion of the population with intakes below the average requirement comprises those with inadequate intakes. For example, the usual zinc intakes of a group of 50 men range from 4.7 to 15.5 mg/d. The estimated average requirement for zinc for adult men is 5.4mg/d.4 Those men with intakes below 5.4mg/d have inadequate intakes. In this sample, only one man had a zinc intake below 5.4mg/d. Thus, the prevalence of inadequate zinc intakes is 2%. This method works well when nutrient intakes and requirements are independent, when the distribution of requirements is symmetrical, and when the variability in intakes is greater than the variability in requirements. If one wants to evaluate the adequacy of intake of an individual, the likelihood inadequacy can be estimated from the usual nutrient intake expressed as a multiple of the Estimated Average Requirement. The requirements must be normally distributed and the variance of the requirements known for this method to work well. f the intake is times the EAR (estimated average requirement), there is little likelihood that the intake is inadequate. However, if the intake is times the EAR, the intake is highly likely to be inadequate. For a zinc EAR of 5.4mg/d, the difference between 1.2xEAR and 0.8xEAR is only 2.2mg. Thus, one needs a very precise, accurate estimate of usual zinc intake in order to estimate the likelihood that zinc intakes are inadequate. This degree of accuracy is difficult, if not impossible, to achieve. The Estimated Average Requirement (EAR) is essential for estimating the adequacy of nutrient intakes of individuals and groups. For this reason, the EAR is core component of the set of nutrient standards included in the Dietary Reference Intakes.

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4. ESTIMATION OF TOLERABLE UPPER INTAKE LEVELS

OF NUTRIENTS Today, individuals get nutrients from natural food sources, from fortified foods, and from vitamin-mineral supplements. These multiple sources of nutrients increase the risk of consuming a level that could cause an adverse health effect. The Food and Nutrition Board, therefore, decided to include a UL, tolerable upper intake level, as one of the DRIs to use as a guide for evaluating the possibility of over consumption. A risk assessment model is used to derive the UL.5 First, a hazard identification process is conducted to identify the adverse health effects that have been caused by the nutrient under consideration. Second, a dose-response assessment is completed. This includes selection of a data set, identification of a critical endpoint with its NOAEL (no observed adverse effect level) or LOAEL (low observed adverse effect level), and an assessment of uncertainty. Potential sources of uncertainty include inter-individual variation in sensitivity, extrapolation of experimental animal to human, and the severity and incidence of the observed effect at the LOAEL, if a NOAEL is not available. If one wanted to estimate a UL for zinc, the first step would be to identify an adverse health effect of high zinc intakes. One possibility might be the reduction in copper absorption. The LOAEL for the effect of zinc on copper absorption is 60mg/d.6 Since this is a LOAEL, rather than a NOAEL, and since inter-individual variation is high, the uncertainty factor may be 2. The UL, therefore, would be the LOAEL divided by 2, 30mg zinc/d. 5. SUMMARY New knowledge about trace element metabolism and changes in dietary habits dictate a need for a new approach in setting dietary standards. The Food and Nutrition Board of the Institute of Medicine has developed a new paradigm for setting dietary standards that involves the use of functional endpoints and multiple reference intakes for each age and gender group. Application of this paradigm to trace element dietary standards will be difficult because of the following limitations: Lack of specific, sensitive, functional indicators of dietary requirements for trace elements. Little information on the functional or homeostatic response to chronically low or high trace element intakes. Limited knowledge of the requirements of children, infants, and pregnant and lactating women. Absence of a clear model for how to deal with trace element interactions. Future research in trace element nutrition should be designed to address these limitations. REFERENCES Harper, A.E. 1987, Evolution of Recommended Dietary Allowances—New Directions? Annual Review of Nutrition. 7:509–537. Institute of Medicine. 1997, Dietary Reference Intakes for Calcium, Phosphorus, Magnesium, Vitamin D, and Fluoride. Washington D.C.: National Academy Press.

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Institue of Medicine. 1998b, Dietary Reference Intakes for Thiamin, Riboflavin, Niacin, Vitamin B6, Folate, Vitamin B12, Pantothenic Acid, Biotin, and Choline. Washington D.C.: National Academy Press. World Health Organization. 1996, Trace Elements in Human Nutrition and Health. Geneva: World Health Organization. Institute of Medicine. 1998, Dietary Reference Intakes: A Risk Assessment Model for Establishing Upper Intake Levels for Nutrients. Washington D.C.: National Academy Press. Mertz, W., Abernathy, C.O., and Olin, S.S. 1994, Risk Assessment of Essential Elements. Washington, D.C.: ILSI Press.

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TRACE ELEMENT ADDITION TO FOODS Technological and Nutritional Aspects

Denis Barclay

Addition of trace elements (TE) to staple and processed foods is a key approach in any comprehensive strategy for the prevention of TE deficiencies (Institute of Medicine, 1998). Costs involved are generally low, more than one deficient nutrient can be added to the chosen vector (Barclay, 1998). TE addition is not only nutritionally effective, but also cost-effective. Furthermore, modification of existing food habits is not required, and population coverage can be high, and the risk of toxicity is low. According to a recent World Bank publication (1994), “no other technology offers as large an opportunity to improve lives . . . at such a low cost... and in such a short time”. However, recent estimates of world-wide prevalence of micronutrient deficiencies including TE, show that much progress remains to be achieved. The failure of food fortification to achieve its full potential in prevention of nutritional deficiencies is due to a variety of factors. The lack of adequate data in most regions of the world, on nutrient intakes and status and dietary habits makes identification of appropriate food vector(s) problematical (ILSI Europe, 1998). Depending on the geographical location, other important factors may include microbial infection or parasitic infestation in the host, regulatory issues, cost constraints and the need for comprehensive quality assurance procedures. In addition, two inter-related factors, firstly, the bioavailability of added TE and interactions between them, and secondly, the negative effects of TE addition on the organoleptic qualities, stability and shelf life of fortified foods, can be barriers to the success of food fortification in deficiency prevention (Clydesdale, 1991). The prime example is iron, which can catalyse cereal lipid oxidation leading to rancidity, form complexes with cereal or cocoa polyphenols leading to unacceptable colour changes, or by simply giving an unpleasant metallic flavour to foods and beverages. Obviously, such organoleptic penalty must be avoided; on one hand, the consumer will not purchase the product and will therefore not receive the necessary additional nutrients, and on the other hand, the manufacturer will suffer loss of sales and may abandon fortification. The most

Address all correspondence to: Dr. Denis Barclay; Nestlé Research Centre, PO Box 44, Vers-chez-les-Blanc, 1000 Lausanne 26, Switzerland; telephone: +41 21 785 8626; fax: +41 21 786 8563; email: [email protected]. Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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often employed solution is to add an “inert” Fe compound such as elemental Fe, or an alternative Fe compound having acceptable bioavailability in the product without introducing organoleptic penalty. Encapsulated Fe compounds such as ferrous sulphate coated with partially hydrogenated soybean oil can also provide an acceptable solution in some products. Another problem posed by addition of trace elements such as Fe and Cu to foods is the accelerated degradation of vitamins in the presence of moisture, light and oxygen, the most sensitive being vitamins C, A, D, B1 and B12 (Ottaway, 1993). Again, use of alternative Fe compounds or encapsulation of Fe can provide a solution. A wide variety of Fe fortification compounds is available (Hurrell, 1984); water or weak acidsoluble compounds, the bioavailability of which are generally high, and insoluble or inert compounds such as elemental Fe whose bioavail-abilities are generally low. The relative costs of these compounds vary about ten-fold. The choice of the appropriate Fe compound depends primarily on the nature of the food vector, and is often a compromise between organoleptic effects and bioavailability. Hurrell (1989) carried out of studies in adults in order to identify Fe fortification compounds for infant cereals that may be appropriate from both the bioavailability and organoleptic standpoints. Compared to ferrous sulphate, the relative bioavailability of ferrous fumarate was 100%, whereas those of the other compounds tested ranged from about 40 to 90%. Since no such data was available for infants, Kastenmayer (1999) recently carried out similar studies in 6–12 month old infants comparing the bioavailability of the commonly used compound ferric pyrophosphate with that of ferrous fumarate. The results confirmed those of Hurrell showing that the bioavailability of ferrous fumarate was 3 fold higher than that of ferric pyrophosphate. Increasing the ascorbic acid : Fe ratio from 10 : 1 to 20 : 1 (wt : wt) gave no further significant enhancement of Fe absorption. Based on these results in adults and infants, a number of infant cereals are now fortified with ferrous fumarate. Over recent years, encapsulated Fe fortification compounds and other alternative Fe compounds have been developed. Before considering them for use, bioavailability data are necessary. Their bioavailability was tested (Couzy, personal communication) using the Hb repletion test in rats (Williams, 1984; Forbes, 1989), compared to ferrous sulphate. Encapsulation of ferrous sulphate with dehydrogenated soybean oil resulted in only a 20% reduction in bioavailability. This compound is now used in various products such as milk powders and infant formulas. The relative Fe bioavailability of Fe bis-glycinate was 59% relative to ferrous sulphate using this assay. Another study in infants (Fox, 1998) showed that the bioavailability of the Fe bis-glycinate in infant cereals with low and high phytic acid levels was similar to that of ferrous sulphate. This compound behaves as other soluble Fe compounds, and our laboratory tests showed that it provokes unacceptable organoleptic alteration of products containing cereals or polyphenols, similar to ferrous sulphate. Compared to iron, the number of zinc fortification compounds is quite small. In the USA, only 5 compounds are found in the food supply, and their use has been steadily increasing over the last three decades. Zn oxide has become the most widely used Zn compound in the USA. Only 6 Zn compounds are permitted for use in foods for infants and children in the EU. Again, compared to iron, there is little information on the relative bioavailability of Zn compounds. This may be partially due to the fact that the extent of Zn deficiency is unknown because of the lack of adequate status indicators. Another possible explanation is that there has been much less drive to discover improved Zn fortification compounds since they cause little organoleptic alteration of foods. There is no data in humans comparing the bioavailability of inorganic Zn compounds. In cattle, the

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bioavailabilities of the most commonly used compounds Zn sulphate and Zn oxide were similar (Rojas, 1996), whereas in poultry and pigs, Zn sulphate was two to three times more available than Zn oxide (Wedekind, 1990, 1992, 1994). Zn sulphate is the most often used Zn fortification compound in infant and clinical nutrition products. In the 1986 Ecuadorian National Nutrition Survey (Freire, 1988) the highest levels of childhood stunting and the highest prevalence of low serum Zn were observed in the Andean Region. This information raised the question of the existence of widespread growth-limiting Zn deficiency in this region. An intervention study was carried out to clarify this issue and to determine the effect of a Zn-fortified food on child growth in the Andean region of Ecuador (Dirren, 1994). Daily supplemental Zn (l0mg/d) vs. placebo of pre-school children for 12 months significantly increased serum Zn levels and longitudinal growth. These results confirmed the presence of a growth limiting Zn deficiency in these children. However, in a parallel study in similar children, 12 months consumption of a Zn-fortified cereal product (extra 5 mg/d of Zn) did not increase serum Zn or growth vs. placebo (Dirren, personal communication). Longitudinal growth over 12 months of the two groups of children consuming cereals either with or without added Zn was similar to that of the group receiving 10 mg/d of supplemental Zn. Two possible explanations are that the amount of Zn provided by the cereal was insufficient, or that the bioavailability of the Zn in the cereal was low. Further investigation of this question is warranted. In general, selenium fortification concerns only those foods that provide the majority or the totality of daily food intake, such infant formulas and clinical nutrition products. Two Se compounds are authorised in Europe and the USA for fortification of infant formulas, sodium selenite and sodium selenate. The most commonly used compound is sodium selenite. To date there is very little information regarding the relative bioavailability of these compounds or on their physiological effects in human nutrition. Australian draft food legislation prepared in 1997 proposed to make Se fortification of infant formulas compulsory. In order to prepare for this and to enable selection of the best compound, the bioavailability in a milk formula of the two most likely Se fortification compounds, sodium selenite and sodium selenate (Van Dael, 1999), was compared using a stable isotope technique in healthy men. Absorption of selenate was significantly higher than that of selenite. However, urinary excretion of selenate was also significantly higher, with the end result that retention levels were similar for the two compounds. It was concluded from that study they were equally efficient for addition to milk formulas from the bioavailability standpoint. A major difficulty in the addition of Se to foods such as infant formulas is that of achieving adequate homogeneity in the final product. This is of particular importance given the relatively low safety margin between Se requirements and toxicity levels. For example, it has been proposed that infant formulas provide at least 10 but no more that of Se per day (Levander, 1989). In order to increase the Se content of an infant formula by the corresponding fortification level is only of sodium selenite per kg of powder. For comparison, Fe fortification of such a product requires addition of ferrous sulphate at a level 1,000 times higher. One possible approach to ensure adequate homogeneity is to prepare an initial diluted Se premix which is then mixed with the mineral premix containing Fe, Zn, Cu etc. before final addition to the product. Se addition to foods in this way must be strictly controlled using adequate sampling procedures and analytical methodology. It has been recognised for some time that interactions between minerals and trace elements at the intestinal level can have significant effects on their bioavailability (WHO,

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1996). In 1981, Solomons et al measured the effects of increasing levels of medicinal iron on Zn bioavailability by measuring the 4-hour change in plasma Zn levels in human adults. They reported that Fe : Zn weight ratios as low as 1 : 1 markedly lowered Zn bioavailability and that higher Fe levels inhibited Zn uptake even further. Since Fe : Zn fortification weight ratios are typically in the range of 1 : 1 to 2 : 1 in infant formulas for example, this raised the question of whether such fortification levels of Fe affect Zn bioavailability from food to the same extent. Again, this is of potential importance for foods that may provide the majority or the totality of daily food intake such as infant formulas and clinical nutrition products. Davidsson (1995) studied the effects of Fe fortification on Zn absorption from an infant formula and an infant cereal in 40 healthy adults. The formula contained 0.1 mg Zn per 100ml of formula; the cereal contained 1 mg Zn per l00g. Zn absorption from these foods with and without added was determined using an isotopic technique. The Fe fortification levels used were similar to those in commercial food products. Zn absorption from the test meals showed no significant differences related to their Fe content. It was concluded from that study that normal levels of Fe fortification in infant cereals and formulas do not diminish Zn absorption. A similar study in infants by Fairweather-Tait (1995) showed that Fe fortification did not inhibit Zn absorption from an infant cereal. In summary, in order to have a beneficial impact on consumer health and nutrition, the addition of trace elements to foods must be based on reliable information concerning the dietary habits and nutritional requirements and status of the target consumer. The chemical form of the fortification micronutrients must be chosen in order to have maximal bioavailability whilst not giving rise to unacceptable organoleptic changes.

REFERENCES Barclay D., 1998, Multiple fortification of beverages. Food Nutr Bull, 2:168–171. Clydesdale F.M., 1991, Mineral additives. In: Nutrient Additions to Food, J.C. Bauernfeind and P.A. Lachance, eds. Pages 87–107. Food & Nutrition Press, Inc. Davidsson L., Almgren A., Sandstrom B., and Hurrell R.F., 1995, Zinc absorption in adult humans: the effect of iron fortification. Br J Nutr, 74:417–425. Dirren H., Barclay D., and Gil-Ramos J. el al., 1994, Zinc supplementation and child growth in Ecuador. In: Nutrient Regulation during Pregnancy, Lactation, and Infant Growth. L.H. Allen, J.C., King, and B. Lönnerdal, eds. Pages 215–222. Plenum Press. Fairweather-Tait S.J., Wharf S.G., and Fox T.E., 1995, Zinc absorption in infants fed an iron-fortified weaning food. Am J Clin Nutr, 62:785–789. Forbes A.L., Adams C.E., and Arnaud M.J. et al., 1989, Comparison of in vitro, animal, and clinical determinations of iron bioavailability: International Nutritional Anemia Consultative Group Task Force report on iron bioavailability. Am J Clin Nutr, 49:225–238. Fox T.E., Eagles J., and Fairweather-Tait S.J., 1998, Bioavailability of iron glycine as a fortificant in infant foods. Am J Clin Nutr, 67:664–648. Freire W., Dirren H., and Mora J.O. et al., 1988, Diagnostico de la situación Alimentaria, Nutricional y de Salud de la población ecuatoriana menor de cinco años (DANS). CONADE, MSP, Quito. Hurrell R.F., 1984, Bioavailability of different iron compounds used to fortify formulas and cereals: technological problems. In: Iron Nutrition in Infancy and Childhood. A. Stekel, ed. Pages 147–178. Academic Press. Hurrell R.F., Furniss D.E., and Burri J. et al., 1989, Iron fortification of infant cereals: a proposal for the use of ferrous fumarate or ferrous succinate. Am J Clin Nutr, 49:1274–1282. ILSI Europe, 1998, Addition of nutrients to foods: nutritional and safety considerations. ILSI Europe, Brussels. Institute of Medicine, 1998, Prevention of micronutrient deficiencies. National Academy Press, Washington.

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Levander O.A. Upper limit of selenium in infant formulas. J Nutr; 119:1869–1872. Kastenmayer P., Szajewska H., and Clough J. et al., 1999, Iron bioavailability in infants from an infant cereal fortified with feric pyrophosphate or ferrous fumarate. TEMA-10, Evian, France. Ottaway P.B., 1993, Stability of vitamins in foods. In: The Technology of Vitamins in Foods, P.B. Ottaway, ed. Pages 90–113. Chapman & Hall. Rojas L.X., McDowell L.R., and Martin F.G. et al., 1996, Relative bioavailability of zinc methionine and two inorganic sources fed to cattle. J Trace Elements Med Biol, 10:205–209. Solomons N.W. and Jacob R.A., 1981, Studies on the bioavailability of zinc in humans: effects of heme and nonheme iron on the absorption of zinc. Am J Clin Nutr, 34:475–482. Van Dael P., Fay L., and Longet K. et al., 1999, Selenium bioavailability from a selenium fortified milk-based formula. TEMA-10, Evian, France. Wedekind K.J. and Baker D.H., 1990, Zinc bioavailability in feed-grade sources of zinc. J Anim Sci, 68:684–689. Wedekind K.J., Hortin A.E., and Baker D.H., 1992, Method for assessing zinc bioavailability: efficacy estimates for zinc-methionine, zinc sulfate, and zinc oxide., J Anim Sci, 70:178–187. Wedekind K.J., Lewis A.J., Giesemann M.A., and Miller P.S., 1994, Bioavailability of zinc from inorganic and organic sources for pigs fed corn-soybean meal diets., J Anim Sci, 72:2681–2689. WHO., 1984, Trace-element bioavailability and interactions. In: Trace elements in human nutrition and health. WHO, Geneva. Williams S., 1984, ed. Official method of analysis of the AOAC. 14th ed. Arlington, VA; AOAC. World Bank. Enriching lives. Washington, 1994.

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TRACE ELEMENTS IN INTRAVENOUS NUTRITION

A. Shenkin Department of Clinical Chemistry University of Liverpool Liverpool, L69 3GA

1. ESSENTIAL TRACE ELEMENTS The provision of an adequate amount of essential inorganic micronutrients (trace elements) is an integral part of all regimens for intravenous nutrition (IVN). Many of the trace elements now recognised as being essential for human nutrition have been better characterised as a result of studies in patients depending totally upon their intravenous, or parenteral intake (that is total parenteral nutrition—TPN), especially for prolonged periods. The key features of an essential trace element are that its removal or inadequate supply in the diet is associated with reproducible structural or biochemical changes, and that these are reversible on provision of the element. This has been convincingly demonstrated in IVN for zinc, copper, selenium, iron, molybdenum, and chromium. In addition, there is good evidence of biochemical essentiality of iodine and of manganese, and the nutritional benefits of fluoride on bones and teeth. Cobolt is also recognised as being essential, although all requirements seem to be met by supply of vitamin alone. Individuals at Risk of Deficiency By the time a patient commences IVN, he may already have developed a whole body depletion of one or more essential nutrients. The extent of this will depend on a number of factors: The nutritional state of the patient on admission to hospital. The pre-existing illness may have caused a period of anorexia, or inadequate digestion or absorption of nutrients. The duration and severity of inadequate nutritional intake whilst in hospital, as a result of surgery or other treatment. Any increased losses through small bowel fistula/aspirate (rich in zinc), biliary fluid (rich in copper) or burn exudate fluid (rich in zinc/copper/selenium). Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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Moreover, some individuals will have an increased daily requirement, partly to keep up with increased losses, and partly to meet metabolic requirements—these are particularly important when patients become anabolic after a period of catabolism or when normal growth occurs in a child.

2. REVIEW OF TRACE ELEMENTS IN IVN A brief review of essential trace elements follows, in terms of (i) signs of deficiency, (ii) requirements intravenously, and (iii) tests used for monitoring and assessing status. 2.1. Zinc The best documented signs of zinc deficiency in IVN is the typical rash affecting face, flexures and perianal region. Moreover, zinc deficiency may cause diarrhoea, reduced weight gain, poor wound healing (Kay et al., 1975), and impaired immune function (Aggett, 1988; Solomons, 1998). The requirement for adult patients receiving IVN has been estimated to be at least per day (Wolman et al., 1979), but per day is frequently given to allow for increased requirements (Shenkin, 1988). Because of the effects of the acute phase response (APR), many patients receiving IVN have a low plasma zinc (Shenkin, 1995). Assessment and monitoring of patients with sepsis or following surgery is therefore particularly difficult. Interpretation of plasma zinc requires repeated measurements, not only of the plasma zinc itself, but also of albumin and C-reactive protein (CRP). In specialist laboratories, neutrophil zinc may provide a better indication of zinc status in septic patients (Goode et al., 1991). 2.2. Copper Copper deficiency has most frequently been found in children, although adult cases have also been documented. The main features are a hypochromic anaemia, sometimes with pancytopenia (Dunlap et al., 1974). In children, periosteal haematoma and painful bone disease may occur (Karpel and Peden, 1972). The requirement for adult patients during IVN has been variously suggested to be between 5 and per day (Shike, 1983; Shenkin, 1988). Although theoretically a dose of per day may seem high in the long-term, most patients receiving home IVN do not receive their supplements 7 days a week (more frequently 3–5 days per week) and hence the risk of excess supply is reduced (Malone et al., 1989). The APR causes an increase in plasma copper due to increased caeruloplasmin synthesis and hence the failure to see an expected rise in serum copper following trauma or in sepsis, or low serum copper in a stable patient may be evidence of an inadequate supply. 2.3. Selenium In adults, selenium deficiency in IVN has been associated with painful skeletal myopathy (Van Rij et al., 1979) or cardiomyopathy (Johnson et al., 1981), and in children also with pseudoalbunism and microcytosis (Vinton et al., 1988). There may also be nail changes with whitening of the nail bed. Despite a high prevalence of biochemical selenium deficiency during IVN lacking in selenium, in only a small proportion of

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cases is this associated with any of the above signs or symptoms (Shenkin et al., 1986). This adds further weight to the argument that some other agent, such as a viral infection, is necessary to induce symptoms. A wide-range of intakes has been used intravenously, and most studies would suggest that about selenium/day intravenously is adequate to maintain selenium status in patients with basal requirements, but a higher intake such as selenium per day may be necessary in some patients (Malone et al., 1989). Plasma selenium falls in an acute phase reaction (Nicholl et al., 1998), but nonetheless it is usually a good marker of adequacy of intake. RBC or platelet glutathione peroxidase gives a better marker of body stores. 2.4. Chromium There have been a few cases reported of chromium deficiency, characterised by glucose intolerance, impaired nitrogen balance, and peripheral neuropathy (Jeejeebhoy et al., 1977). Chromium requirements have been estimated to be approximately per day intravenously. However, IVN solutions contain a variable amount of chromium as contaminant, and hence it is very rare to find clinical deficiency—indeed, most patients receiving IVN have plasma levels above the reference range. (Moukarzel et al., 1992; Shenkin et al., 1986) This IV estimate may therefore be high. Provided renal function is normal, this excess chromium is excreted in urine, and there have been no reports of harmful effects resulting from this overprovision. Chromium status can be monitored using plasma chromium, provided care is used in collection of blood samples through a plastic catheter rather than a metal needle which would contaminate the specimen. 2.5. Molybdenum There has been only one well-documented case of molybdenum deficiency, characterised by an intolerance to sulphur containing amino acids in the IVN regimen (Abumrad et al., 1981). The main symptoms induced by these amino acids were tachycardia, flushing, and visual disturbance. The estimated IV requirement is per day, based upon the normal oral intake. There have however been no studies of different intravenous doses. Molybdenum status can be monitored by activity of the molybdenum containing enzymes, xanthine oxidase and sulphite oxidase—molybdenum deficiency was associated with high urinary hypoxanthine and sulphite levels. 2.6. Manganese There have been no well-documented cases of manganese deficiency during IVN. The best study of a controlled manganese deficient oral diet led to development of anaemia and hypercholesterolaemia (Friedman et al., 1987). Manganese requirements in IVN appear to be low, being sufficient to maintain plasma and whole blood manganese content (Malone et al., 1989) and lower amounts of have also been recommended (National Advisory Group, 1998). Care must be taken in patients with cholestatic liver disease since manganese is usually excreted in the bile.

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Manganese status is best assessed by whole blood or RBC manganese, where the concentration is much higher than in plasma, and hence contamination during collection is less of a problem (Taylor et al., 1996). 2.7. Iron Iron deficiency anaemia during IVN is a common occurrence because of the difficulty of maintaining an adequate intravenous supply. The minimum requirement is about per day, but higher amounts are required in those with additional blood loss or who have frequent venepunctures. Since provision of iron may lead to compatibility problems within the nutritional formulation, provision of iron by intermittent iron infusion or by blood transfusion may be necessary. Iron status is best measured by bone marrow stainable iron, but in practice serum ferritin together with an estimate of the acute phase response may give an indication of iron status, or the newly introduced test of soluble transferrin receptor may prove to be helpful (Ferguson et al., 1992).

3. CURRENT CONTROVERSIES IN TRACE ELEMENTS IN IVN Optimisation of Intake A number of preparations designed for IVN are now available. These have largely been formulated with the aim of preventing deficiency states. Given the current knowledge of trace element requirements, provided such a preparation is available (there are still some countries where no suitable preparation is licensed), daily provision should prevent clinical deficiency states from developing. However, prevention of deficiency is no longer seen as the best end point for assessment of adequacy (Gaby et al., 1991), since micronutrients can be related to many aspects of tissue function. Studies are now being undertaken to determine the functional benefit of different intakes. In particular, some studies are addressing the relationship between micronutrient status, immune function and outcome. Two important studies have recently been published:

a) Berger and co-workers (1998) reported the results of a prospective randomised double-blind clinical trial of zinc, copper, and selenium supplements in patients with severe burns, the supplement being provided intravenously for the first 8 days. The amount of supplement was designed to replace measured losses of these trace elements, primarily in the burn exudate. Supplementation was associated with reduced incidence of pulmonary infection over the 30 day period following burn. b) Young and co-workers (1996) performed a prospective trial on adult patients with blunt head injury, and evaluated the effect of either or of intravenous elemental zinc per day, for the first 15 days after injury. Thereafter the supplemented group received 22 mg elemental zinc as zinc gluconate by the oral route. In the supplemented group, the mortality rate was 4 out of 33, whereas in the non-supplemented group the mortality rate was 9 out of 35. The mechanism for this beneficial effect is not clear.

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Over-Provision and Toxicity Over-provision of trace elements can occur either as a result of contamination of IV nutrients or by supplementation with an excess amount. The most disturbing contaminant has been aluminium, initially as part of protein hydrolysates, but subsequently particularly in calcium and phosphate supplements (Koo et al., 1986). Excess provision has been associated with a painful metabolic bone disease (Klein, 1998). Most modern solutions should now contain acceptable levels of aluminium. For many years, manganese was provided intravenously at a dose of approximately per day, and no obvious harmful effects were found. However, since this caused abnormally high blood manganese concentrations (Shenkin et al., 1986), more recent preparations usually contain per day. However, some individuals still receive higher amounts, and this has been identified as a cause of neurological disturbances in a few cases, with Parkinson-like condition, and also associated with manganese deposition in the brain. This has been seen in both children (Fell et al., 1996) and adults (Reynolds et al., 1998). One intriguing controversy is the extent to which high manganese levels may promote cholestasis, in addition to the well recognised effect of cholestasis reducing manganese excretion.

Stability of Trace Elements in Parenteral Nutrition In general, the trace elements are fairly stable, but some examples of instability are recognised. For example some amino acid formulations contain cysteine, and this can partially degrade during sterilisation to produce small amounts of hydrogen sulphide. After addition of trace elements including copper, insoluble copper sulphide precipitates. The precipitate is more likely in multilayered bags which are less gas permeable (Allwood et al., 1998). Iron phosphate precipitates have also been reported in high phosphate containing regimens, after storage for periods of greater than a few days. This precipitate can be prevented by formulating mixtures with vitamins in multi-layered bags (Allwood et al., 1998). The reduction of selenite to elemental selenium by high concentrations of ascorbic acid has been suggested, but this is unlikely to occur in clinical practice since it only occurs in acid solution (Ganther and Kraus, 1989). Most of these minor incompatibilities can be prevented by delaying addition of these trace elements until immediately before the final mixing of the bag and its infusion into the patient.

4. SUMMARY The regular infusion of trace element mixtures containing zinc, copper, selenium, chromium, and manganese, should mean that clinically obvious trace element deficiencies are now rare events. The main focus of current research is to determine those clinical situations where patients might benefit from a higher or lower level of supply, either by more sensitive and specific methods of biochemical assessment, or by new methods of assessing tissue function and outcome.

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REFERENCES Abumrad, N.N., Schneider, A.J., Steel, D., and Rogers, L.S., 1981, Amino acid intolerance reversed by molybdate therapy. Am. J. Clin. Nutr. 34:2551–2559. Aggett, P.J., 1989, Severe zinc deficiency, in: Zinc in Human Biology, (C.F. Mills, ed), pp. 259–279, Springer Verlag, London. Allwood, M.C., Martin, H.M., and Greenwood, M., et al., 1998, Precipitation in trace elements in parenteral nutrition mixtures. Clin. Nutr. 17:223–226. Bates, C.G., Greine, G., and Gegenheimer A., 1984, Precipitate in admixtures of new amino acid injection. Am. J. Hosp. Pharm. 41:1316. Berger, M.M., Spertini, R, Shenkin, A., Wardle, C., Wiesner, L., Schindler, C., and Chiolero, R.L., 1998, Trace element supplementation modulates pulmonary infection rates after major burns: a double-blind, placebo-controlled trial. Am. J. Clin. Nutr. 68:365–371. Dunlap, W.M., James, G.W., and Hume, D.M., 1974, Anaemia and neutropenia caused by copper deficiency. Ann. Intern. Med. 80:470–176. Ferguson, B.J., Skikne, B.S., Simpson, K.M., Baynes, R.D., and Cook, J.D., 1992, Serum transferrin receptor distinguishes the anaemia of chronic disease from iron deficiency anaemia. J. Lab. Clin. Med. 119: 385–390. Friedman, B.J., Freeland-Graves, J.H., and Bales, C.W., et al, 1987, Manganese balance and clinical observations in young men fed a manganese deficient diet. J. Nutr. 117:133–143. Gaby, S.K., Bendich, A., Singh, V.N., and Machlin, L.J., 1991, Vitamin Intake and Health—A Scientific Review. pp. 1–217, Marcel Dekker, New York. Ganther, H.E. and Kraus, R.J., 1989, Chemical stability of selenious acid in total parenteral nutrition solutions containing ascorbic acid. JPEN. 13:185–188. Goode, H.F., Kelleher, J., and Walker, B.E., 1991, The effects of acute infection on indices of zinc status. Clin. Nutr. 10:55–59. Jeejeebhoy, K.N., Chu, R.C., and Marliss, E.B., et al., 1977, Neuropathy reversed by chromium supplementation in a patient receiving long-term parenteral nutrition. Am. J. Clin. Nutr. 30:531–538. Johnson, R.A., Baker, S.S., and Fallon, J.T., et al., 1981, An occidental case of cardiomyopathy and selenium deficiency. New. Engl. J. Med. 304:1210–1212. Karpel, IT. and Peden, V.H., 1972, Copper deficiency in long-term parenteral nutrition. J. Pediat. 80:32– 36. Kay, R.G., Tasman-Jones, C., and Pybus, J., et al, 1976, A syndrome of acute zinc deficiency during total parenteral alimentation in man. Ann. Surg. 183:331–340. Klein, G.L., 1998, Metabolic bone disease of total parenteral nutrition. Nutrition. 14:149–152. Koo, W.W., Kaplan, L.A., Horn, J., Tsang, R.C., and Steichen, J.J., 1986, Aluminium in parenteral Nutrition solution—sources and possible alternatives. JPEN. 10:591–595. Malone, M., Shenkin, A., Fell, G.S., and Irving, M.H., 1989, Evaluation of a trace element preparation in patients receiving home intravenous nutrition. Clin. Nutr. 8:307–312. Moukarzel, A.A., Song, M.K., Buchman, A.L., Vargas, J., Guss, W., McDiarmid, S., Reyen, L., and Ament, M.E., 1992, Excessive chromium intake in children receiving total parenteral nutrition. Lancet. 339:385–388. National Advisory Group on Standards and Practice Guidelines for Parenteral Nutrition, 1998, Safe Practices for Parenteral Nutrition Formulations. JPEN. 22:49–61. Nicholl, C., Herdman, J., Sattar, N, O’Dwyer, P.J., O’Reilly, D. St. J., Littlejohn, D., and Fell, G.S., 1998, Changes in the concentration of plasma selenium and selenoproteins after minor elective surgery: further evidence for a negative acute phase response. Clin. Chem. SS44 8:1764–1766. Reynolds, N., Blumsohn, A., Baxter, J.P., Houston, G., and Pennington, C.R., 1998, Manganese requirement and toxicity in patients on home parenteral nutrition. Clinical Nutrition. 17(5):227–230. Shenkin, A., 1988, Clinical aspects of vitamin and trace element metabolism. Baillieres. Clin. Gastroenter. 2:765–798. Shenkin, A., 1995, Trace elements and inflammatory response: implications for nutritional support. Nutrition. 11:100–105. Shenkin, A., Fell, G.S., Halls, D.J., Dunbar, P.M., Holbrook, I.B., and Irving, M.H., 1986, Essential trace element provision to patients receiving home intravenous nutrition in the United Kingdom. Clin. Nutr. 5:91–97. Shike, M., Roulet, M., Kurian, R., Whitwell, J., Stewart, S., and Jeejeebhoy, K.N., 1981, Copper metabolism and requirements in total parenteral nutrition. Gastroenterology. 81:290–297.

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Solomons, N.W., 1998, Mild human zinc deficiency produces an imbalance between cell mediated and humoral immunity. Nutr. Rev. 56:27–28. Taylor, A., Branch, S., Halls, D.J., Owen, L.M.W., and White, M., 1998, Atomic spectrometry update— clinical and biological materials, food and beverages. J. Anal. Atom. Spectrum. 13:57R–106R. Van Rij, A.M., Thompson, C.D., McKenzie, J.M., and Robinson, M.F., 1979, Selenium deficiency in total par– enteral nutrition. Am. J. Clin. Nutr. 32:2076–2085. Vinton, N., Dahlstrom, K., Strobel, C, and Ament, M. 1988, Macrocytosis and pseudoalbinism: manifestations of selenium deficiency. J. Paediat. 111:711–717. Wolman, S.L., Anderson, G.H., Marliss, E.B., and Jeejeebhoy, K.N., 1979, Zinc in total parenteral nutrition: requirements and metabolic effects. Gastroenterology. 76:458–467. Young, B., Ott, L., Kasarskis, E., Rapp, R., Moles, K., and Dempsey, R.J., et al, 1996, supplementation is associated with improved neurologic recovery rate and visceral protein levels of patients with severe closed head injury. J. Neurotrauma. 13:25–34.

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TRACE ELEMENT INTAKE AND BALANCE IN ADULTS IN CENTRAL EUROPE

Anke, M., Glei, M., Dorn, W., Müller, R.*, Vormann, J.**, Müller, M., Jahritz, M., Seifert, M., Holzinger, S., Drobner, S., Röhrig, B.,

Rother, C., Angelow, L., and Latunde-Dada, G. O. Friedrich Schiller University Institute of Nutrition D-07743 Jena, Dornburger Str. 24 *Society of Ecology and Environmental Chemistry Ltd Hohenwindenstr. 13, D-99086 Erfurt **Protina Pharmazeut Ltd D-8573 Ismaning Germany

1. INTRODUCTION In the 1980s and 1990s, the sufficient trace element supply of humans was not guaranteed everywhere and for all persons all over the world. At the beginning of the 1980s, the intake of the ultratrace elements lead, aluminium, cadmium and mercury reached amounts which were dangerous to health locally or in individual groups of the population whereas there were no data on the intake of other elements (e.g. uranium, barium, vanadium, rubidium) in Central Europe. Therefore, the trace element supply of adults was investigated with the help of the duplicate method in order to register deficiencies or exposures and to prevent impairment of health. In these investigations, particular attention was paid to time (1988, 1992, 1996 with a local or international offer of foodstuffs), sex, age, body weight, performance (nursing), season and eating habits.

2. MATERIAL AND METHODS 21 test populations from Germany (19) and Mexico (2) were available for the investigations. They consisted of at least 7 women and 7 men at the age of between 20 and 70 years. If possible, 10 women and 10 men should participate per study and 2 women and 2 men per decade in order to register the influence of age reliably. The test persons Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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collected the duplicates of all consumed foods, beverages and sweets on 7 consecutive days. All of them were asked not to change their eating habits. The test persons kept a daily record of the consumed foods which allowed the calculation of the mineral intake (basket method) and thus, the comparison of this method with the findings of the duplicate method. In 1992 and 1996, the excretions in the form of urine and faeces and—in 2 populations the milk as well—were collected every day and analyzed. The analysis of the trace elements was carried out after dry ashing at 450 °C and the dissolution of the ashes in 2.5% HCl. Special disintegration procedures were necessary for the determination of iodine, selenium, arsenic and mercury. Iron, zinc, manganese, copper, molybdenum, strontium, barium, titanium, chromium and vanadium were measured with ICP-OES, uranium with ICP-MS, iodine according to the Sandell-Kolthoff procedure, selenium and arsenic with AAS hydride technique, lithium and rubidium with AAS and the standard addition procedure and mercury with the atom absorption cold vapour method and nickel, lead and cadmium with flameless AAS.

3. RESULTS OF THE INVESTIGATIONS Energy, Nutrient and Ash Intake of Adults The trace and ultratrace element intake of humans is varied by the extent of the food and drink consumption, sex, season, physical strain and performances (pregnancy, nursing period) and the form of diet. The reunification of Germany and the consequent complete change of the food and beverage offer due to the transition from the local production and marketing to an international offer in the supermarket did not change the quantity of the dry matter consumption. Women and men consumed 2 to 4% less dry matter in 1996 than in 1988. Women with mixed diets eat 300g dry matter/day and men 380 g on the average of the week. Vegetarians (ovo-lacto vegetarians) had a mean higher dry matter consumption of 29 and 27% than people with mixed diets. The energy concentration in the dry matter consumed by vegetarians is lower and forces them to a more extensive consumption. The energy, protein, fat and carbohydrate intake does not differ between people with mixed diets and vegetarians. Vegetarians of both sexes eat about 30% more bulk material and 38% more ashes than people with mixed diets. The vegetarians’ higher consumption of inorganic food components is important for the trace and ultratrace element supply although the macro elements (Na, K, Ca, Mg, P, S, Cl) form 99% of the consumed ashes. Trace Element Intake of Adults In the case of an Fe bioavailability of 10%, the individual basic Fe requirement of adults is indicated by the WHO (Anonymous 1996) with 8mg/day for young women, 7mg/day for nursing mothers, 6.5mg/day for elderly women and 6mg/day for. The iron intake decreased continuously and significantly between 1988 and 1996. An iron offer of 6 to 7 mg/day meets the requirement of young women and that of 7 mg/day the requirement of nursing women. The supplementation of 10mgFe/day did not improve the iron balance. The faecal iron excretion increased by the supplemented Fe amount. The iron intake of young women must be paid much attention to since it can individually amount to <6mg, the iron offer may be subjected to a further decrease via foodstuffs and although

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vegetarians take in 60% more iron than people with mixed diets, the bioavailability of this iron is poor (Table 1). The zinc consumption of people with mixed diets also decreased significantly and steadily between 1988 and 1996 without reaching the individual basic requirement of women of 4mg/day and that of men of 5mg/day when the bioavailability of zinc in the foodstuffs is moderate. However, the zinc intake of 7mg/day in women and of 9mg/day in men recommended by the WHO is not reached. In a placebo-controlled double blind study, the supplementation of l0mgZn/day did not improve the zinc balance of women with 6.5mgZn/day. The supplemented zinc left the body via faeces. The zinc content in the blood serum and the superoxide dismutase content in the full blood did not change due to the zinc supplementation (Anke et al., 1999). Vegetarians took in one third more zinc than people with mixed diets in Germany in 1996. The balance experiments, however, show that, due to the high phytin content in the consumed food, the bioavailability of the zinc taken in by them is much worse. They excreted renally significantly less zinc than people with mixed diets. Compared to the duplicate method, the calculation of zinc—just like the trace element intake—(basket method) overestimates the zinc intake of adults highly significantly. It postulates a zinc intake which is 40% too high. The zinc supply in all forms of diets must be paid attention to. On the average of the week, 10% of women with mixed diets and 3% of men in Germany do not meet their zinc requirements of 4 and 5 mg/day. Both sexes did not reach the recommended zinc intake of 7 and 9mg/day in 1996 as well. Contrary to iron and zinc, the copper supply improved in Germany between 1988 and 1996. The individual basic requirement of women of 0.6mg/day and of men of 0.7mg/day was met in any case in 1996 and the recommendations of the WHO of 1.0 and 1.2mgCu/day were met (Anonymous 1996). The iodine supply of adults improved via the higher iodine content in eggs and milk due to the iodination of the mineral mixtures of farm animals with l0mgI/kg and via the supplementation ofcooking salt for individual and commercial cooking, without reaching the I consumption of registered in Mexico. The individual basic iodine requirement of body weight is met in Germany in 1999 and the recommended intake of will soon be reached. The stru-uma of babies has disappeared. The iodine content in mother’s milk in the 5th week of nursing reached almost It decreases insignificantly during the nursing period. In the case of an iodine intake of 100 or on the average of the week, the supplementation of to young, pregnant and nursing women did not take effect either on their TSH level nor on their

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concentration. However, the I offer did not normalize their free content of tri-iodothyronine in the blood serum. This was only possible after the administration of in a placebo-controlled double blind study. The thyroxin deiodinase—1 represents a selenium protein which contains an atom Se per molecule in its centre. In the case of a mean Se intake of this seleno-protein is no longer formed in sufficient amounts in women. The selenium supply of people with mixed diets (and vegetarians) improved by about 60% in Germany between 1988 and 1996. This does not mean that the individual basic selenium requirement of 16 and for women and men is reached in each individual person (Anonymous 1996). Women with a selenium intake of on the average of the week had a negative selenium balance. The individual selenium requirement is higher and should be increased to 20 and However, the intake of 12 to on the average of the week allowed a normal GSHPx production. The supplementation of did not improve their level. These persons apparently disintegrate selenium from body reserves and this allows a normal GSHPx production. The recommended selenium intake of 30 and was reached. However, this does not exclude individual selenium deficiency and can restrict the change of thyroxin into tri-iodothyronine. The individual basic requirement of 1–2 mg manganese/day, of 25 to nickel/day and of molybdenum/day of adults is met by nutrition (Table 1). Deficiency symptoms were not registered in these 3 trace elements. There is, however, the great danger of a nickel allergy in nickel-sensitive women. It occurs after the filling of the nickel depots. Ultratrace Element Intake of Adults The biological importance of ultratrace elements has grown within the last years since the essentiality of some of them (Anke et al., 1998a) has been discussed or their toxicity played a more important role. After the aluminium intoxications in dialysis patients, which were due to technological failures and which have been removed in the meantime, the importance of the aluminium intake has increased. The aluminium intake decreased between 1988 and 1996 by about 6mg/day to about 3mg/day, which is a satisfying trend. On the other hand, the danger of acidification and of the increased aluminium uptake of plants has not been banned yet. The game from acidified habitats has increased aluminium concentrations in the tissues. The strontium intake decreased from 1988 to 1996 continuously. There are, however, local “strontium exposures” via drinking water and home-made or locally produced beverages, which can be considerable. Rubidium and lithium, whose essentiality is no longer generally denied and whose contribution to the “well-being” and reproduction of several species is accepted, are also transferred to the food chain in very different amounts. Both elements may be essential (Anke and Angelow 1995a). On an average, their intake is high with <1.5mgRb/day and 700 to According to the experience made in Germany, a uranium exposure is most likely possible via drinking water and particularly via mineral water. Mineral waters in Germany contain between 25 and They contribute decisively to the uranium

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intake of humans and they can expose babies to uranium if they are used for the production of formulae. The barium intake (1.5mg/day) has been reduced to one third since 1988. Only <10% of this element are taken in via beverages. The arsenic intake of adults in Germany is subjected to extreme variations, as it is demonstrated by the considerable standard deviation, and varies between 21 and between the test populations. The cause of this high variation range has not yet been definitely clarified. The above-mentioned amounts of arsenic are harmless for humans. In all probability, arsenic belongs to the elements which are also esssential. A minimum intake of 12 to seems necessary for humans (Anke et al., 1987). Titanium is added as titanium oxide to cat’s and dog’s food in order to obtain a brighter colour. It was used in the food industry for the same reason. A titanium intake of 80 and is harmless for humans. It meets the titanium requirement of adults if such a requirement exists. The titanium intake still amounted to 207 and in 1988. The individual chromium requirement of humans amounts to and was met in all test persons. Chromium deficiency need not be reckoned with in Germany. Apart from nursing mothers, the chromium balance was positive. Chromium exposures may occur in chromium-sensitive men (chromium allergy). The lead, cadmium and mercury intake of humans has been reduced during the last 10 years. This is true for all 3 elements (Table 2). The drastic restrictions on lead and cadmium emissions and the ban on mercurycontaining fungicides show positive effects. This does not come true for the use of amalgam for tooth cement. Test persons with amalgam fillings excreted more mercury than they took in. This is also true for persons who had their amalgam fillings removed. They apparently excreted the amalgam accumulated in the body before over a longer period of time (Anke et al., 1998b, Müller 1993). In all probability, vanadium is essential. Adults need and men take in much higher amounts than women because several beverages and particularly beer contain much vanadium.

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SUMMARY The iron and zinc intake decreased continuously between 1988 and 1996 whereas the copper, iodine and selenium intake was improved. Like the zinc and iron intake, the selenium, iodine and copper offer must be individually improved. This is particularly true for selenium in which an intake of takes effect on the tri-iodothyronin level in the blood serum. Under these conditions, a selenium supplementation improved the iodine status. The manganese, nickel and molybdenum offer of humans met the requirements. Fortunately, the aluminium, strontium, barium, titanium, lead, cadmium and mercury intake of adults with mixed diets has decreased and is at a harmless level. The purchase in supermarkets overcomes remarkable differences. These elements can get into individual households via herbs and vegetables cultivated in house gardens. Some elements (strontium, rubidium, lithium, vanadium and uranium) are abundantly offered via water and home-made beverages. The geological origin of the habitat, technological processes and the amalgam in tooth cement take effect on the human intake of ultratrace elements.

REFERENCES Anke, M. and Angelow, L. 1995, Rubidium in the food chain. Fresenius J. Anal. Chem. 352, 236–239. Anke, M., Dorn, W., Gunstheimer, G., Arnhold, W., Glei, M., Anke, S., and Lösch, E. 1998a, Effects of trace and ultratrace elements on the reproduction performance of ruminants. Vet. Med., Czech. 43, 272–282. Anke, M., Glei, M., Arnhold, W., Müller, M., Schäfer, U., Seifert, M., Hartmann, E., and Gunstheimer, G. 1998b, Environmental Pollution and its health effects on people in Germany. In: International Symposium on Itai-itai Disease, Environmental Cadmium Pollution and Countermeasures, May 13–16, 1998b, Toyama, Japan, 36–37. Anke, M., Glei, M., Winnefeld, K., Arnhold, W., Vormann, J., Röhrig, B., Jaritz, M., Holzinger, S., LatundeData, O., and Hartmann, E. 1999, Supplementierung und Therapie mit Zink, in: Schriftenreihe der Gesellschaft für Mineralstoffe und Spurenelemente e. V., (Meiner, D. ed), pp. 98–109, Wiss. Verlagsgesellschaft mbH. Anke, M., Krause, U., and Groppel, B. 1987, The effect of arsenic deficiency on growth, reproduction, life expectancy and disease symptoms in animals, in: Trace Substances in Environmental Health, (Hemphill, D.D. ed), pp. 533–550, 21. University of Missouri, USA. Anonymous, World Health Organisation General 1996, Trace Elements in Human Nutrition and Health. WHO Geneva. Müller, M. 1993, Cadmiumaufnahme und Cadmiumausscheidung Erwachsener nach der Marktkorb- und Duplikatmethode. Diss. Friedrich-Schiller-Universität Jena, Biologische Fakultät, Deutschland.

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NUTRIENT RISK ASSESSMENT Implications for Food Fortification Policy1

Mary R. L’Abbé2, Kevin A. Cockell2, Sheila Dubois3, and William H. Ross3 1

Publication Number 523 of the Bureau of Nutritional Sciences Bureau of Nutritional Sciences, and 3 Bureau of Biostatistics and Computer Applications Food Directorate, Health Protection Branch Health Canada, Ottawa ON Canada K1A 0L2 2

1. INTRODUCTION Nutrient risk assessment (NRA) and food fortification are two separate activities, which are intimately linked when changes to food fortification policies are being considered. As consumers and others recognize that increasing the intake of some nutrients may provide health benefits beyond their traditional role in the prevention of nutrient deficiency diseases, the demand for and interest in fortified foods increases. When fortifying foods with vitamins and mineral nutrients, we must also be concerned with “How much is too much?” Nutrient Risk Assessment (NRA) is a model which can be used for many nutrient related activities, one of which can be evaluation of food fortification scenarios. Inherent in establishing food fortification policies is the need to determine how to target the fortification to those who need the intervention while avoiding potential adverse effects, such as excessive or imbalanced intakes in other groups and choosing between mandatory or voluntary fortification programs. Whatever program is instituted, NRA is an important step in determining the efficacy of a proposed action in meeting a public health objective while ensuring the safety of the food supply.

Research Centre, Ross Ave Ottawa, ON Canada K1A 0L2 telephone: 613-957-0924; fax: 613-941-6182; email: «Mary_L’[email protected]» Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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2. PUBLIC HEALTH BENEFITS OF FOOD FORTIFICATION Governments have policies concerning the addition of nutrients to foods for a number of reasons. They provide a uniform set of principles, which when applied, ensure a rational and consistent approach to the addition of nutrients to foods. Food fortification policies can be used to achieve the public health objectives of maintaining and improving the over-all nutritional health of the population, improving the nutritional quality of the national food supply, and of addressing the nutritional needs of specific population sub-groups. Their application can avoid uncoordinated nutrient addition which could lead to the coexistence of excessive and inadequate intakes in the population and create nutrient imbalances for some individuals. Under the Codex General Principles for the Addition of Essential Nutrients to Foods (Codex Alimentarius, 1994), the basic principles or rationales for addition of vitamins and minerals to foods are related to specific public health benefits. These have been summarized in Table 1. In the Dietary Reference Intakes (DRIs) reports on calcium and related nutrients (National Academy of Sciences, 1997) and on the B vitamins (National Academy of Sciences, 1998), the basic definition of nutrient adequacy was expanded to include specific indicators or biomarkers related to reduction in the risk of chronic degenerative diseases or disorders. This is in contrast with earlier definitions of adequacy which were usually limited to the prevention of classical nutritional deficiency diseases or deficient states. To meet the challenges posed by the widening definition of nutrient requirements, food fortification policies need to be developed which recognize and address these broader public health objectives. Additional food fortification approaches could include specific initiatives which would: Improve the nutritional quality of the food supply to meet the widened definition of nutrient adequacy, as established in the recent DRIs, rather than simply to prevent deficiencies Increase the nutrient density of foods targeted to particular groups or sub-groups (i.e. a wider variety of foods for infants, seniors etc.), and/or Increase the variety of food products which are good sources of particular nutrients. There are other potential reasons for fortifying foods, which may have little demonstrable public health benefit but may provide other benefits to the food industry or consumers. Examples of such reasons could include fortifying foods to provide an alternative form of vitamin and mineral supplements, using food fortification as a marketing feature to meet consumer demands or “perceived” needs, to address nutrient fads, to enable foods to make a “health claim”, or using food fortification to encourage food industry innovation or to enhance trade opportunities.

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3. NUTRIENT RISK ASSESSMENT In the context of Health Canada’s review of its policies on food fortification, the goals of Nutrient Risk Assessment (NRA) are two-fold: (a) to identify options that permit greater flexibility in the addition of nutrients to foods, and (b) to ensure at the same time that food consumption patterns do not result in nutrient intake levels that are excessive, imbalanced or potentially hazardous to health. A NRA might be initiated to assess the potential risks associated with a proposed fortification plan or to assess the risks associated with high levels of exposure created though a potential fortification error. Since a specific risk assessment framework for reviewing scientific evidence on health risks associated with non-deficient levels of nutrient intakes did not exist, an ad-hoc working group was established in the Food Directorate at Health Canada to examine this question. The HPB Model of Risk Determination (Health Canada, 1993), which is usually applied to establishing safe intakes of other substances such as environmental contaminants or Pharmaceuticals, was adapted for this purpose. An outline of the four-step Nutrient Risk Assessment Model (L’Abbé et al., 1997) is shown in Fig. 1. The steps include hazard identification, hazard characterization, exposure evaluation and risk characterization. Hazard characterization, exposure evaluation and risk characterization together are also referred to as risk estimation. A similar approach has been used by the Food and Nutrition Board of the National Academy of Sciences in their nutrient risk assessments which were used for determining tolerable upper intake levels (ULs) in the DRI reports (National Academy of Sciences, 1998). 3.1. Hazard Identification Hazard Identification includes the search for and identification of all potential adverse effects of a nutrient through a systematic and thorough review of the literature.

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These can be associated with direct effects attributable to the nutrient under consideration or indirect effects mediated through an interaction with other nutrients, drugs or food components. In the NRA model developed at Health Canada (L’Abbé et al., 1997), potential adverse effects under consideration were then prioritized according to the following criteria: (a) the weight of the evidence that the adverse biological effect was causally associated with the nutrient, (b) the burden of illness, which includes (i) the prevalence or size of the population affected or at risk and (ii) the severity of the adverse biological effect, (c) public perceptions of risk, which are influenced by the context in which they occur and may be quite different from actual risk, and (d) consideration that some adverse effects may be related to more than one causative agent or dietary factor, and that modification of the other factor may be the preferred approach for other public health reasons. 3.2. Hazard Characterization The second phase of Nutritional Risk Assessment is Hazard Characterization which can also be referred to as dose-response modelling. It involves quantitation of the probability of occurrence of the potential adverse health effect identified during Hazard Identification across a range of nutrient exposure levels. Hazard characterization can sometimes involve statistical modelling to determine the dose level at which the adverse effect is likely to occur. During hazard characterization, one must first ascertain that at high intake levels, the nutrient under question is actually being absorbed, so that there would be an increased “internal exposure” level. 3.3. Exposure Evaluation Exposure Evaluation involves development of nutrient exposure profiles for the total population as well as for the population subgroups identified as “at risk” during the hazard identification phase. Exposure evaluations should include all oral sources of the nutrient under assessment including ingestion in foods (as naturally-occurring nutrients, additives or as fortificants), from supplements or from other over-the-counter drugs. Exposure profiles should include data on the proportion of the population that is exposed, and the level of exposure among those exposed, by appropriate age and sex groups where possible. In evaluating population exposures, current intake (exposure) levels and the effects of permitting the addition of a specific nutrient to a specific food product(s) and/or food groups, singly or in combination, versus unrestricted addition of the nutrient to the food supply should determined. NRA must consider the total food supply rather than an assessment of fortification of individual foods. 3.4. Risk Characterization Risk characterization, the final phase of nutrient risk assessment, combines the data from hazard characterization and exposure evaluation. Risk characterization involves determining the margin of safety between the requirement level and the UL, the likelihood and severity of the adverse effect at the population level, and the exposure characteristics of the population, as described above, under the different test fortification scenarios. As an example, for calcium which has a narrow margin of safety between requirement and UL, fortification of a single or a few food products would be unlikely to increase intakes above the UL, while unrestricted addition of calcium to many foods

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may readily do so, particularly in some groups such as adolescent males who may consume large amounts of many food products. A risk evaluation of food fortification with folate has also been completed (McCourt et al., 1998).

4. EVALUATING PUBLIC HEALTH EFFECTIVENESS OF FOOD FORTIFICATION Determining how well the desired public health benefit can be achieved for the target group while minimizing risks to other population groups is the primary goal of any public health agency. Food fortification programs can be evaluated according to several criteria related to their effectiveness in meeting public health objectives. National food fortification policies and regulations should be consistent with national public health objectives, priorities and education programs. Foods which are fortified should be consumed by the target group expected to obtain the public health benefit. The intake of fortified foods should be relatively stable and uniform, and the level of added nutrient should be in sufficient quantity in a food, such that the food may be considered to be a significant source of the nutrient (usually at least 5–10% of the RDA per serving). Finally, fortification policies should minimize adverse effects in other groups who are not expected to derive the benefits of the fortification, which underscores the importance of NRA in establishing food fortification policies.

REFERENCES Codex Alimentarius, 1994, Joint FAO/WHO Food Standards Programme, Codex Alimentarius Commission, Volume 4, Rome, p. 9. Health Canada, Health Protection Branch, 1993. Health Risk Determination: The challenge of Health Protection. L’Abbé, M.R., Cockell, K.A., Dubois, S., and Ross, W.H., 1997, Calcium Risk Assessment Parts I and II: Hazard Identification and Hazard Characterization, Food Directorate, Health Protection Branch, Health Canada. Executive Summary is available on the Health Canada website at: «www.hc-sc.gc.calfood-aliment/english/subject_area/nutrition/executive_summary_risk.html» McCourt, C., Turner, L., Lee, N., and Junkins, B., 1998, Risk Evaluation of Fortification Measures to Respond to the Evidence for a Protective Effect of Folate Against Neural Tube Defects, Food Directorate, Health Protection Branch, Health Canada. National Academy of Sciences, Institute of Medicine, Food and Nutrition Board, Standing Committee on the Scientific Evaluation of Dietary Reference Intakes. Dietary reference intakes for calcium, phosphorus, magnesium, vitamin D, and fluoride. Washington DC: National Academy Press; 1997. National Academy of Sciences, Institute of Medicine, Food and Nutrition Board, Standing Committee on the Scientific Evaluation of Dietary Reference Intakes. Dietary reference intakes for thiamin, riboflavin, niacin, vitamin B6, folate, vitamin B12, pantothenic acid, biotin, and choline. Washington DC: National Academy Press; 1998. National Academy of Sciences, Institute of Medicine, Food and Nutrition Board, Subcommittee on Upper Reference Levels of Nutrients. A risk assessment model for establishing upper intake levels for nutrients. Washington DC: National Academy Press; 1998.

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VANADIUM—AN ESSENTIAL ELEMENT FOR ANIMALS AND HUMANS?

Anke, M., Illing-Günther, H., Gürtler, H.*, Holzinger, S., Jaritz, M., Anke, S.*, and Schäfer, U. Friedrich Schiller University Jena

Institute of Nutritional Dornburger Str. 24, D-07743 Jena *University of Leipzig D-04123 Leipzig, Germany Institute of Veterinary-Physiological Chemistry Semmelweisstr. 4

The vanadium content (V) of the 16-km thick earth’s crust is calculated differently—l00mg/kg on an average. In spite of its 22nd rank on the frequency list, its biological essentiality was not paid much scientific attention to. Therefore, the V transfer in the food chain from soil via flora and fauna to humans and its essentiality were systematically investigated.

MATERIAL AND METHODS After the dry ashing of the biological material at 450 °C in 10 to 30 g dry matter or 400ml fluid, the determination of V in the investigated 7,845 samples was carried out by means of ICP-OES (Spectroflame D, Spectro-Analytical Instruments) and the line position of 292,402. The precision of the V measurement was checked with the reference material IAEA H-9 Mixed Human Diet.

RESULTS Essentiality of Vanadium for the Flora The geological origin of the material for soil formation and, thus, the natural or anthropogenic V offer influences the V content of the flora depending on species, age and parts of plants. The effect of the origin of the soil on the V content was investigated Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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with the help of rye, wheat, acre red clover and meadow red clover when rye was in blossom. The region with the highest vanadium content in plants was equated with 100 and the other regions were related to it. The soils of the lower strata of new red sandstone, loess and granite produced a V-rich vegetation, boulder clay, shell lime weathering soils, keuper weathering soils, new red sandstone weathering soils and phyllite weathering soils deliver average V amounts into the flora. Those of diluvial sands, gneiss, moors and alluvial riversides (60) are significantly V poorer. The V content in the individual plant species from the same region correlated with r 0.75 to 0.90 and showed the suitability of the widespread indicator plants. Anthropogenic V emissions are indicated even years after the closure of the V source as it was demonstrated in the vicinity of a cement and phosphate enterprise. The V content in the investigated species decreased significantly with increasing age. The V proportion of the flora decreased to a third of the initial value from the beginning of May to the middle of June. The V concentrations in several plant species are varied by their leaf—stalk ratio. Leaves stored the main V amount. Bulges of stalks and roots or tubers accumulated significantly less V. Seeds and fruit proved to be particularly V-poor. They deliver <10% of the V amount registered in leafy vegetables. The perennial winter grazing of game proved to be extremely V-rich. The essentiality of V for highly developed plants is debatable whereas it seems to be essential for algae because V supplementation increased their biomass production. The enzyme bromium peroxidase of brown sea algae and the chlorine peroxidase of the mushroom Curvularia inaequalis need V for activation. Vanadium Content in Plant Foodstuffs Seeds, cereal products, bread, cakes and pastries, tubers and fruit generally have a low V content (5 to 25 and dry matter, resp.). Mushrooms and leafy vegetables as well as herbs contain much higher levels of V (100 to dry matter). The V content in 112 foodstuffs of plant origin (n 1352) and 49 comparable ones (n 196) from ecological production showed that—as a rule—ecowheat flour, ecobread, ecofruit and ecovegetables delivered less V than conventionally produced ones. The missing phosphate fertilization which delivers V to the plants becomes noticeable in the ecoproducts. In contrast to this fact, ecologically produced sugar contained more V than conventionally refined sugar. The use of this sugar delivers much V to several kinds of pasta, bread, cakes and pastries. Therefore, ecologically produced chocolate and sweets are V-richer than conventionally produced ones. The Essentiality of Vanadium for the Fauna and Humans The essentiality of V for the fauna was investigated in goats, rats and hens (Anke et al., 1989, Nielsen and Uthus 1990). In goats, V levels of feed dry matter reduced feed intake, growth, reproduction performance, the protein and fat concentration of milk, and life expectancy highly significantly after repeated intrauterine V depletion. Skeleton damage occurred, the creatinine and triglyceride concentrations and the y-glutamyl transferase activity in blood plasma were significantly reduced. The V concentration in most organs, tissues and the milk of goats is not homeostatically regulated. This is the case for all animal species investigated (mice, voles, shrews, wild boars, fallow deer, moufflons, red and roe deer.

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In humans, the V content in the liver was not affected by age (from birth till >80 years) or sex. Its V content was in correspondence with that of the investigated animal species. However, from babies and toddlers to >80-year-old people, the V content in kidneys, prostate gland and rib decreased significantly to 5–30% of the amount registered in babies. Both sexes showed V depletion by the time of the onset of puberty (Table 1). Vanadium Content in Animal Foodstuffs and Beverages Like the milk of other species, mother’s milk does not contain much V. Therefore, babies mainly rely on intrauterine V deposits. The different formulae for the nutrition of babies offer between 4 and dry matter. Like cow’s milk, most formulae deliver dry matter. Mother’s milk contained more V with dry matter than cow’s milk in 1996. The V content in mother’s milk should be subjected to further investigations in order to prepare baby food according to physiological principles. Depending on the species, meat and fish contain little V (20 to dry matter). The V content in hen’s eggs varied by the 42fold between 1988 and 1996. The cause of this V accumulation in hen’s eggs may be the high V content in the calcium carbonate fed to hens in 1988 or the V emission when heating oil is burnt. The V amount accumulated in eggs does not take adverse effects on human health. Ecologically produced animal foodstuffs can contain significantly less V than conventionally produced ones. Beverages deliver most of the vanadium taken in. The main V supplier is beer which contained on the average of different brands and origins. Vanadium Intake of Humans After V-deficiency experiments with <10 to ration dry matter over 15 generations in goats had led to reduced feed intake and growth, to worse success of the first insemination and a worse conception rate with an increased abortion rate, to a sex ratio shifted to the female side, skeleton damage and reduced life expectancy, it cannot

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be excluded that minimum V amounts are also essential for humans. The V-poor nutrition of rats increased the weight of their thyroid gland. It was derived from these findings that a V-depending halogen peroxidase may exist in mammals as well (Nielsen and Uthus 1990). The V intake was investigated with the duplicate method in 19 test populations from Germany and Mexico. A total number of 139 women and 125 men at the age of between 20 and 69 years collected duplicates of all consumed foodstuffs, sweets and beverages on 7 consecutive days, i.e. 1848 duplicate samples were available. The postulated V requirement of adults of is met by foodstuffs (Table 2). On average, men take in double the V amount ingested by women. The higher intake results from the higher beer consumption of men The region took a significant effect on the V intake. This may be due to the different V offer in the drinking water. The V intake decreases with increasing age. Nursing mothers with mixed diets take in less V than other women. Independent of the type of diet, 94 to 98% of V are excreted via faeces and 6% via urine. Nursing mothers excreted 4% of V via urine, 16% via milk and 80% via faeces. Their V balance was negative whereas that of people with mixed diets was balanced and that of vegetarians slightly positive (Table 3).

SUMMARY According to the present level of knowledge and a given essentiality of this ultratrace element, V deficiency in flora, fauna and man need not be reckoned with in practice. It seems that there is no danger of a V overload via foodstuffs in Central Europe. However, the V emissions due to burning V-rich fossile fuels must be taken into consideration internationally.

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LITERATURE Anke, M., Groppel, B., Gruhn, K., Langer, M., and Arnhold, W. (1989) The essentiality of vanadium for animals. In: Anke, M. et al. (eds): 6th Int. Trace Element Symp. Vol. 1. Mo, V. Jena, Friedrich-SchillerUniversität, 17–27. Nielsen, F.H. and Uthus, E.O. (1990) The essentiality and metabolism of vanadium. In: Chasteen N.D. (ed): Vanadium in Biological Systems, Dortrecht, Netherlands, Kluver, 51–62.

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EFFECT OF IRON FORTIFICATION OF INFANT WEANING FOODS ON MINERAL ABSORPTION

Martínez C., López G., Ros G., Fox T.*, and Fairweather-Tait S. J.* Area de Nutrición y Bromatología Facultad de Veterinaria Universidad de Murcia Campus Espinardo 30071-Murcia. España *Institute of Food Research Norwich Research Park Colney. Norwich NR4 7UA, UK

Interactions among trace minerals and between trace minerals and other nutrients are commonly of a competitive nature. In the gastrointestinal tract, trace minerals may interact intraluminally or compete for carrier sites. Interactions among iron, copper and zinc, and possibly other metal ions have been described for a variety of conditions. The need for iron in infants is particularly high between 6 and 12 months of age. Iron fortification is one way of increasing the iron levels in the diet of growing infants. This study was designed to examine whether the type of iron fortificant used in weaning foods has an effect on utilisation of other nutrients, particularly zinc, copper and manganese. Six month old infants (n = 16) were recruited from the Child Health Register in Norwich and divided into two groups of 8. Infants in the first group were given two meals daily of approximately 100 g of a commercial homogenised weaning food using an haem iron concentrate as fortificant. In the second group ferrous sulphate was added as fortificant. A seven-day metabolic balance was performed in each baby to compare Zn, Cu, Mn, K, Ca, Mg, and P absorption of the two groups of subjects. Duplicate samples of every meal and nappies were collected daily from homes. Stools were separated from the diaper and autoclaved. Food samples and stools were freeze-dried and ground to fine powder. Bulked samples were prepared by mixing the whole 7 day collection, dry-ashing and then analysing for mineral composition by Inductively Coupled Plasma. Net absorption was calculated as intake minus faecal excretion.

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AN APPROACH TO ASSESSING THE SAFETY OF TRACE ELEMENT SUPPLEMENTATION

D. H. Shrimpton Council for Responsible Nutrition 63 Hampton Court Way Thames Ditton, Surrey KT7 OLT United Kingdom

A rational is proposed for assessing for populations upper safe levels of consumption of trace elements because, for some individuals and for some trace elements, the daily intake for optimising health may be greater than their RDAs. Two considerations are the reliability of the published data and the extent to which cariation in a national population may be greater than that of the populations studied, especially in respect of their sensitivity to adverse effects. A risk assessment model has been proposed in the USA by the Food and Nutrition Board to derive an uncertainty factor relating to the quality of publishd data for establishing NOAEL and LOAEL values. A model is proposed here to take account of risk from variation in the population to the responses from which NOAEL and LOAEL data has been established. In this model a term is introduced that will provide for assurance against excess consumption within populations in a manner that is comparable with that provided by the RAD for assurance against deficiency. This term is the Population Upper Safe Level calculated from the ratio of the differences between LOAEL and NOAEL and of NOAEL and RDA. The greater the difference between the LOAEL and the NOAEL—ls—and the greater the difference between the NOAEL and the RDA—up—the greater is the confidence in the appropriateness for populations of the use of the NOAEL as an upper sage level without modification. The ratio of these two differences—ls/up—is termed the Population Factor (PF) and the Population Upper Safe Level = PF × NOAEL where PF = ls/up The following population factors (PF) are proposed: When ls/up = <1.0 PF = 25% When ls/up = 0.10, PF= 100% When ls/up =>1<10, where n = a whole integer 1 to 9. The Population Upper Safe Levels so calculated are for chronic daily intake from all sources. These limits are not necessarily applicable to acute consumptions. 228

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STRONTIUM TRANSFER IN THE FOOD CHAIN OF HUMANS

M. Anke, M. Seifert, M. Jaritz, E. Lösch, and E. Hartmann Institute of Nutrition and Environment Friedrich Schiller University D-07743 Jena, Dornbuger Str. 24 Germany

The biological essentiality of Sr has not or insufficiently been investigated. Amoebae species living in sea water use Sr instead of Ca for the formation of the skeleton. Sr deficiency experiments in animals have not yet been carried out. Sr is assumed to have an anticarious effect. The toxicity of Sr in humans has been controversially discussed. Therefore, the interest in this ultratrace element used to be concentrated almost exclusively on the isotope. The Sr content in the flora is determined by the Sr offer (geological origin of the soil-forming rocks and anthropogenic emissions). Syenite and granite weathering soils and moor and loess sites produce an Sr-rich vegetation, alluvial riverside soils and Muschelkalk weathering soils an Sr-poor one. Leafy dicotyledons store much Sr, monocotyledons little Sr. The thickenings of roots and stems accumulate average Sr amounts; fruit, seeds and tubers are Sr-poor. The fauna reflects the Sr offer very well via the Sr content in the body or in tissues. Most Sr is incorporated in the skeleton. Milk and cheese deliver more Sr with 0.2 to 40mg/kg dry matter. Leafy vegetables and herbs contain most Sr (10 to > 100mg/kg dry matter), fruit store 2.5 to 30mgSr/kg dry matter, cereal products 0.3 to 7mgSr/kg dry matter, bread 0.3 to 4.5mgSr/kg dry matter, potatoes 2 to 5mgSr/kg dry matter. The habitat takes a significant effect on the Sr intake via the Sr content in the local drinking water. In accordance with their higher dry matter intake, men took in 23% more Sr than women. Adults take in one third more Sr in summer than in winter (vegetable consumption). Vegetarians took in 50% more Sr than people with mixed diets. In the case of vegetarian diets, the bioavailability of Sr is reduced due to the phytin acid. Vegetarians only excreted 6% of the consumed Sr renally, people with mixed diets 14%. The seeming Sr absorption of people with mixed diets reached 25 and 19%, resp., that of vegetarians only amounted to 0.3 and 7%, resp. On an average, the Sr intake of people with 229

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mixed diets decreased by one quarter between 1988 and 1996 and amounted to 1.8 and 2.2mg/day in women and men in 1996. According to the present level of knowledge, this Sr intake is not dangerous for humans. The Sr content in humans decreases with increasing age highly significantly.

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CONTENT OF THE MINERAL ELEMENTS IN THE LIGHT DIETS USED FOR ALIMENTATION OF THE PENSIONER HOUSE RESIDENTS IN WARSAW

A. Klos and J. Bertrandt Military Institute of Hygiene and Epidemiology 4 Kozielska St. 01-163 Warsaw Poland

Residents of the Pensioner House are the people aged 60–100. Occurence of the different diseases caused by inappropriate nutrition is observed among this group. Symptoms of many diseases may be mitigated by properly composed diet. It is known that proper nutrition is based on the diets contained adequate energy value and nutritive elements content including mineral ones. Analyse of 40 daily light diet menus planned for residents of the Pensioner House nutrition showed that copper content was 1.75 mg what made 87.5% of the minimum of the Polish standard for the people aged over 60. The zinc content figured out at 16.6mg, magnesium 381.2 mg what made 103.8% and 103.0% of the recommended norm respectively. Only the manganese content met the requirements in full and figured out at 4.93 mg. The highest copper and manganese content was noted in the diets planned in the winter, zinc in the autumn and magnesium in the spring. Content of m/a elements in the diets given for consumption trended downwards. Copper, zinc and magnesium content did not meet the recommended standards and figured out as follows: 67.0%, 89.6% and 81.5%. Only manganese supply was in accordance with the norms and indicated 4.93 mg. The biggest supply of all examined elements in the diets was in the summer and autumn. In this time the supply of the copper covered 71.0%, zinc 96.1% and magnesium 87.5% of the recommended norm. Manganese content met the requirements.

ACKNOWLEDGMENTS This study was supported by a Research Grant No 148/115C-TOO97 “Zywienie” from National Scientific Commitee (KBN) in Poland. 231

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DETERMINATION OF MINERAL LEVELS IN WALNUT CULTIVARS AFTER MICROWAVE MINERALISATION

F. Lavédrine, A. Ravel, A. Villet, V. Ducros, and J. Alary LBSO, Université J. Fourier Grenoble, France

Mineral composition of two walnut cultivars (Franquette and Hartley) originating both in France and California was determined. It included antioxidant trace elements: copper, zinc, and selenium. Microwave assisted mineralisation was followed by atomic absorption spectrometry for all minerals except selenium which was quantified by GCMS, and phosphorus by a colorimetric method. Three elements presented major level differences linked to variety or origin. For origin, potassium levels of the French walnuts were higher (Franquette: 487 and Hartley: 466 mg per 100g) than those of the Californian walnuts (Franquette: 358 and Hartley: 372 mg per 100g). Inversely, sodium level was higher in the Californian Hartley than in the French Hartley (6.7 vs. 0.6 mg per 100g). For variety, high levels of magnesium were observed for the Franquette cultivars (French: 191 and Californian: 202 mg per 100g) while the Hartley levels were lower (French: 129 and Californian: 134 mg per 100g). The same observation could be made for zinc, but the differences were weaker. No statistical differences were observed for copper and selenium levels.

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AVERAGE INTAKE OF TRACE ELEMENTS IN MILITARY FOOD SERVICES IN FRANCE

P. Le Francois and D. Argaud SCERCAT, 11 bis rue de Groussay F 78120 Rambouillet, France

A food consumption survey by weighing has been performed during three consecutive days in three restaurants of French land forces, in Clermont Ferrand, Lyon and in the district of Var. The survey concerned 331, 527 and 195 persons, respectively. The quantity of food used, the average portions served and the wastage on plates have been weighed to assess mean daily consumption of food per man. Trace element intake has been assessed with the food composition table of minerals of CIQUAL (Lamand et al., 1996). The results of average intake of trace elements are summarized on the following table:

Average intake of iron is on excess of the RDA while manganese, copper, zinc and selenium intakes are below the RDAs. Food iron is mainly supplied by cereal products (30%), meat and fish (29%) and vegetables (24%). 56% of manganese are derived from cereal products, 22% from vegetables. 34% of copper comes from cereal products, 29% from vegetables. Meat con233

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tributes 44% zinc and 55% selenium intakes. These results are discussed with those of other authors. They will be confirmed by chemical determination of duplicate meals.

REFERENCES Dupin H., Abraham J., and Giachetti I., 1992. Apports nutritionnels conseillés pour la population française. Tec & Doc éditeur, Paris. Lamand M., Tressol J.C., Ripert J.I., Favier J.C., and Feinberg M., 1996. Répertoire général des aliments. Tome 4. Table de composition minérale. Tec & Doc éditeur, Paris.

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USE OF HAEM IRON CONCENTRATE IN THE FORTIFICATION OF WEANING FOODS

Martínez C., Ros G., Periago M. J., and López G. Area de Nutritión y Bromatología Facultad de Veterinaria Universidad de Murcia Campus Espinardo 30071-Murcia España

Iron deficiency is particularly prevalent in infants and is a major nutritional problem in the world today. Food fortification is generally considered to be the best longterm strategy to combat iron deficiency, however there are technical problems in the choice of the iron compound as those of high relative bioavailability, such as iron sulphate, often provoke unacceptable organoleptic changes. Haem iron is less likely to be affected by inhibitors of iron absorption. Bovine haem concentrates have been used to fortify infant foods such as cereals, milk and biscuits but there is no published information on its utilisation in homogenised weaning foods. In fact, most of them are iron fortified with inorganic salts in order to improve the contribution to iron needs of infants. The purpose of this study was to clarify if the fortification of homogenised weaning foods with a porcine haem concentrate is technologically feasible by evaluating changes in haem iron content and sensory attributes under different storage conditions throughout a period of 8 months. A commercial homogenised weaning food (“Chicken and lamb with vegetables”) and an experimental weaning food obtained by the addition of 0.5% of porcine haem concentrate to the commercial one, were selected for investigation. Final iron content were 0.71 mg/100g for the non-fortified weaning food (NFWF) and 1.46mg/100g for the haem iron fortified weaning food (FWF). Both types of samples were stored at room temperature and also at 37 °C for 8 months. It has been established that food processing, storage, chemical treatment and cooking alters the haem and non haem content of food. In our study a marked change was found for non-haem iron content, which increased significantly with the time of storage in both samples at room temperature and at 37 °C. Therefore, haem iron decreased with the time of storage, resulting that at the third month of the experiment, all haem iron in NFWF changed to non haem iron. However, at the end of the study, FWF still possessed a 17% and a 21% of haem iron for samples stored at room temperature and 37°C respectively. Appearance, flavour, aroma, texture 235

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and overall acceptance were performed by a panel of 12 trained judges using a 9-pint rating scale (9 = excellent, 1 = very poor). There was no significant difference (p < 0.05) in appearance, flavour, aroma and overall acceptance due to the addition of haem iron. Only colour ratings were significantly lower for FWF at the first stage of the experiment. After that, and over all the period of storage, judges gave similar scores for both samples. In summary FWF obtained higher ratings for flavour, aroma and texture while in NFWF colour and appearance were better scored. Colour of samples was also tested using a Chromameter Minolta CR-200 to determine the and values as well as the hue angle and chroma data transformations. Following this instrumental measurement, significative changes were found for value depending on the storage temperature.

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MINERAL CONTENT IN FOUR SPANISH FISH AFTER BONE ADDITION

Isabel Martínez, María Jesús Periago, and Marina Santaella y Gaspar Ros Area de Nutrición y Bromatología Faculty of Veterinary University of Murcia 30071 Murcia, Spain

Flesh fish is considered a good mineral source of Ca and P, but it should be considered that the splinters as well as fish bony skeletal are a better source of these elements, many time not taken into account (NRC, 1991). Because bone tissue, in its stage of highest crystallisation, is constituted by the hidroxiapatite salt, which has Ca and P in a 2.15/1 ratio (w/w) (Russell et al., 1986), its addition to the flesh could contribute to a high Ca and P support. Fish can not be eaten with bone in its natural stage so it can not be chewed or digested. However it is technically possible to process some fishes with bone by careful prior homogenization, obtaining a fish purée which could be incorporated to some manufactured foods, increasing the Ca and P contents and the Ca/P ratio of the meal. Four commercial types of Mediterraneam fish, (Bleu whiting (Micromesistius poutassou, R), Little hake (Merluccius merluccius, L), Hake (Merluccius merluccius, L) and Sole (Solea vulgaris vulgaris, Q)) commonly consumed in Span were analysed in this study. Though, hake and little hake are scientificaly the same fish spice, they are sold as two different commercial types depending on their size. From each type of fish two samples were obtained, the first one was an homogenized flesh sample and the second one was an homogenized of flesh with bone. The aim of the present study was to ascertain the influence of incorporating fish bone on the Fe, Zn, Cu, Mn, Mg, Ca, P, Na, and K content in these samples, to evaluate in later studies their incorporation as ingredient of fish-based foods. The levels of trace elements (Fe, Zn, Cu, and Mn), expressed as mg/100g on a wet basis, were quite low whitin all the samples assayed, not being affected by bone addition. After bone addition, Ca content increased significantly in all samples, ranging from 350.74 mg/100g for bleu whiting to 476.35 mg/100g in sole. About P, high content was observed in flesh samples (from 420.76 mg/100g for hake to 604.28 mg/100 g for bleu whiting) increasing also after bone incorporation (from 731.08 for mg/100g for hake to 1249 mg/100g for sole), and givin a better Ca/P ratio Na and K contents did not 237

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show signifant changes. In summary, the content of Ca and P were increased after the bone addition to the flesh due to the high content of these elements in bony tissue, which coul be interesting since a nutritional point of view to use these samples in fish based foods to fortificate Ca and P contents.

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TRACE ELEMENTS IN AUSTRIAN FOOD

A. Sima, M. Wilplinger, S. Zöchling, S. Heumann, U. Schaller, and W. Pfannhauser Institute of Bio- and Food Chemistry Graz University of Technology A-8010 Graz, Petersgasse 12/II Austria

The aim of this study was to determine the supply of the Austrian population with the essential trace elements Cr, Cu, Mo, Ni, Se and Zn by food produced exclusively in Austria. Therefore 12 locations distributed all over Austria were selected, each area being large enough to offer all ingredients used for the diets from locally production. The daily pooled samples were analyzed for their content of the essential trace elements and correlated with the soil. According to this study the Austrian people are optimally supplied with Cu and Zn while there is a slight deficiency of Cr, Mo, Ni and Se. Depending on the location, significant differences in the concentration were observed for all elements except Zn. A distinct variation in content of the daily diets was found for Zn and Se. High resp. low contents of the diet were reflected in tendentious high resp. low contents of soil. But this correlation could not be statistically verified.

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TRACE ELEMENT (Cu, Fe, Zn) INTAKES IN CUBA T. Verdura1, J. Arnaud2, P. Fleites3, M. Chassagne4, A. Favier3, R. Perez-Cristia3, J. Barnouin4, and the SECUBA group 1

Instituto Finlay, Ave 27, n°19805 La Lisa, La Habana, Cuba 2 Laboratoire de Biochimie c CHUG, BP 217, 38043 Grenoble Cedex 9 France 3 Centro National de Toxicologia Ave 31 y calle 114, La Habana Cuba 4 Laboratoire d’Ecopathologie INRA, 63122 Saint Genès Champanelle France

The presented results are part of a multidisciplinary prospective study: Seguridad alimentaria y Buena Alimentacion in Cuba (SECUBA). The aims of this study were to determine the potential toxic and nutritional health risks in Cuba and to evaluate the influence of lifestyle and seasons on these risk factors. The present paper is focused on trace element intakes and their seasonal variations. One hundred and ninety nine healthy middle-aged men (30–50 years) living in Havana were randomly selected from the La Lisa health centres. Subjects were studied at four different periods over one year [March–April 1995 (period 1), June–July 1995 (period 2), October 1995 (period 3) and January–February 1996 (period 4)]. May corresponds to the benning of the rainy season and October to the transition period between rainy and dry seasons. The protocol was approved by the Cuban Ministry of Public Health and all subjects gave their informed written consent. Food and beverage intakes were assessed at the four periods by 7 consecutive days of food record. Nutrient intakes were calculated using the Cuban NUTRISIS food composition table. Although very low energy intakes, total protein, copper and iron intakes were within the reference ranges (Table 1). Depending on the period, zinc intakes were lower than 10 mg/d in 70 to 80% of subjects; copper intakes were lower than 1.5 mg/d in 4.8 to 15.8% of subjects and iron intakes were lower than 7 mg/d in 4.8 to 10.4% of subjects. Iron 240

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(p = 0.009) and zinc (p = 0.037) intakes were the highest in period 4. Copper intakes were higher in rainy season than in dry season (p = 0.005). Highly signigicant correlations between trace element intakes and energy or protein intakes at each period were observed. These results suggest that Cubans may be at risk of zinc deficiency.

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DISTRIBUTION OF TRACE ELEMENTS AND MINERALS IN PULP AND PEEL OF APPLES AND OF PERSIMMONS Z. Zachwieja1, J. Piotrowicz1, M. Folta1, H. Barton1, S. Gorinstein2, M. Zemser2, and S. Traktenberg3 1

Department of Food Chemistry and Nutrition Jagiellonian University School of Medicine Medyczna 9, 30-688 Cracow Poland 2 Department of Pharmaceutical Chemistry Hebrew University Ein Karem, Jerusalem, 91120 Israel 3 Kaplan Hospital Hebrew University Rehovot, Israel

The nutritional importance of elements is under increasing interest due to their influence on several biochemical reactions e.g. antioxidant processes. For this preliminary study two popular kinds of fruit from Israel (persimmons) and Poland (apples) were selected and 10 trace elements and minerals were analyzed. Fruits were purchased at fruit market: Persimmons (Diospyros Kaki Thunb) in Jerusalem (1997), apples (Malus sylvestris Mill. var Lobo) in Kraków (Poland, 1998). Fruits were washed in distilled water, then peeled. Samples (separately, pulp and peel) were lyophilized. The samples ca. 0.8g of lyophilizated fruits were mineralized in a microwave oven MDS 2000 (CEM) with addition of 5ml of conc. nitric acid (ultrapure, Merck). The concentration of elements were estimated using an atomic absorption spectrometer (Perkin Elmer 5100 ZL), using the flame method for Cu, Zn, Fe, Na, K, Mg, Ca and a flameless one for Mn, Pb, Cd. The mean concentrations of elements in whole fresh fruits and the ratio of their content in peel to pulp (given in parentheses) were as follows: 242

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– persimmons: Mn 107(2.7), Zn 13.9(4.1), Cu 9.76(4.6), Pb 0.32(12), Cd 0.13(3.1) in ug/100g Na 4.91(0.9), K 254(1.1), Mg 8.22(1.7), Ca 9.35(4.6), Fe 101(3.7) in mg/100g – apples: Mn 30.7(2.9), Zn 18.1(2.3), Cu 24.0(2.3), Pb 0.72(9.0), Cd 0.20(2.9) in ug/100g Na 0.59(0.8), K 81.9(1.4), Mg 5.02(3.3), Ca 4.28(2.6), Fe 94.3(4.5) in mg/100g The values of the element content obtained for the whole fruit were in agreement with tabulated data (Scherz and Senser, 1994). There were no significant differences between the element contents in persimmons and apples except for Mn, K and Na, for which higher contents were found in persimmons. The peel to pulp element ratios were generally similar in both kinds of fruit investigated and were ca. 1 for mono-valent metals (Na and K) and 2–4 for bivalent metals. For toxic metals this ratio was 3.0–3.1 for Cd; that is, the same as for bivalent metals, whereas it was significantly higher (9.0–12) for Pb. This suggests that in both kind of fruits from two different countries an additional contamination with lead (e.g. from traffic) occurred during the final stages of growing, picking, transportation and/or trade.

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MINERAL ELEMENTS IN THE DIET PLANNED FOR CONSUMPTION FOR RESIDENTS OF THE PENSIONER HOUSE IN WARSAW

J. Bertrandt and A. Klos Military Institute of Hygiene and Epidemiology 4 Kozielska St., 01-163 Warsaw Poland

The concept of the proper nutrition is to deliver the adequate amount of nutritive elements, contained in the food products, required by human organism for its best working. It is known that insufficiency or surplus of the energy and nutritive elements may lead to different health disturbances. Mineral elements make the group of compounds, which are not synthesised by organism and have to be delivered together with food. It is accepted that for the proper nutrition minimum 14 mineral elements are required. The content of the selected mineral elements in the diet planned for consumption in the Pensioner House was estimated. Based on 40 daily menus the content of macroelements such as calcium, phosphorus, magnesium, sodium and potassium and microelements: iron, zinc, manganese, copper, selenium and chrome was calculated. Content of m/a elements in the diets was calculated using software FOOD and the “Tables of the trace elements content in food products”. It was stated that from 11 examined elements only copper content did not meet the recommended standard for people aged over 60, and made 98.6% of its minimum. Content of the other mineral elements met the requirements of the norm. It should be mentioned that diets planned for consumption contained too big amount of sodium, exceeding the recommended minimum what is unfavourable from medical point of view.

ACKNOWLEDGMENTS This study was supported by a Research Grant No 148/115C-TOO97 “Zywienie” from National Scientific Research Commitee (KBN) in Poland.

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ZINC IS SCARCE AND EXPENSIVE

N. Darmon and A. Briend Groupe Nutrition-Santé CNAM-ISTA, 2 rue Conté, 75003 Paris France

OBJECTIVE To assess the nutritional constraints associated with poverty, special attention being given to the cost of trace elements in the diet.

METHODS Current food prices were added to a food composition table giving more than 30 nutrients for about 100 food items. Scarce nutrients were defined as those present either in a limited number of foods or in low concentration compared to the French nutritional recommended daily intakes. Scarce nutrients were found by analysis of the nutrient distribution table. Expensive nutrients were defined as those having either a high individual cost (estimated cost to cover the daily recommendation with the various food items), or a high additional cost (compared to a diet providing at a minimal cost the basic needs in energy and proteins), or a high marginal cost (compared to a diet complying with all nutritional recommendations at the lowest cost). Cost-minimisation of the diets was achieved by linear programming.

RESULTS Our analysis selected some of the nutrients as both scarce and expensive and zinc was at the top of this list. Although zinc can be found in more than 80% of the foods, it is present in most of them at a very low concentration compared to the needs. In addition, zinc is expensive because it has both a high individual, a high additional and a high marginal cost. 245

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CONCLUSION These results suggest that nutritional risks associated with poverty can simply be linked to the cost of the foods and that it may be more difficult for low-income individuals to cover their zinc needs. This may have important health implications since zinc deficiency is hard to diagnose and zinc plays a key role for growth, immunity, and skin and intestinal mucosae protection.

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ESTIMATED DAILY INTAKES AND CONCENTRATIONS OF ESSENTIAL TRACE ELEMENTS IN INFANT FORMULAS

I. Navarro, J. I. Alvarez, and A. Martín University of Navarra Fac. of Sciences. Department of Chemistry Pamplona, E-31.080 Spain

The need for essential trace elements has been relatively well studied by growth infant. This study has been carried out to determine the concentration of essential trace elements (iron, zinc, copper, manganese and selenium) in the majority of infant formula commercialized in Spain. The infant formulas investigated include powder or liquid, such as dairy and soya formulas. A scrupulous and systematic approach was adopted to minimize every possible source of elemental contamination from the sampling step on wards. Digestion of samples was attempted with subboiling nitric acid in close system an microwave oven. The concentration of iron, zinc and copper was determined by Atomic Absorption Spectrometry while for manganese and selenium the Inductively Coupled Plasma Emission Spectrometry was used. The results obtained are similar to what was found by other authors in European and American formulae. The theoretical intake of lactating infant has been established with the different types of infant formulas studied and stages of lactation in relationship to the Recommended Dietary Allowances (RDAs) for trace elements. The investigated formulas provide an intake of zinc, manganese and selenium that don’t reach the RDAs to the first months of neonate life.

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CLINICAL EVALUATION OF “DESIGNER EGGS” AS A SOURCE OF ESSENTIAL MICRONUTRIENTS FOR HUMANS

P. Surai and A. MacPherson SAC, Auchincruive Ayr, Scotland, UK

Many essential micronutrients are in short supply in the normal Scottish diet and this may well account, at least in part, for our dismal health record. For example inadequate intakes of n-3 polyunsaturated fatty acids, particularly docosahexaenoic acid (DHA), adversely affect cardiovascular and renal function and the development and normal function of the retina and brain in neonates. DHA is also important for maintaining correct membrane fluidity and permeability. Vitamin E, selenium and the carotenoids are important antioxidants and contribute significantly to the body’s defences against free radical attack and hence to its ability to counteract many disease processes. Our research and that of other workers have shown that there is often an inadequate intake of these nutrients. For example our dietary selenium intake now provides less than half our recommended daily requirement. This has been reflected in a marked decline in plasma selenium concentration in the population of the West of Scotland. Similarly poor intakes of vitamin E and the carotenoids result from our generally low consumption of green vegetables. While all these nutrients can be obtained in tablet or capsular forms from Pharmacists or Health shops it is generally held that their supply in normal dietary components is preferable. This was the motivation for the development of our “designer egg” and this ethically approved, clinically controlled study with informed consent was designed to discover if it is an effective means of providing them in terms of their availability and acceptance. Laying hens were fed a supplemented diet which resulted in an egg providing 15mg vitamin E, 200 mg DHA, 30 µg selenium and 1 mg lutein which will thus supply from 40–100% of the adult male daily reference nutrient intake of three of these parameters. No reference value has yet been fixed for lutein. The main carotenoid in the designer egg was lutein (>65%) with a much lower proportion of zeaxanthin and traces of other carotenoids. In the table eggs carotenoid concentration was significantly lower (3–4 fold) and composition was notably different with lutein, citranaxanthin and carotenoic acid 248

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being the main carotenoids comprising more than 80% of total. Vitamin E concentration in the table eggs was more than 20 times lower than in the designer eggs. Boiling designer eggs did not change the concentration of lutein and losses of α-tocopherol were less than 5%. Our results also indicated that a combination of these nutrients in the egg increased lipid stability against peroxidation. In an in vitro experiment accumulation of malondi– aldehyde in the “designer” yolk as a result of spontaneous or Fe-stimulated peroxidation was significantly lower than in the normal table eggs. Forty subjects were recruited, stratified by age and sex, and randomly allocated either to a “designer” or a commercial table egg per day. The treatments were blinded. Blood samples were collected and blood pressure recorded prior to the start and at the end of an 8 week experimental period. No significant changes in blood pressure or in total or HDL-cholesterol were recorded. Plasma vitamin E, lutein and DHA were all significantly elevated in subjects consuming a designer egg/day but plasma selenium concentrations were unaffected. This may reflect the presence of free thiol groups which would react with selenium to form insoluble selenides. Regular consumption of this product would make a significant contribution to meeting the daily requirements of these essential micronutrients and could have beneficial effects in reducing the risk of some of the major diseases.

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PLASMA LEVELS OF SELENIUM, SELENOPROTEIN P, AND GLUTATHIONE PEROXIDASE AND THEIR CORRELATIONS TO FISH INTAKE AND SERUM LEVELS OF THYROTROPIN AND THYROID HORMONES—A STUDY ON LATVIAN FISH CONSUMERS L. Hagmar1, M. Persson-Moschos2,3, B. Åkesson3, and A. Schütz1 1

Department of Occupational and Environmental Medicine University Hospital, Lund 2 Department of Applied Nutrition and Food Chemistry, and 3 Division of Biomedical Nutrition Chemical Center, University of Lund Lund, Sweden

The bioavailability and metabolic fate of selenium from dietary fish in humans is not well known. The present study was made to assess the importance of dietary selenium from fish for the levels of selenoproteins in plasma in subjects with a larger variation in selenium status than in previously studied cohorts. Pronounced changes in thyroid hormone metabolism occur in experimental selenium deficiency, mainly because iodothyronine deiodinases are selenoproteins. The functional effects of low selenium status on thyroid function in humans have not been well characterized. Another objective was to study the relationships between different markers of selenium status and thyroid hormone function. In a cross-sectional study sixty-eight Latvian men (age 24–79 years) were recruited among coastal fishermen and inland subjects from Latvia. None of the subjects were on selenium medication or had any known endocrine disease. In the whole study group the concentration of selenium in plasma ranged from 0.30 to 1.56 µmol/l selenoprotein P levels from 0.54 to 2.21 a.u., and the concentration of glutathione peroxidase from 1.20 to 5.73 mg/l. The number of fish meals per month was correlated with plasma selenium, selenoprotein P and glutathione peroxidase (r = 0.63, r = 0.62 and r = 0.50, respectively; P < 0.001). Plasma selenium was correlated with selenoprotein P and glutathione 250

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peroxidase (r = 0.88 and r = 0.67, respectively; P < 0.001), and also selenoprotein P and glutathione peroxidase were correlated (r = 0.63, P < 0.001). When the subjects were divided into three groups according to fish consumption (0–3, 4–20 or 21–50 fish meals/month), the mean plasma selenium level in the high fish consumers was 81% higher than in low fish consumers. The corresponding figures for selenoprotein P and glutathione peroxidase were 68% and 45%, respectively. Organic mercury in erythrocytes was significantly correlated with fish intake, plasma selenium, selenoprotein P and glutathione peroxidase. Serum creatinine was significantly correlated with plasma selenium and selenoprotein P, but surprisingly not with glutathione peroxidase. TSH in serum was inversely correlated with plasma selenium and selenoprotein P, but not with glutathione peroxidase or fish intake. Thyroid hormone levels were not correlated to plasma selenium, selenoproteins, or fish intake. It can be concluded that in this study selenium from fish intake had a marked impact on all variables on selenium status that were studied. No impact of selenium status on T3 and T4 levels was observed. The slightly negative correlation of selenium status to TSH levels might indicate a higher TSH secretion at low selenium status. The study was supported by grants from the Swedish Environmental Protection Agency, the Medical Faculty, Lund University, and the Påhlsson Foundation. BÅ was supported by FAIR CT95.0771.

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INTERLABORATORY COMPARISON STUDIES INTO DETERMINATION OF METALS CONTENT IN FOODS, ORGANIZED BY THE NATIONAL INSTITUTE OF HYGIENE

Krystyna Starska, Maria Wojciechowska–Mazurek, Elzbieta Brulinska–Ostrowska, and Kazimierz Karlowski National Institute of Hygiene Department of Food Research Chocimska 24, 00-791 Warsaw Poland

Department of Food Research of the National Institute of Hygiene systematically since 1991 organizes proficiency studies in determination of the content of Pb, Cd, Cu, Zn and Hg in food. These tests are designed mainly for laboratories of the Voivodship Sanitary-Epidemiological Stations (Laboratories) which perform continuous supervision over the health quality of food. The tests are based on the proficiency testing procedure developed by the Ministry of Agriculture, Fisheries and Food, Food Science Laboratory, Norwich (UK) and comprises preparation and distribution to participants 2–3 samples per year, containing either standard Pb, Cd, Cu, Zn and Hg solutions with addition of possibly interfering inorganic salts solutions, or food products; statistical analysis of results, preparation of reports and organization of consulting and training sessions. Principles of organizing the tests and assessment of results are in conformity with the ISO 5725-1994(E) standard and “The International Harmonized Protocol for the Proficiency Testing of (Chemical) Analytical Laboratories” prepared by ISO/IUPAC/ AOAC.1,2 Participating laboratories perform tests using identical materials and routine testing methods. In their routine work, the Laboratories are recommended to use Flame AAS methods developed and validated by the National Institute of Hygiene.

Address all correspondence to: Krystyna Starska, National Institute of Hygiene, Department of Food Research, Chocimska 24, 00-791 Warsaw, Poland; telephone: +4822 8494051 ext. 362; fax: +4822 8497445; email: [email protected]

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Control samples distributed to participants are prepared by the National Institute of Hygiene with consideration of elemental concentrations and matrices corresponding to samples routinely tested by the laboratories, verified as to their uniformity and stability. Participation in tests is voluntary; results are fully confidential. Proficiency of laboratories is evaluated on the basis of their “z-score”calculated for each laboratory separately, taking into account their individual result, the “true” value for each analyte concentration and standard deviation calculated after statistically eliminating outliers in the given population.

EXPERIMENTAL WORK IN 1998 The following samples for proficiency testing were distributed to participating laboratories in 1998: Round 1/98: standard Pb, Cd, Cu, Zn and Hg solutions in with addition of possibly interfering inorganic salts in quantities most commonly present in mineralizates of main food products constituting typical daily diet in Poland. Two fortified levels were used. Round 2/98: samples of daily diets reconstituted by National Institute of Hygiene according to data concerning food consumption in Poland, published by Central Statistical Office, and fortified with appropriate amounts of Pb, Cd, Cu, Zn and Hg ions. Of the 52 participating in 1998 laboratories, 81% to 98% attained a satisfactory result, depending on the element. Number of laboratories with satisfactory z—scores values in each respective round in 1998 are given in the Table.

REFERENCES ISO 5725-1994(E) Accuracy (Trueness and Precision) of Measurement Methods and Results. Thompson, M. and Wood, R., 1993, ISO/IUPAC/AOAC International Harmonized Protocol for the Proficiency Testing of (Chemical) Analytical Laboratories, Journal of AOAC Int., 76, 926–940. Garfield, P.M., 1991, Quality Assurance Principles for Analytical Laboratories, AOAC. Wernimont, G.T., 1990, Use of Statistics to Develop and Evaluate Analytical Methods, AOAC, Arlington.

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BIOAVAILABILITY OF TRACE ELEMENTS IN HUMAN DIET

Susan J. Fairweather- Tait Institute of Food Research Norwich Research Park Colney, Norwich NR4 7UA Norfolk, UK

1. IMPORTANCE OF BIOAVAILABILITY 1.1. Definition Biovailability is the term used to describe the proportion of trace elements in the diet that are utilised for normal metabolic functions. This information is essential for the derivation of dietary recommendations for trace elements whereby physiological requirements are converted into dietary intakes. However, since it is not possible to determine the utilisation of most trace elements, assessments of bioavailability are usually made from absorption studies, using stable or radioisotope tracers (Sandstrom et al., 1993). Iron is an exception in that bioavailability can be measured directly either using haemoglobin repletion studies in anaemic subjects or haemoglobin incorporation of isotopically labelled iron. There is a pressing need to investigate and prioritise the numerous functions of trace elements in the body, many of which are still unknown or only partly characterised, in order to select the most appropriate end-point to be used to assess bioavailability. This important research objective will be made easier when the findings of the human genome project are published.

1.2. Selection Criteria Most trace elements are inefficiently and variably absorbed (Fairweather-Tait, 1998) (Table 1), depending on a variety of diet and host-related variables. Interest in trace element bioavailability is focused on trace elements that are associated with a public health problem, either in terms of inappropriate supply or evidence that high intakes may be protective against chronic disease. These include iron, zinc, copper, selenium and iodine. The dietary supply of the last two trace elements depends on geographical location since the concentration found in the food chain is directly related to the soil content. Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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Although different countries do not necessarily have the same problems, iron deficiency is relatively common throughout both industrialised and developing countries, probably because humans have developed very effective controls for preventing iron from entering the body as there are no mechanisms for excreting excess iron. The dietary intake of trace elements is changing quite dramatically in western countries in that an increasing number of people are taking vitamin and mineral supplements, thereby increasing their intake of certain trace elements very significantly. There is also greater activity in the area of food fortification with the expanding market for functional foods. Bioavailability of trace elements used for supplements and food fortificants is an important consideration with respect to efficacy. Also it is vital that we understand and acknowledge any potential adverse effects that high intakes of one or more trace element may have on the absorption or metabolism of other trace elements and hence the health of individuals. When the dietary supply of a trace element is limited and/or individuals have an elevated physiological requirement, for example in periods of growth, improving bioavailability is one of the recognised strategies for increasing the dietary supply. This requires a detailed knowledge of factors that modulate trace element absorption (Table 2), in particular dietary enhancers and inhibitors that are relatively easy to manipulate (Fairweather-Tait and Hurrell, 1996). This approach is particularly relevant to developing countries where diets are monotonous with a lower trace element bioavailability and fortified processed foods are virtually absent.

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2. AVAILABILITY FOR ABSORPTION 2.1. Dietary Modulators Absorption of most trace elements takes place in the small intestine, and the form present in the lumen will dictate the amount that is potentially available for absorption. Thus, liberation of the trace element from the food matrix and subsequent chemical changes that take place in the intestinal tract, as determined by the presence of constituents of the diet and volume and composition of intestinal secretions, are the first step in the bioavailability pathway. 2.1.1. Luminal Events. One mode of action of dietary enhancers and inhibitors is to elicit a change in the efficiency of absorption through the formation of complexes with the trace element in the GI tract. This prevents it from participating in further chemical reactions within the digestive chyme e.g. precipitation as insoluble oxides, particularly in the case of iron, with the rise in pH as the digesta is moved from the acid environment of the stomach into the duodenum when the conditions are close to neutral. If the binding of the trace element within the complex is very tight, then the trace element may not be released to the transport protein associated with the enterocytes of the small intestine. The substance would then be classed as an inhibitor, e.g. phytate (myo-inositol hexa phosphate) with iron and zinc. Conversely, if the binding constant of the compound is lower than that of the transport protein, then the trace element will be released from the complex and because it has been protected from binding with other compounds or from precipitation, the net result will be an increased absorption. Substances classed as enhancers include ascorbic acid (with iron) and meat protein (with iron and zinc). The effect of absorption modulators is dependent on chemical reactions and is not consistent between trace elements (Fairweather-Tait, 1996), for example, ascorbic acid increases iron but reduces copper absorption. Information on trace element speciation is central to predicting trace element absorption (Fairweather-Tait, 1999). Haem iron and non-haem iron are absorbed by two independent pathways and information on quantities of these forms of iron in the diet are needed to assess its bioavailability. A change in valency may affect the absorption of some trace elements. Non-haem iron in the ferrous (Fe2+) form is more soluble than ferric (Fe3+) iron at pH 4 and higher. Thus reducing compounds such as ascorbic acid increase iron bioavailability in a dose-related manner over the range 25–1,000 mg (Cook and Monsen, 1977). A similar situation applies with cuprous (Cu+) and cupric (Cu2+) copper but in this case the reduced form may have a lower bioavailability (Wapnir, 1998). Competitive interactions also occur between trace elements, particularly the divalent cations, both with regard to binding with macromolecules and low molecular weight ligands and also non-specific transport protein involved in carrier-mediated uptake (Gunshin et al., 1997). However, the antagonism is only of practical significance when the intake of one of the metals is particularly high (as with supplements and fortified foods) and the intake of the other is at the lower limit of requirement. The interactions of greatest practical significance are between (a) zinc and copper where increasing the intake of zinc from 5–20 mg/d requires a 60% increase in copper intake to maintain balance (Wapnir, 1998), (b) iron and zinc where interactions are important when the total ionic species in any one meal exceeds 25 mg (Solomons, 1986) which can occur when iron supplements are taken with a meal but not with iron-fortified foods (Fairweather-Tait,

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1995), and (c) iron and copper, particularly in relation to iron-fortified infant formulas (Haschke et al., 1986; Lonnerdal and Hernell, 1994). Calcium has been shown to reduce non-haem and haem iron absorption from test meals to a similar extent. Since haem iron is absorbed intact and by an independent route, the mechanism probably takes place in the cell or basolateral membrane. The relative decrease or increase in iron absorption on addition of 25 mg phytate or 50 mg ascorbic acid to a meal is the same in meals with or without added calcium, again indicating that the effect is not luminal (Hallberg et al., 1991; Hallberg et al., 1992). Calcium reduces iron absorption in a dose-related manner from 40–300 mg when it reaches a plateau. Thus no effect will be seen with meals containing >300mg calcium, as supported by results of longer-term supplementation studies. For example, giving 400 mg calcium three times daily with meals for 6 months did not reduce body iron stores in adults consuming a relatively high (950mg/d) calcium diet (Fairweather-Tait and Minihane, 1998). 2.1.2. Systemic Events. Speciation affects selenium bioavailability because different forms are transported intact by independent pathways. Thus selenomethionine is utilised for non-specific protein synthesis whereas inorganic selenium is incorporated into selenoproteins. The dietary form of other trace elements is unimportant with respect to systemic metabolism. Dietary constituents can also exert an affect on trace element bioavailability through systemic pathways, such as changing the quantity excreted via urine or faeces or by altering the efficiency of reabsorption of trace elements secreted into the GI tract. Homeostasis plays a key role in determining the bioavailability of trace elements. A dose-response effect is well established for some trace elements whereby increased intake results in a fall in percentage absorption but an increase in the absolute quantity absorbed and homeostasis is maintained through increased excretion. Since the retention of trace elements from the diet is dictated to a large extent by physiological requirements, bioavailability is not constant and only partly characteristic of the food or diet under investigation, being primarily dependent on the genetic profile and stage of development of the individual, and other host-related variables.

3. METHODS OF MEASUREMENT 3.1. Availability for Absorption It should be possible to predict the availability of trace elements for absorption from speciation measurements in digesta, but this approach is not feasible, being invasive and associated with numerous technical difficulties. Alternative in vitro methods have been developed but it is very difficult to model the action of the human alimentary tract, particularly in view of the fact that it is a dynamic system. At present, the most promising technique is a cell culture system using Caco-2 cells, a human colon adeno-carcinoma cell line. This model system accurately predicts the effect of phytate on iron and zinc uptake (Han et al., 1994) and can be used to study the availability of iron from food homogenates following simulated digestion (Glahn et al., 1996).

3.2. In vivo Studies The bioavailability of trace elements can be assessed using stable or radioisotopes to label foods/diets intrinsically or extrinsically. The experimental approaches include (a)

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calculating absorption as the difference between intake and faecal excretion (apparent absorption) or intake minus faecal plus urinary excretion (true absorption), (b) direct measurements of whole body retention using a whole body counter (only applicable to studies employing gamma isotopes), (c) plasma appearance techniques, and for iron only (d) haemoglobin incorporation or repletion (requires iron deficient individuals). The latter is a direct measure of bioavailability and equivalent techniques, based upon some measure of utilisation, are needed for the other trace elements.

3.3. Whole Diets Much of the information on mineral bioavailability has been obtained from singlemeal studies. However, the results are not always predictive of longer-term studies in that they tend to exaggerate the effects seen in whole diets (Cook et al., 1991), therefore greater emphasis should be given to improving our understanding of the mechanisms underlying adaptive responses. The development of functional markers of mineral utilisation is an important research task, as not only would it increase the portfolio of experimental approaches that can be used to study mineral bioavailability but it would also facilitate/validate the interpretation of results from short-term studies with isotopes.

REFERENCES Cook, J.D. and Monsen, E.R., 1977, Vitamin C, the common cold, and iron absorption, Am. J. Clin. Nutr. 30:235–241. Cook, J.D., Dassenko, S.A., and Lynch, S.R., 1991, Assessment of the role of nonheme-iron availability in iro n balance, Am. J. Clin. Nutr. 54:717–722. Fairweather-Tait, S.J., 1995, Iron-zinc and calcium-Fe interactions in relation to Zn and Fe absorption, Proc. Nutr. Soc. 54:465–73. Fairweather-Tait, S.J., 1996, Bioavailability of dietary minerals, Biochem. Soc. Trans. 24:775–780. Fairweather-Tait, S.J., 1998, Trace element bioavailability, in: Role of Trace Elements for Health Promotion and Disease Prevention, Bibl. Nutr. Dieta (B. Sandstrom and P. Walter, eds.), pp. 29–39, Karger, Basel. Fairweather-Tait, S.J., 1999, The importance of trace element speciation in nutritional sciences, Fres. J. Anal. Chem. 363:536–540. Fairweather-Tait, S. and Hurrell, R.F., 1996, Bioavailability of minerals and trace elements, Nutr. Res. Rev. 9:295–324. Fairweather-Tait, S. and Minihane, A.M., 1998, Effect of calcium supplementation on daily nonheme-iron absorption and long-term iron status, Am. J. Clin. Nutr. 68:96–102. Glahn, R.P., Wien, E.M., Van Campen, D.R., and Miller, D.D., Caco-2 cell iron uptake from meat and casein digests parallels in vivo studies: use of a novel in vitro method for rapid estimation of iron bioavailability, J. Nutr. 126:332–339. Gunshin, H., MacKenzie, B., Berger, U.V., Gunshin, Y, Romero, M.F., Boron, W.F., Nussberger, S., Gollan, J.L., and Hedeger, M.A., 1997, Cloning and characterization of a mammalian proton-coupled metalion transporter, Nature 388:482–488. Hallberg, L., Brune, L., Erlandsson, M., Sandberg, A.S., and Rossander-Hulten, L., 1991, Calcium: effect of different amounts on nonheme-iron and heme-iron absorption in humans, Am. J. Clin. Nutr. 53:112–119. Hallberg, L., Rossander-Hulten, L., Brune, M., and Gleerup, A., 1992, Inhibition of haem-iron absorption by calcium, Br. J. Nutr. 69:533–540. Han, O., Failla, M.L., Hill, A.D., Morris, E.R., and Smith, J.C., 1994, Inositol phosphates inhibit uptake and transport of iron and zinc by a human intestinal cell line, J. Nutr. 124:580–587. Haschke, F., Ziegler, E.E., Edwards, B.B., and Fomon, S.J., 1986, Effect of iron fortification of infant formula on trace mineral absorption, J. Ped. Gastro. Nutr. 5:768–773. Lonnerdal, B. and Hernell, O., 1994, Iron, zinc, copper and selenium status of breast–fed infants and infatns fed trace element fortified milk-based infant formula, Acta Paed. 83:367–373.

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Sandstrom, B., Fairweather-Tait, S., Hurrell, R., and van Dokkum, W., 1993, Methods for studying mineral and trace element absorption in humans using stable isotopes, Nutr. Res. Rev, 6:71–95. Solomons, N.W., 1986, Competitive interaction of iron and zinc in the diet: consequences for human nutrition, J. Nutr. 116:927–935. Wapnir, R.A., 1998, Copper absorption and bioavailability, Am. J. Clin. Nutr. 67(suppl):1054S–1060S.

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THE INFLUENCE OF VARIOUS FOOD INGREDIENTS AND THEIR COMBINATIONS ON IN VITRO AVAILABILITY OF IRON AND ZINC IN CEREAL BASED VEGETARIAN MEALS

V. V. Agte, K. V. Tarwadi, and S. A. Chiplonkar Agharkar Research Institute G. G. Agarkar Road, Pune 411004 India

1. INTRODUCTION Vegetarian meals have a poor bioavailability of iron rather than the poor content which is also true for zinc (Agte et al., 1999; MacPhail and Bothwell, 1992). Both, the amount of food intake as well as types of food ingredients in the meal decide the amount of bioavailable mineral. A large number of studies have been conducted in past to identify the action of individual foods and their active ingredients. Adding orange juice or ascorbic acid to a breakfast has more enhancing effect than adding the same to lunch or dinner on iron bioavailability (Hallberg et al., 1992). This may be due to the fact that breakfast is a simple meal having two or three foods while lunch or dinner contains many more food items eaten at the same time. The present investigation was therefore to study the influence of food ingredients and their combinations on iron and zinc bioavailability in vegetarian meals and compare their performance with non-vegetarian meals.

2. MATERIALS AND METHODS Materials Meals were chosen from reported human absorption studies, NNMB reports, and other literature (Chiplonkar et al., 1993; Ensminger et al., 1994) representing different regions as consumption during breakfast, lunch and dinner. Meals comprised of a variety of cereals, legumes, vegetables, fruits, milk products, oils, sugar, leafy vegetables and ready to eat items from market. Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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Methods Foods were prepared in a traditional manner as observed in each region. For example, for Indian meals, pressure cooking, cereal flours prepared rice and legume as unfermented pancake called as “Roti”, vegetables were cooked with traditional spices, jaggery and oil. Other meals were prepared using canned foods, bakery products from super market for international customers. Individual cooked food items were homogenised and analysed in triplicates for dialysability of zinc and iron under simulated gastrointestinal conditions and using isotopic tracer technique i.e. and as previously reported Chiplonkar et al., 1999). Calorific value of meals was estimated by proximate principle analysis. Bioavailable zinc and iron density was computed as:

Statistical Methods Differences between group means of regions, cereal types or food ingredients were tested by multiple comparison test. Effect of increasing legume contents in meals on bioavailable iron and zinc was tested by regression. Analyses were carried out using SPSS under Windows, version 6.0.

3. RESULTS Zinc contents of the meals were observed to vary in a narrow range (3.64– 4.44mg/l,000kcal) with Indian meals having the highest value and African the lowest (Table 1). Iron contents of Indian and Latin American meals showed significantly higher iron levels than the other regions (p < 0.05). Values of and were significantly lower in Asian meals than the other four regions (p < 0.05) and also lower than nonvegetarian meals (p < 0.05). Although average for pooled vegetarian data was lower than that of nonvegetarian meals the differences were not statistically significant due to large variation within the group (p > 0.1). Further, the differences between vegetarian and nonvegetarian meals were significant only for iron content and and not for zinc content. With respect to zinc contents all cereals were on par except pearl millet (Table 2). Iron contents were highest in pearl millet and lowest in maize and rice. Values for pearl

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millet were significantly (p < 0.01) higher than wheat for all the four parameters. Maize was the highest followed by finger millet, pearl millet, sorghum, rice and wheat for Similarly for pearl millet was the highest, then were finger millet, sorghum, wheat, maize, and rice in decreasing order. Addition of milk or fruit showed no significant change in zinc and (p > 0.1) nor in iron content and values (p > 0.05). Inclusion of green leafy vegetable (GLV), or animal food had significant enhancing effect on both and (Table 3). Contribution of legume more than 5% in a multi-ingredient vegetarian meal was found to have a negative effect on both and (Fig. 1). Interestingly, the variation

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in percent bioavailability was inversely dependant on the number of food ingredients with strong buffering effect in multi-ingredient vegetarian meals that prevented further enhancing or inhibitory effect of certain foods ingredients (Fig. 2).

4. DISCUSSSION In vitro technique has been validated previously by us.5 Meals were chosen from national level databases and the values of bioavailability were obtained by preparing each of the 290 meals in the traditional way in a single laboratory under identical conditions. Thus these data are a valuable frame for relative comparisons between regions, food types and to rank various meals using and Our data showed that if millets could be consumed instead of wheat, maize and rice as the staple, then for the same level of energy consumption, millets would give 3 times more bioavailable iron than rice and 1.5 times more than wheat and maize. In particular a sedentary adult man consuming 2,400 kcal as rice diet will get 1.056 mg of bioavailable iron and 1.824 mg of bioavailable zinc which is 40 and 70% of the requirement respectively. Nevertheless if the same energy is consumed as pearl millet diet then he gets 1.2 times iron than required and 80% of zinc requirement. The value for wheat seems to be low due to inclusion of refined wheat products in some of the meals. Adding a legume will enhance the protein quality but will decrease and One important message obtained from the data is that the enhancing factor containing meal should have least number of food ingredients in order to exert significant enhancing effect.

ACKNOWLEDGMENT Authors wish to thank Director ARI, for providing necessary facilities. This work is part of the project funded by Department of Science and Technology, Government of India (SP/SO/B39/94).

REFERENCES Agte V.V., Gokhale M.K., Paknikar K.M., and Chiplonkar S.A. 1995, Assessment of pearl millet Vs rice based diets for bioavailable trace metals. Plant Foods for Human Nutrition, 48:149–158. Chiplonkar S.A., Agte V.V., and Gokhale M.K. 1993, Zinc, copper and iron contents in cooked foods and esti– mates of their daily intakes in young hostel residents. Ind J Med Res, 98(B):283–289. Chiplonkar S.A., Agte V.V., Tarwadi K.V., and Kavedia R. 1999, In vitro dialysability using meal approach as an index for zinc and iron absorption in humans. Biol Trace Elem Res, 67, 249–256. Ensminger A.H., Ensminger M.E., Konlander J.E., and Robson J.R.K. 1994, Food and Nutrition Encyclopae– dia, CRC Press, Boca Ratan, Ann Arbor, London. Hallberg L., Rossander L., and Brune M. 1992, Prevention of Iron Deficiency by Diet. In: Nutritional Anemias, Nestle Nutrition Workshop series, Vol 30 (S.J. Fomon and S. Zlotkin ed.) pp. 169–181, Vevey/raven Press Ltd. New York. MacPhail P and Bothwell T.H. 1992, The Prevalence and Causes of Nutritional Iron Deficiency Anemia, In: Nutritional Anemias, Nestle Nutrition Workshop series, Vol 30 (Ed.: S.J. Fomon and S. Zlotkin) pp. 1–12, Vevey/Raven Press Ltd. New York.

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IS INCREASING ASCORBIC ACID INTAKE FROM FOODS AN EFFECTIVE STRATEGY FOR IMPROVING DIETARY IRON BIOAVAILABILITY? AN EVALUATION USING DIETARY ALGORITHMS, IRON ISOTOPES, AND A FOOD-BASED COMMUNITY INTERVENTION TRIAL IN RURAL MEXICO

Lindsay H. Allen, Olga Garcia Obregon, Margarita Diaz, Steven Abrams, Suzanne Murphy, and Jorge L. Rosado Program in International Nutrition University of California Davis, California 95616 Department Nutritional Physiology Instituto Nacional de la Nutricion Tlalpan, 14000 Mexico D.F. Children’s Nutrition Research Center 1100 Bates Street, Houston Texas 77030

1. INTRODUCTION More than 50% of women and children living in rural Mexico have iron deficiency or iron deficiency anemia. Their staple diet is based predominantly on tortillas made from whole maize, and beans. These staples are relatively high in iron so that intakes of iron by rural Mexican adults average about 18 mg/d. However, the intake of phytate is over 4,000 mg/d for adult women and 5,000 mg/d for men, as well as other inhibitors of iron absorption such as polyphenols. In addition, the diet contains only small amount of enhancers of iron absorption such as ascorbic acid (AA) and meat, fish or poultry. Thus the rural Mexican diet is high in iron but its bioavailability is very poor, contributing to the high prevalence of anemia and iron deficiency in this population. Increasing ascorbic acid intake has the potential to improve iron absorption and status in this population, because iron intake is high and its bioavailability is low. From Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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published research it is known that increasing AA intake by 25–50 mg per meal approximately doubles iron absorption. However, in none of the clinical trials that tested the effect of increasing AA on iron status was there a significant improvement in iron stores. Those trials lasted from 2 weeks to 2 months. There is no information on the efficacy of this approach for improving iron status at the community level, or using locally-available foods. Testing this efficacy was the purpose of the research presented here.

2. METHODS AND RESULTS Identifying the Source of Ascorbic Acid The strategy used was to first identify food sources of AA in the rural Mexican diet. In a previous study we had collected over 20, 24-h recalls on each of 100 adult women that could be used for this purpose. Foods were selected on the basis of their AA content, frequency of consumption, cost, seasonal availability, and if they contained at least 5 mg AA per usual serving. The best approach was determined to be increasing AA intake from limes in the form of agua de limon, a beverage often served with meals in rural Mexico and prepared with fresh lime juice, water and sugar. No other foods were found to meet our selection criteria. Assessment of the Impact of Ascorbic Acid on Iron Absorption Using Radioisotopes in Non-Anemic Males The next step was to estimate the effect of different doses of AA as agua de limon on iron absorption using radioisotopes with healthy male volunteers at UC Davis. The volunteers were fed meals containing typical Mexican foods (beans, maize tortillas and salsa) that provided similar amounts of phytate, nonheme iron and AA to those in the main meals consumed in the community. Adding 25 mg to 1 meal had no significant effect on iron absorption but when it was added to each of 2 meals iron absorption doubled. Comparison of Results from Radioisotope Studies with those from Simulation Models The impact of AA on iron absorption in the radioisotope studies was compared to that estimated by three simulation models (Murphy et al., 1992; Allen and Ahluwalia, 1997; Tseng et al., 1997). The model published by Tseng et al. came closest to the actual absorption measured in the isotope experiments. Evaluation of the Efficacy of Ascorbic Acid on Iron Absorption Over Two Weeks, Using Stable Isotopes in Iron-Deficient Mexican Women A second study was then conducted in rural Mexico to confirm that the effect of this dose persisted over a 2-week period, during which stable iron isotopes were fed with two meals a day. Again, 25 mg AA as agua de limon given to iron deficient, non-anemic women at each of 2 meals a day doubled iron absorption.

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Community Trial of the Effectiveness of Increasing Ascorbic Acid Intake for Improving the Iron Status of Iron Deficient Mexican Women The above isotope trials all led to the conclusion that increasing AA intake as limeade may be a practical approach to improve iron status in an iron deficient population. The final step was therefore to test the long-term impact of increasing AA intake from agua de limon on the iron status of iron deficient Mexican women in the community, and assess the acceptability of this approach. The study took place in two communities in the same rural region as the previous isotope study, the Solis Valley, 170 km NW of Mexico City. Participants were 36 irondeficient women. The sample size was calculated to detect a significant ferritin increase of 2 ug/L and the expected increase was 15 ug/L. The experimental group received 25 mg of AA as limeade, twice a day, 6 d/wk for 8 mo. The total volume was 500 mL/serving. The control group received 300 mL of a lime-flavored beverage, without any citric or AA, with the same frequency as the experimental group. The AA content was determined in fresh lime juice every day so that exactly 25 mg per container was delivered twice a day, on ice, to each subject’s home. The women knew the beverages had to be consumed with the two main meals of the day, and actual consumption time was checked at each visit. Morbidity data was collected 3 times/week for 8 months by the field workers. Diet intake data was collected using a 3-d, 24 h recall at both the planting and the pre-harvest season. Parasites in fecal samples were monitored twice. Biochemical analyses included Hb, and plasma ferritin (cut-off <12 ug/L), transferrin receptors (TfR), AA, and CRP. In addition, socioeconomic status, medical history and acceptability data were collected using questionnaires. No variables were significantly different between groups at baseline. Neither body weight nor Hb concentration changed significantly during the 8 mo. of the study. However, AA concentration was significantly higher in the experimental group at 2, 4, 6 and 8 months confirming that subjects were consuming the limeade, that AA was absorbed, and the subjects in this group were consuming more AA than in the control group. Ferritin concentrations were only different between groups at 4 and 6 mo, but not at 8 mo. Plasma TfR concentrations were not significantly different between groups at any time point during the 8 months, and TfR:ferritin was significantly lower in the AA group only at 4 mo. Based on mean ferritin and TfR concentrations, both groups remained iron deficient at the end of the study; all subjects were severely depleted of both functional and storage iron in both groups, and remained so throughout the study. The lack of significant differences between groups could not be attributed to the intake of total iron, non-heme or heme iron, or phytate, or to non-dietary factors such as parasites, reported menstrual blood losses or parity. The group receiving ague de limon perceived the beverage to be more acid and volume to be too, but only at baseline. More than 80% of the women in the experimental group said they would not continue to drink the beverage with the same frequency as they did during the intervention, compared to 17% of women in the control group.

3. DISCUSSION In summary, we conclude that limeade was the only food-based strategy in this community that could have provided the necessary amount of AA to be consumed with meals. However, this food-based intervention was not enough to overcome the factors causing

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chronic iron deficiency in this population of iron-deficient women. Moreover, the women would not have been willing to continue consuming this amount of AA as limeade. It is difficult to improve iron status by increasing AA intake alone, even in this potentially optimal situation where the intakes of nonheme iron and phytates were high, usual AA intakes low, and blood loss due to parasites is minimal. Nevertheless it may be possible to give higher amounts of AA in other locations, and if AA intake is increased where foods are fortified with iron this would be expected to have a greater impact on iron status.

ACKNOWLEDGMENTS This study was supported by UC Mexus and USAID-OMNI.

REFERENCES Allen, L.H. and Ahluwalia, N., 1997, The Practical Application of Knowledge Concerning Dietary Iron Bioavailability in Humans, USAID, Washington D.C. Murphy, S.P., Beaton, G.H., and Calloway, D.H., 1992, Estimated mineral intakes of toddlers: predicted prevalence of inadequacy in village populations in Egypt, Kenya and Mexico. Am. J. Clin. Nutr. 56:565– 572. Tseng, M.H., Chakraborty, H., Robinson, D., Mendez, M., and Kohlmeier, L., 1997, Adjustment of iron intake for dietary enhancers and inhibitors in population studies: bioavailable iron in rural and urban residing Russian women and children. J. Nutr. 127:1456–1468.

78a

IRON INTAKE FROM MIXED MEALS IN CAMEROON

A. Bell, F. Ndigui, and P. Effa Research Centre for Food and Nutrition P.O. Box 6163, Yaounde Cameroon

1. INTRODUCTION Anemia due to iron deficiency is a common problem in Cameroon (Cornu et al., 1985). A previous study (Bell et al., 1993) has shown that despite high levels of dietary intake (mean 28 mg/d), the prevalence of iron deficiency is high (37%); suggesting that the level of absorbed iron may be less than 1 mg/d for most Cameroonian feeding patterns. In order to assess the role of dietary habits, iron availability from common meals was investigated.

2. MATERIALS AND METHODS 2.1. Materials On the basis of data from a food survey (Bell et al., 1993), seven snacks and meals were reconstituted: fritters + beans; bread + avocado; rice + peanut sauce with smoked fish (PSF); ripe plantain + PSF with bitter leaves (ndole); cassava tuber + cowpeas (koki); fufu (fermented cassava, FC) + PSF with okra; bobola (FC) + PSF with Corchorus leaves (keleng-keleng). The composition of meals and mean intakes are shown in Table 1.

2.2. Methods Total iron levels were determined with flame AAS after wet ashing. Dialyzable iron was measured afer extrinsic tag using the standardized protocol (Flair, 1992) based on the method of Miller et al. (1981). Differences on iron intake from mixed meals vs single foods were statistically analyzed using the paired t-test. Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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3. RESULTS AND DISCUSSION Results of total iron intake and per cent dialyzable iron are illustrated in Table 2. 3.1. Total Iron Intake Per Day from Mixed Meals Total iron intake increased (by 58 up to 321%) when mixed meals were eaten instead of single foods (except fritters and bread). Values ranged from 8.3 (bread + avocado) to 42.1 mg (fufu + PSF with okra) per total meal consumed per day. 3.2. Dialyzable Iron in Staple Foods and Mixed Meals Per cent dialyzable iron ranged from 0.3 (beans) to 12.4% (bobola). It ranged from 1.0 (bobola + keleng-keleng) to 6.8% (cassava + koki) for mixed meals. Only bread, rice and fermented cassava-based foods showed values above 5%; confirming the assertion that iron availability from african diets is low. Except ripe plantain + ndole and cassava + koki, per cent dialyzable iron decreased in mixed meals: by 75% for fritters + beans (P = 0.166); 40.5% for bread + avocado (P = 0.151); 67.8% for rice + PSF (P = 0.017); 44.4% for fufu + PSF with okra (P = 0.051); and 91.9% for bobola + keleng-keleng (P = 0.001). High levels of phytate in peanut and of contamination iron in open air-dried fish and nuts could explain low iron availability in sauces (Guiro and Hercberg, 1988). In contrary, factors such as presence of organic acids

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in bitter leaves, soaking of cassava tubers and fermentation of dough could explain higher iron dialyzability in ndole, fufu, bobola, fritters and bread. Soaked cowpeas enhanced iron solubility of cassava.

4. CONCLUSIONS These results suggest that Cameroonian dietary patterns can result in lower intake of iron from mixed meals than from staple foods such as rice, fermented cassava-based foods, fritters and bread.

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ACKNOWLEDGMENTS This study is part of a research project (CMR/2/002) financed by IAEA, Vienna.

REFERENCES Bell, A., Rikong-Adie, H., and Hagbe, B., 1993, Iron and zinc intakes and status of an urban population in Cameroon, in: Bioavailability ’93: Nutritional, chemical and food processing implications of nutrient availability, proceedings part 1 (U. Schlemmer, ed.), pp. 248–252. Cornu, A., Pondi-Njiki, O., and Agbor-Egbe, T., 1985, Anémie et malnutrition protéino-énergétique modérée chez l’enfant dans la province du Nord Cameroun, Rev. Sci&Tech. (Sér. Sci. Sté) 2:47–66. Flair concerted action n° 10, 1992, The measurement of micronutrient absorption and status. In-vitro determinations, Agro-industrial Research. Guiro, A. and Hercberg, S., 1988, Iron exchangeability from pearl millet and Senegalese pearl millet meals, Nutr. Repts. Int. 38:231–237. Miller, D.D., Schricker, B.R., Rasmussen, R.R., and Van Campen, D., 1981, An in-vitro method for estimation of iron availability from meals, Am. J. Clin. Nutr. 34:2248–2256.

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BIOAVAILABILITY OF CALCIUM AND ZINC FROM VARIOUS INFANT FORMULAE WITH AND WITHOUT THICKENING AGENTS

Douwina Bosscher, Kristien Van Dyck, Harry Robberecht, Micheline Van Caillie-Bertrand, and Hendrik Deelstra University of Antwerp (UIA) Department of Pharmaceutical Sciences Laboratory of Food Sciences Universiteitsplein 1, B-2610 Antwerp (Wilrijk) Belgium Koningin Paola Kinderziekenhuis Algemeen Ziekenhuis Middelheim (AZM) Department of Pediatric Gastroenterology and Nutrition Lindendreef 1, B-2020 Antwerp Belgium

1. INTRODUCTION Viewpoints on how a baby has to be fed have changed considerably over the last 10 years. Although we recommend human milk as the ideal standard, more than 70% of the babies in Belgium are formula fed from 12 weeks on (ONE, 1998). These adapted formula approximate the composition of human milk as much as possible. For babies suffering from regurgitation however, thickened formulae are recommended. Because regurgitation is a very common complaint in infancy, many babies are fed with thickened feeds from the first months after birth through the second year of age. These commercial formulae may contain different fibers which reach the colon undigested in their original form. They play a major role in the gastro-intestinal tract and may affect the bioavailability of micronutrients and essential trace elements, like calcium and zinc (Ha et al., 1989; Zemel and Zemel, 1985). It is clear that especially during infancy an adequate calcium and zinc intake is of paramount importance (NRV, 1996).

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2. MATERIALS AND METHODS 2.1. Materials Pooled mature human milk was studied. The infant formulae were kindly provided by the manufacturers. During the first months of infancy, first age infant formulae are used. From the age of four to six months on, babies are switched to second age infant formulae. The high viscosity formulae, AR (anti-regurgitation) -formulae, are thickened with locust bean gum (AR-1b) or pregelatinized rice starch (AR-rs). 2.2. Methods The method used is a modification (Shen et al., 1994; Bosscher et al., 1998a) of the continuous flow dialysis in vitro model as developped by Minihane, Fox and Fairweather-Tait (1993) adapted to the upper gastro-intestinal tract of infants younger than six months of age and children from six months on (Bosscher et al., 1998b). The method consists of two phases: a gastric stage and an intestinal stage. Prior to the gastric stage hydrochloric acid (6mol/L) was added to lower the pH of the food sample. To simulate the gastric conditions of infants younger than six months of age the pH was set at 4, while for children from six months on the gastric pH was two. Pepsin was added (3 ml) and the food sample was placed for two hours at 37 °C in a shaking waterbath. The intestinal stage was performed in a stirred cell (Amicon, Beverly, USA), with a dialysis membrane of 1,000 MWCO, and takes two hours and thirty minutes. During the first thirty minutes a gradual pH adjustment from acid to neutral will occur (Shen et al., 1994). After thirty minutes, a pancreatin-bile mixture (15ml) is added to the neutralized food sample and dialysis is continued for another two hours. Calcium and zinc content of the reagentia, samples and dialysate fractions were determined by flame atomic absorption spectrometry (Perkin Elmer, Aanalyst 300, Norwalk, USA). Prior to analysis four portions of about 0.5g of the samples were subjected to a destruction process as described by Robberecht et al. (1994). 2.3. Statistical Analysis The availability data are given as mean values ± SD. Mean availability from non-thickened versus thickened infant food was compared by Student’s t-test for two populations with homogeneous variances. The difference was considered significant at p < 0.05. 3. RESULTS Results of availability study for calcium and zinc are illustrated in Tables 1 and 2. The relative availability of calcium (13.1± 0.8%) and zinc (13.1 ± 0.7%) from human milk proved to be as good or even better than from all the first age infant formulae tested. Availability of calcium and zinc from non-thickened first and second age infant formulae tends to be significantly better in comparison with the corresponding first (calcium: 9.4 ± 0.7%, zinc: 3.2 ± 0.3%) and second age (calcium: 11.9 ± 0.8%, zinc: 6.8 ± 0.4%) products thickened with indigestible carbohydrate (locust bean gum). On the contrary, first age (calcium: 16.6 ± 0.9%, zinc: 4.5 ± 0.4%) and second age infant formula (calcium:

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14.6 ± 0.6%, zinc: 13.5 ± 1.2%) thickened with digestible carbohydrate (pregelatinized rice starch) does not influence the availability.

4. DISCUSSION From this in vitro model, it appears that thickening infant formula powders with indigestible carbohydrate (locust bean gum) reduces availability of calcium and zinc. On

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the contrary, incorporation of digestible carbohydrate (pregelatinized waxy rice starch) as thickening agent in infant formulae does not influence or, in some cases, increases availability of calcium and zinc in comparison with infant formulae of normal viscosity. It is well known that fibers have the capacity to exchange cations. The cation exchange capacity of fiber serves as a reservoir, exchanging polyvalent metal ions for hydrogen at low pH, and being replaced with new cations as they become available when saliva and ingesta are mixed (Van Soest and Jones, 1988). The elemental net-intake depends on the concentration of the element in foodstuffs and the bioavailability of the element. This in turn is greatly influenced by the physical and chemical properties of the different food components. Also the maturation state of the gastro-intestinal tract of the infant will play a major role in determining the bioavailability of micronutrients from foods. It is clear that both in vitro methods used in this study, to simulate the upper gastro-intestinal tract of infants younger than six months of age and children from six months on, are important tools in evaluating the bioavailability of mineral and trace elements from foods (Bosscher et al., 1998b).

ACKNOWLEDGMENTS This study was performed with the financial support of the “Flemisch Institute to promote the scientific-technological research at the industry (IWT)”.

REFERENCES Bosscher, D., Van Dyck, K., Robberecht, H., Van Caillie-Bertrand, M., and Deelstra, H., 1998a, Bioavailability of calcium and zinc from cow’s milk based versus soya-based infant food, Int. J. Food Sci. Nutr. 49:277–283. Bosscher, D., Van Caillie-Bertrand, M., and Deelstra, H., 1998b, Biobeschikbaarheid van nutriënten: optimalisatie in vitro modellen voor kinderen jonger dan drie jaar, Tijdschrift voor Gastro-Enterologie 36:17–27. Ha, Y, Thomas, R., Dyck, L., and Kunkel, M., 1989, Calcium binding of two microalgal polysaccharides and selected industrial hydrocolloids, J. Food Sci. 54:1336–1340. Minihane, A., Fox, T., and Fairweather-Tait, S., 1993, A continuous flow in vitro method to predict bioavailability of Fe from foods, in: Bioavailability ’93: Nutritional Chemical and food processing implications of nutrient availability, proceedings part 2 (U. Schlemmer, ed.), pp 175–179, Bundesforschungsanstalt fur Ernarhung, FRG. Nationale Raad voor de Voeding (NRV), 1996, Voedingsaanbevelingen voor België, Sint-Martens-Latem: G. De Backer, p 77. ONE, 1998; http: //www.ONE.be/donnee/allait 2.htm. Shen, L., Luten, J., Robberecht, H., Bindels, J., and Deeelstra, H., 1994, Modification of an in vitro method for estimating the bioavailability of zinc and calcium from foods, Z. Lebensm. Unters. Forsch. 199:442–445. Van Soest, P. and Jones, L., 1988, Analysis and classification of dietary fibre, in: Trace element Analytical Chemistry in Medicine and Biology, (P. Bratter and P. Schramel, eds.), p 351, W. De Gruyter, New-York. Zemel, M. and Zemel, P., 1985, Effects of food gums on zinc and iron solubility following in vitro digestion, J. Food Sci. 50:547–550.

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ABSORPTION OF SELENIUM FROM BIOSYNTHETICALLY LABELLED FOODS IN HUMANS 1

1

1

1

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C. Atherton , T. Fox , S. Fairweather-Tait , J. Dainty , J. Lewis , 2 2 2 M. Baxter , H. Crews , and N. Langford 1

Institute of Food Research Norwich NR4 7UA UK 2 MAFF CSL Food Science Laboratory Norwich NR4 7UQ, UK

1. INTRODUCTION The absorption and utilization of a mineral element is greatly dependent on the chemical form in which it is present. Selenium is found either as an inorganic form, such as selenite or selenate, or as an organic form, such as selenomethionine or selenocysteine. Foods normally contain the organic forms of selenium, with the inorganic forms only entering the diet as supplements or contaminants (Reilly, 1996). In general, organic forms of selenium have a higher absorption than selenite (Reilly, 1996). There are differences in absorption between the inorganic forms: the transport of selenite across the intestinal brush border occurs through simple diffusion, but selenate appears to be taken up by two specific transport mechanisms, a cotransporter and a exchange mechanism (Wolffram, 1995). The organic forms, selenomethionine and selenocysteine, are absorbed by an amino acid active transport mechanism, using the same pathway as their sulphur containing analogues (Wolffram, 1995). Human studies have shown that selenomethionine has an absorption of more than 90% (Swanson et al., 1991), which is similar to the apparent absorption of selenate, also higher than 90% (Thomson and Robinson, 1986). Selenite, in comparison, has a lower apparent absorption, ranging from 30% (Young et al., 1982; Moser-Veillon et al., 1992) to about 60% (Thomson and Robinson, 1986). In the present study we measured the apparent absorption of selenium from different dietary sources. Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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2. METHODS

2.1. Labelling of Foods Stable isotopes were used to biosynthetically label selenium in different foods. Winter wheat (Triticum aestivum) and garlic were grown hydroponically in a gravel bed with a water recirculation system containing all essential nutrients for growth. Isotopically enriched selenium was added to the nutrient solution as selenious acid. In order to label the wheat, the stable isotope was added at a concentration of 0.1 ppm at influorescence. After 9 months the wheat was harvested and ground to flour. Garlic was labelled by adding at a concentration of 2.5 ppm and harvested after a 30 week period. Cod fish, between 2–3 years of age, were housed in large plastic tanks through which sea water was continuously pumped. Isotopic labelling was carried out by feeding each fish one sprat (that had been injected with of enriched selenious acid) twice per week After a period of 18 months, the cod were killed, filleted and the meat was grilled, homogenized and frozen at –40°C, ready for human consumption. Brewer’s yeast (Saccharomyces cerevisiae) was grown in 1 litre batches in YEPD broth, together with 1.25 mg in the form of selenious acid. The flasks were continuously shaken at 120 rpm (30°C) for 72 hours, after which the broth was centrifuged at 10,000 rpm to retrieve the yeast cells. The yeast was washed several times in 0.9% saline solution and then freeze dried. 2.2. Protocol Ten adult volunteers (five men and five women) took part in the feeding study. All subjects were aged between 20 and 55 years, healthy, non-smokers and had passed a pre-study biochemical screening test. Each subject was asked to consume one test meal of each of the 4 intrinsicallylabelled foods, with a wash-out period of at least 6 weeks between each test meal during which the subject returned to their habitual diet. On the test day, after an overnight fast, a standard low selenium breakfast was provided consisting of apple juice, a bowl of cereal with semi-skimmed milk and a slice of toast with jam (approx. selenium content of The labelled foods were fed at lunch, together with other foods, as an integral part of a meal (cod was fed as fish cakes together with potatoes and okra, wheat was fed as wheat porridge containing milk, yeast as marmite on toast and garlic as garlic bread). At the same time each subject was given an oral dose of of selenite as selenious acid, labelled with in a drink of water. Subjects carried out complete faecal collections starting immediately after the test meal for the next 8 days. A standard low selenium dinner was also supplied to each subject on the test day, consisting of a slice of pizza, a mixed salad, a yoghurt and a flapjack biscuit (approx. selenium content of 2.3. Sample Preparation Faecal samples were frozen, bulked as 24 hours samples, autoclaved and freezedried. The freeze-dried powder was then ground, mixed and subsampled. Faecal samples were digested in a pressurised microwave system and analysed by ICP-MS (Perkin Elmer SCIEX Elan 6,000) for isotope enrichment and total selenium content. Luminal

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disappearance or apparent absorption of the labelled selenium was calculated using a faecal monitoring technique where the difference between oral dose and faecal excretion is measured.

3. RESULTS Isotopic abundances and total selenium for each biosynthetically labelled food were measured by ICP-MS (Table 1). The apparent absorption of selenium from the labelled food and selenite is shown in Fig. 1. Apparent absorption of selenite was significantly different (P < 0.01) on each occasion, with the exception of the selenite fed with the wheat and yeast. Apparent absorption was higher for wheat (82.6% ± 0.89SEM) and garlic (80.4% ± 3.92) than for the cod (59.7% ± 1.40) and yeast (43.8% ± 2.10) (Fig. 1). The apparent absorption between each of the labelled foods was found to be significantly different (P < 0.01), with the exception of wheat and garlic.

4. DISCUSSION Incorporation of the isotope into the plant foods was higher than in the fish, which may be due to the fact that plants, unlike animals, do not have specific physiological requirements for selenium and therefore uptake of the isotope is not controlled.

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Selenite absorption was found to be variable and influenced by constituents of the diet. These findings are supported by work carried out by Christensen et al. (1983), who demonstrated that when selenite was fed with food its absorption was markedly decreased. As selenite was consumed in its free ionic form and is absorbed by passive diffusion, it is more susceptible to chemical reactions occurring in the gut lumen, which depend on the chemical nature of the food with which it was fed. Hence selenite cannot not be used as a reference dose when fed with a test meal. The differences in absorption of selenium present in the foods can probably be explained by its chemical form. Speciation of the different foods is currently being investigated and will provide more information on the effect of the chemical form of selenium on absorption. There was a very small inter-individual variation in apparent absorption within each of the foods, which supports findings of other researchers (Mutanen, 1986; Diplock, 1987), who suggest that there is no homeostatic control of absorption from organic selenium compounds.

ACKNOWLEDGMENTS This work was supported by the Ministry of Agriculture, Fisheries and Food, the Biotechnology and Biological Sciences Research Council and the European Union.

REFERENCES Christensen, M.J., Janghorbani, M., Steinke, F.H., Istfan, N., and Young, V.R., 1983, Simultaneous determination of absorption of selenium from poultry meat and selenite in young men: application of a triple stable-isotope method, Br. J. Nutr. 50:43–50. Diplock, A.T., 1987, Trace elements in human health with special reference to selenium, Am. J. Clin. Nutr. 45:1313–1322. Moser-Veillon, P.B., Mangels, A.R., Patterson, K.Y., and Veillon, C., 1992, Utilization of two different chemical forms of selenium during lactation using stable isotope tracers: an example of speciation in nutrition, Analyst 117:559–562. Mutanen, M., 1986, Bioavailability of selenium, Ann. Clin. Res. 18:48–54. Reilly, C., 1996, Selenium in health and disease, Blackie Academic and Professional, London. Swanson, C.A., Patterson, B.H., Levander, O.A., Veillon, C., Taylor, P.R., Helzlsouer, K., McAdam, P.A., and Zech, L.A., 1991, Human [74Se]selenomethionine metabolism: a kinetic model, Am. J. Clin. Nutr. 54:917–926. Thomson, C.D. and Robinson, M.F., 1986, Urinary and fecal excretions and absorption of a large supplement of selenium: superiority of selenate over selenite, Am. J. Clin. Nutr. 44:659–663. Wolffram, S., 1995, Mechanismen der intestinalen absorption von selen, Medizinische klinik 90(S1): l–5. Young, V.R., Nahapetian, A., and Janghorbani, M., 1982, Selenium bioavailability with reference to human nutrition, Am. J. Clin. Nutr. 35:1076–1088.

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TISSUE ZINC UPTAKE AS A MEASURE OF THE RELATIVE BIOAVAILABILITY OF SUPPLEMENTAL ZINC SOURCES FOR DOMESTIC ANIMALS

C. B. Ammerman, M. Sandoval, P. R. Henry, R. C. Littell, and R. D. Miles Departments of Animal Science Statistics, and Dairy/Poultry Sciences University of Florida Gainesville, Florida 32611-0900 USA

1. INTRODUCTION Zinc is an essential micromineral element for domestic animals and the frequently used dietary ingredients do not always supply adequate zinc to meet the animals’ requirement for the element. Thus, supplemental zinc sources are required and the bioavailability of the zinc in those sources is important (Baker and Ammerman, 1995).

2. MATERIALS AND METHODS A series of studies has been conducted at the University of Florida in which elevated dietary levels of zinc from supplemental sources were fed to broiler chickens and growing lambs and the increase in tissue zinc was used as an indicator of zinc bioavailability. In initial studies with day-old chicks, supplemental dietary zinc concentrations up to l,500 ppm were used and tissues including tibia, liver and kidney were tested for sensitivity as indicated by zinc accumulation (Henry et al., 1987). Two bioavailability studies were conducted with chicks (Sandoval et al., 1997) in which reagent grade or feed grade zinc sources were fed at dietary concentrations up to 900 or l,200 ppm for 20 or 21 days. Sandoval et al. (1997) conducted studies with growing wether lambs in which as much as 2,100 ppm supplemental dietary zinc as either reagent grade or feed grade sources were fed for about 21 days. A standard curve assay was used in the studies with a single point Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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addition of the test material. Six lambs or 3 pens of 7 chicks each were used per treatment.

3. RESULTS AND DISCUSSION Results of the bioavailability studies are summarized in Table 1. All numbers are presented as relative bioavailability values expressed in relation to the response obtained with reagent grade zinc sulfate which was set at 100. The most sensitive response criterion to elevated dietary zinc in poultry was tibia zinc. Relative bioavailability estimates for poultry based on bone zinc were 78, 77 and 46% for reagent grade zinc carbonate, zinc oxide and zinc metal, respectively (P < 0.05). In a similar study with chicks, relative values were 99, 87 and 54% for feed grade zinc sulfate and feed grade zinc oxides A and B, respectively (P < 0.05). As opposed to the results obtained with the young chick, accumulation of zinc in the liver was the most sensitive response criterion to elevated dietary zinc in growing sheep (Henry et al., 1996). Other tissue responses examined were kidney, spleen, muscle, heart and bone. Multiple linear regression slope ratios of log 10 transformed concentration of zinc in liver gave relative bioavailability estimates for the reagent grade products of, 104 for carbonate, 103 for oxide and 68 for metal. In the second study with feed grade test products, bioavailability values were 83, 80 and 74 for sulfate and oxides A and B, respectively. Utilization estimates obtained with lambs did not differ significantly (P > 0.05). Reagent grade and feed grade supplemental zinc sources ranked numerically similar in relative bioavailability whether estimated with poultry or sheep. The day-old chick was a more sensitive test animal in detecting differences among zinc sources than was the growing lamb averaging approximately 40kg in body weight. Significant linear increases in bone zinc were observed in chicks whereas virtually no increase in bone zinc occurred with supplemental zinc in lambs. Liver zinc was the most responsive tissue to dietary zinc in lambs.

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REFERENCES Baker, D.H. and Ammerman, C.B., 1995, Zinc bioavailability, in: Bioavailability of Nutrients for Animals: Amino Acids, Minerals, and Vitamins, (Ammerman, C.B., D.H. Baker, and A.J. Lewis, eds.), pp. 367–398, Academic Press, San Diego, CA. Henry, P.R., Ammerman, C.B., and Miles, R.D., 1987, Effect of dietary zinc on tissue mineral concentration as a measure of zinc bioavailability in chicks, Nutr. Rep. Int. 35:15–23. Henry, P.R., Littell, R.C., and Ammerman, C.B., 1996, Effect of high dietary zinc concentration and length of zinc feeding on feed intake and tissue zinc concentration in sheep, Anim. Feed Sci. Technol. 66:237–245. Sandoval, M., Henry, P.R., Ammerman, C.B., Miles, R.D., and Littell, R.C., 1997a, Relative bioavailability of supplemental inorganic zinc sources for chicks, J. Anim. Sci. 75:3195–3205. Sandoval, M., Henry, P.R., Littell, R.C., Cousins, R.J., and Ammerman, C.B., 1997b, Estimation of the relative bioavailability of zinc from inorganic zinc sources for sheep, Anim. Feed Sci. Technol. 66:223–235.

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EFFECTS OF WHOLE WHEAT FLOUR AND FERMENTABLE CARBOHYDRATES ON INTESTINAL ABSORPTION OF TRACE ELEMENTS IN RATS

C. Coudray, H. W. Lopez, M. A. Levrat-Verny, J. Bellanger, C. Rémésy, and Y. Rayssiguier Centre de Recherche en Nutrition Humaine d’Auvergne Laboratoire Maladies Métaboliques et Micronutriments INRA Clermont-Ferrand/Theix 63122 Saint Genès Champanelle France

1. INTRODUCTION More than fifty percent of the world’s total food energy is supplied by grain species. They are the major source of starch together with fibers, and they also contain considerable amounts of minerals and vitamins (O’Dell et al., 1972; Stephen, 1994). Because of their high in vitro mineral binding capacity (Claye et al., 1996), dietary fibers in brown bread or wheat bran have been often suspected to impair mineral absorption in animal and man (Torre et al., 1991; Donangelo and Eggum, 1986). Phytic acid (PA) is an active mineral-binding component responsible for mineral absorption impairment (Brune et al., 1992; Morris and Ellis, 1980; Davies et al., 1977; Torre et al., 1991). To improve mineral bioavailability by eliminating fibers and PA, grains are subjected to various types of processing, which may alter the nutritional value of foods (Salovaara, 1993). However, such a refining results in significant nutrient losses, in particular, minerals and vitamins. Twenty years ago, our laboratory has shown that fermentable carbohydrates may improve intestinal absorption of magnesium in rats (Rayssiguier and Rémésy, 1977). Since, many literature data reported a favorable effect of many type of fermentable carbohydrates on the absorption and the status of several minerals including in animals and man (Coudray et al., 1997; Lopez et al., 1998). Fermentable carbohydrates may have a positive effect on mineral absorption, by improving their solubility in the large intestine (Younès et al., 1996). Consequently, in a first study, we re-examined the effect of whole flour and white flour on the absorption and status of trace elements in rats. We then studied the effects of WB, an important component of Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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the whole flour, associated or not to fermentable carbohydrates on trace element absorption (Fe, Zn, Cu).

2. MATERIAL AND METHODS Wheat flours and wheat bran were purchased from a local Massagette Mill, Massagette, France. Casein was purchased from Louis François, St Maur, France. and phytic acid were purchased from Sigma Chemical, St Louis, MO. NaCl was purchased from Merck, Darmstadt, Germany. All other chemicals were of the highest quality available. Distilled water was used throughout.

First Experiment Whole and white wheat flour effect: Twenty male Wistar rats of 150–160 g were adapted to the two experimental diets for 21d. The diets contained 87.5% of French variety wheat flour (white or whole). They were supplemented with 6% casein, 5% peanut oil. Because flours are generally poor in calcium and sodium, the diets were added of 7.5 g/kg and 7g/kg NaCl. No vitamins were added because wheat flour provided sufficient to meet essential requirements. Daily food consumption and body weight were recorded. The feces were collected over the balance period. Second Experiment Wheat bran (WB) and raw potato resistant starch (RS) effect: 40 male Wistar rats, weighing 150–160g, were used. They were fed one of the four diets A: purified diet, B: A+ 20% WB, C: A+ 20% RS, D: A+ 20% WB and 20% RS, for 21 d. Daily food consumption and body weight were recorded. Because WB is very rich in minerals, the four experimental diets were adjusted to the same level of trace elements (mg/kg): 45 Fe, 40 Zn and 5 Cu. During the balance period, rats were individually housed in metabolic cages fitted with urine/feces separators suitable for feces collecting. Sample Treatment and Analysis In both experiments, rats were anaesthetized with sodium pentobarbital and blood was drawn from the abdominal aorta. The cecum with content was removed and weighed. Cecal contents were transferred, and the cecal wall was weighed. Feces were dried and powdered. Phytic acid was measured by a colorimetric procedure, as described by Latta and Eskin (1980). Short Chain Fatty Acids (SCFA) were measured by gas-liquid chromatography (Demigné et al., 1980). Transferrin saturation percent and plasma iron were determined using Ferrimat-Kit and TIBC additif from Bio Mérieux (Lyon, France). To determine Fe, Zn and Cu levels in liver, food and feces, 0.25 to 0.5 g of dried samples were dry-ashed (l0h at 500°) and then extracted at 130° in (2/1) (Merck, Suprapur) until decoloration; final dilution was made in 0.5 M The trace elements were measured after a 5-fold plasma dilution in HCl and trichloroacetic acid for protein elimination. Mineral concentrations were determined by atomic absorption spectrophotometry (Perkin-Elmer 560, Norwalk, CT) in an acetylene-air flame at the following wavelengths: 248 (Fe), 214 (Zn) and 325 (Cu).

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Statistical Analysis Standard procedures were used to calculate means and standard error (SEM). Differences were considered as significant between groups when p < 0.05.

3. RESULTS AND DISCUSSION Whole and White Wheat Flour Effect The whole flour diet, compared to the white flour diet, contained almost 4-fold higher level of fiber and phytic acid. Mineral diet analysis showed that whole flour diet was richer in Fe, Zn and Cu (2-fold) than the white flour diet. The whole flour diet met dietary mineral recommendations for rats, whereas the white flour diet was deficient in all of the measured minerals. The whole flour diet was consumed in significantly larger quantity and the weight gain of rats fed this diet was significantly higher (5.8 ± 0.3 vs. 4.2 ± 0.2 g/d) than that of the rats fed the white flour diet. The daily intake and the apparent absorption percent of Fe were higher with the whole flour diet compared to the white flour diet. Consequently, the daily apparently absorbed Fe amount (mg/d) was more than doubled with the whole flour diet. Plasma Fe level and transferrin saturation percent as well as liver Fe level in rats fed whole flour diet were twice as high as in the white flour group. The daily Zn intake was doubled with the whole flour diet, compared to the white flour diet, but its apparent absorption percent was significantly decreased (–30%) in the first group. However, the daily apparently absorbed Zn amount (mg/d) increased by about 40%, in the rats receiving the whole flour diet compared to those fed the white flour diet. Finally, the Cu intake and the percentage of apparent absorption of Cu were higher with the whole flour diet. Consequently, these rats absorbed 5 fold more Cu than those fed white flour diet. However, plasma or tissue Cu and Zn concentrations were not significantly different between the two groups of rats. This reflects the high control of Zn and Cu homeostasis at low variations in their intake (Kirchgessner, 1993).

Wheat Bran and Resistant Starch Effect Body weight gain and daily food intake were not altered by the diet conditions. The present data indicate that 41% of dietary Fe was absorbed in rats fed the control diet (Table 2). Apparent Fe absorption was not modified by WB ingestion. On the other hand, this absorption was significantly enhanced in the presence of RS in the diet, in presence

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or absence of WB. In rats fed the control diet, about 20% of ingested Zn was apparently absorbed and up to 30% in rats fed RS alone. However, in rats fed WB diet, Zn absorption was significantly depressed. In the presence of RS in the diets, the inhibitory effect of WB on Zn absorption was neutralized and Zn absorption was the highest in rats fed the WB + RS diet. In rats fed the control diet, 16% of dietary Cu was absorbed and this absorption was significantly reduced when WB was added in the diet. However, RS addition significantly increased Cu absorption in rats fed the WB or the control diets. Indeed, the RS but not PA ingestion resulted in a significant enlargement of the cecum and hypertrophy of the cecal wall. RS ingestion led also to significantly high SCFA concentrations with an acidic cecal compared to the other groups, indicating high fermentescibility. Rats receiving WB diets had a significant intake of PA as well as a measurable PA fecal excretion, with a higher breakdown of PA (+30%) in the presence of RS. 4. CONCLUSION If these results can not be directly extrapolated to human nutrition, they show that whole flour or unrefined cereal products ingestion can contribute to improved mineral balance. The cecal fermantation of soluble carbohydrates present in these products may be responsible for such mineral absorption enhancement and adequate mineral balance. Several epidemiological and clinical studies have recently shown growing interest in increasing the consumption of phytic acid-rich products in preventive nutrition. Negative effects of such products on mineral bioavailability may be neutralized when these products are taken together with the other components of the meal. It is thus possible to promote the consumption of whole grains rather than of purified cereal products, to keep functionally active constituents of grains and to optimize the mineral status in humans. Human studies are still needed to confirm these rat results. REFERENCES Brune, M., Rossander-Hultén, L., Hallberg, L., Gleerup, A., and Sandberg, A.S., 1992, Iron absorption from bread in humans: inhibiting effects of cereal fiber, phytate and inositol phosphates with different numbers of phosphate groups. J. Nutr. 122:442–449. Claye, S.S., Idouraine, A., and Weber, C.W., 1996, In vitro mineral binding capacity of five fiber sources and their insoluble components for copper and zinc. Plant Foods Hum. Nutr. 49:257–269.

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Coudray, C., Bellanger, J., Castiglia-Delavaud, C., Rémésy, C., Vermorel, M., and Rayssiguier, Y., 1997, Effect of soluble and insoluble dietary fiber supplementation in healthy young men: apparent absorption and balance of calcium, magnesium, iron and zinc. Europ. J. Clin. Nutr. 51:375–380. Davies, N.T., Hristic, V., and Flett, A., 1977, Phytate rather than fibre in bran as the major determinant of zinc availability to rats. Nutr. Rep. Int. 15:207–214. Demigné, C., Rémésy, C., and Rayssiguier, Y., 1980, Effects of fermentable carbohydrates on volatile fatty acids, ammonia and mineral absorption in the rat caecum. Reprod. Nutr. develop. 20:1351–1359. Donangelo, C.M. and Eggum, B.O., 1986, Comparative effects of wheat bran and barley husk on nutrient utilization in rats. 2. Zinc, calcium and phosphorus. Br. J. Nutr. 56:269–280. Kirchgessner, M., 1993, Homeostasis and homeorhesis in trace element metabolism. In: Trace element in man and animals. Volume 8, (M. Anke, D. Meissner, and C.F. Mills, eds.), pp. 4–21, Verlag Media Touristik. Latta, M. and Eskin, M., 1980, Simple and rapid colorimetric method for phytate determination. J. Agric. Food Chem. 28:1313–1315. Lopez, H.W., Coudray, C., Bellanger, J., Younes, H., Demigné, C., and Rémésy, C., 1998, Intestinal fermentation lessens the inhibitory effects of phytic acid on mineral utilization in rats. J. Nutr. 128:1192– 1198. Morris, E.R. and Ellis, R., 1980, Bioavailability to rats of iron and zinc in wheat bran: response to low-phytate bran and effect of the phytate/zinc molar ration. J. Nutr. 110:2000–2010. O’Dell, B.L., De Borland, A.R., and Koirtyohann, S.R., 1972, Distribution of phytate and nutritionally important elements among the morphological components of cereal grains. J. Agr. Food Chem. 20:718– 721. Rayssiguier, Y. and Rémésy, C., 1977, Magnesium absorption in the caecum of rats related to volatile fatty acids production. Ann. Rech. Vét. 8:105–110. Salovaara, H., 1993, Cereals, in: Encyclopaedia of food science, food technology and nutrition, volume 2 (R. Macrae, R.K. Robinson, and M.J. Sadler, eds.), pp. 768–772, Academic press, London, UK. Stephen, A.M., 1994, Whole grains: impact of consuming whole grains on physiological effects of dietary fiber and starch. Crit. Rev. Food Sci. Nutr. 34:499–511. Torre, M., Rodriguez, A.R., an Saura-Calixto, F., 1991, Effects of dietary fiber and phytic acid on mineral availability. Crit. Rev. Food Sci. Nutr. 30:1–22. Younes, H., Demigné, C., and Rémésy, C., 1996, Acidic fermentation in the caecum increases absorption of calcium and magnesium in the large intestine of the rat. Br. J. Nutr. 75:301–314.

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BIOAVAILABILITY OF DIFFERENT SOURCES OF PROTECTED ZINC

Daniel D. Gallaher*, Cynthia M. Gallaher*, Stephanie Shulman*, Andrea McElhome*, Kyle A. Brokken#, and Gerry Shurson† Departments of Food Science and Nutrition* and Animal Science† University of Minnesota St. Paul, Minnesota 55108 USA and Quali Tech, Inc.# Chaska, Minnesota 55318 USA

1. INTRODUCTION Protected trace elements are inorganic trace elements that are either chelated or complexed to, or encapsulated within an organic molecule. Protected trace elements were developed as a means to improve absorption of trace elements. The organic molecule(s) with which a protected trace element is associated presently fall within one of three groups: a protein hydrolyzate, an amino acid or mixture of amino acids, or a polysaccharide. Although trace elements bound to organic molecules have been commercially available for a number of years, few studies have examined the bioavailability of protected trace elements. Scholmerich et al. (1987) reported that zinc complexed to histidine was absorbed better than zinc sulfate in humans. Barrie et al. (1987) reported that zinc complexed to picolinate was absorbed better than either zinc citrate or zinc gluconate in humans. However, uptake of zinc intestinally perfused into mice did not differ between zinc chloride and zinc methionine (Beutler et al., 1998). The objective of the present study was to compare the relative bioavailability of three protected forms to zinc to an inorganic form of zinc when fed as part of a diet containing marginal concentrations of zinc. Each diet was fed either with or without added phytic acid, in order to ascertain whether the protected forms of zinc would improve bioavailability in the presence of this known inhibitor of zinc absorption.

Address all correspondence to: Dr. Daniel D. Gallaher, Department of Food Science and Nutrition, University of Minnesota, St. Paul, Minnesota 55108 USA; telephone: 612-624-0746; fax: 612-625-5272; email: [email protected] Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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2. MATERIALS AND METHODS 2.1. Methods Male weanling Wistar rats were fed diets containing 12 ppm zinc, which is approximately the minimum zinc level for normal growth in rats. The basal diet had the following composition (g/kg): egg white solids, 200; dextrose, 630.4658; corn oil, 100.0; cellulose, 30.0; vitamin mix (Teklad #40060, Harlan Teklad, Madison, WI USA), 10.0; biotin, 0.004; ethoxyquin, 0.02; mineral mix (Zn-free) (Teklad #81264), 25.6902; chromium potassium sulfate, 0.02; and calcium carbonate, 3.8. Zinc was supplied as either a zinc salt, or one of three different protected sources of zinc— zinc polysaccharide (SQM, Quali Tech, Inc., Chaska, MN), zinc methionine (ZinPro, ZinPro Corporation, Edina, MN), and zinc amino acid complex (Availa Zinc, ZinPro Corporation, Edina, MN). Each diet was fed with or without added phytic acid (300mg/kg diet; phytate : Zn molar ratio of 25:1), a component of cereals and other food products known to reduce zinc absorption. As a positive control, one group was fed 38 ppm Zn, as Diets were fed for three weeks. Body weights and food intake were measured weekly. At the end of three weeks, animals were fasted overnight, anesthetized, and blood removed by cardiac puncture in heparinized syringes. Plasma was collected by centrifugation and stored frozen until analyzed for alkaline phosphatase activity (procedure No. 104, Sigma Chemical Co., St. Louis, MO USA). Erythrocytes were washed in saline and frozen until assayed for superoxide dismutase activity by a modification of the method of Hill (1996). Livers and femurs were subsequently excised, femurs cleaned of adhering tissue, and both tissues weighed and frozen. Livers were wetashed with concentrated nitric acid and femurs dry-ashed at 400°C for 24h, followed by solubilization in 20% HCl. Zinc concentration was determined by atomic absorption spectrophotometry. 2.2. Statistical Analysis The data were analyzed by twoway analysis of variance, with zinc form and presence or absence of phytate as the main effects. The group fed 38ppm zinc are shown as a reference but were excluded from the statistical analysis.

3. RESULTS Growth is one of the most sensitive, possibly the most sensitive indicator of the adequacy of zinc in the diet. Figure 1 shows the effect of the dietary treatments on final body weight. There was a statistically significant effect of zinc form, a trend for a decrease in final body weight with the presence of phytate, and a trend towards a interaction between zinc form and presence of phytate. Animals fed zinc polysaccharide (SQM Zn) had greatest final body weight, which was significantly different from both the zinc sulfate and zinc amino acid complex (Availa Zn) groups; however, the difference from zinc methionine (ZinPro Zn) did not achieve statistical significance. As expected, there was a significant reduction in final body weight in phytate-fed animals compared to the diet without phytate in animals fed zinc sulfate (by Student’s t-test). However, no body weight reduction was evident in phytate-fed animals fed zinc polysaccharide, suggesting that this form of zinc overcomes the inhibitory effect of dietary phytate.

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Bone zinc is also a sensitive indicator of zinc status. Femur weight was not affected by either dietary phytate or the form of zinc fed (data not shown). However, as seen in Fig. 2, animals fed phytate had greatly reduced zinc in their femurs, regardless of the form of zinc fed. Overall, animals fed zinc polysaccharide (SQM Zn) had a greater amount of zinc in their femurs than did those fed either zinc sulfate or the zinc amino acid complex (Availa Zn), with animals fed zinc methionine (ZinPro Zn) having

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an intermediate amount of bone zinc. Thus, in terms of zinc form, the pattern for bone zinc was similar to that of final body weight. Liver weight was not significantly affected by the presence of dietary phytate. However, animals fed zinc polysaccharide had a significantly greater liver weight than those fed either zinc sulfate or the zinc amino acid complex. Liver weight from animals fed zinc methionine had an intermediate value, which was not significantly different from the other zinc forms (data not shown). As shown in Fig. 3, total liver zinc was unaffected by dietary phytate but was significantly affected by the form of dietary zinc. Animals fed zinc polysaccharide (SQM Zn) had a significantly greater amount of liver zinc than animals fed zinc sulfate, zinc methionine (ZinPro Zn), or the zinc amino acid complex (Availa Zn). Erthryrocyte superoxide dismutase (SOD) activity and plasma alkaline phosphatase activity were both measured as potential markers of zinc status. SOD activity was unaffected by either phytate or zinc form (data not shown). Alkaline phosphatase activity was slightly but significantly reduced by phytate, but was not affected by zinc form (data not shown). In summary, in rats fed diets containing 12ppm zinc, those fed zinc polysaccharide (SQM Zn) had significantly greater final body weight, total bone zinc and total liver zinc compared to zinc sulfate or zinc amino acid complex (Availa Zn) as well as greater total liver zinc compared to animals fed zinc methionine (ZinPro Zn).

4. DISCUSSION These results indicate that protected forms of zinc vary in their efficacy; zinc encapsulated within a carbohydrate of alginate appeared superior to zinc sulfate or an amino acid complex of zinc using several different indices of zinc status. The reasons for these differences are not apparent from this study, but may involve differences in the strength of zinc binding to different organic molecules.

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ACKNOWLEDGMENTS This study was performed with the financial support of Quali Tech, Inc. and the University of Minnesota Agricultural Experiment Station.

REFERENCES Scholmerich, J., Freudemann, A., Kottgen, E., Wietholtz, H., Steiert, B., Lohle, E., Haussinger, D., and Gerok, W., 1987, Bioavailability of zinc from zinc-histidine complexes. I. Comparison with zinc sulfate in healthy men, Am. J. Clin. Nutr. 45:1480–1486. Barrie, S.A., Wright, J.V., Pizzorno, J.E., Kutter, E., and Barron, P.C., 1987, Comparative absorption of zinc picolinate, zinc citrate and zinc gluconate in humans, Agents Actions 21:223–228. Beutler, K.T., Pankewycz, O., and Brautigan, D.L., 1998, Equivalent uptake of organic and inorganic zinc by monkey kidney fibroblasts, human intestinal epithelial cells, or perfused mouse intestine, Biol. Trace Elem. Res. 61:19–31. Hill, G.M., 1996, NCR-42 Superoxide dismutase determination procedure. Michigan State University.

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COBALT DEFICIENCY INDUCED HYPERHOMOCYSTEINEMIA AND OXIDATIVE STATUS OF CATTLE

G. I. Stangl, F. J. Schwarz, and M. Kirchgessner Institute of Nutrition Sciences Technical University of Munich 85350 Freising-Weihenstephan Germany

1. INTRODUCTION Ruminants normally do not have any dietary source of vitamin and therefore rely entirely on rumen microorganisms for their supply of this vitamin. Synthesis of vitamin in rumen is dependent on a continuous supply of dietary cobalt, and longterm consumption of diets containing inadequate amounts of cobalt leads to vitamin deficiency in ruminants. Among the function of vitamin as cofactor for methylmalonyl-CoA mutase (EC 5.4.99.2), vitamin is needed for the re-synthesis of methionine by methylation of homocysteine via the methionine synthase (EC 2.1.1.13). Thus, vitamin deficiency is characterized by elevations in the plasma concentration of homocysteine which is apparent from previous studies with vitamin deficient fruit bats (Van der Westhuyzen et al., 1985), pigs (Young et al., 1997), and lambs (Kennedy et al., 1994). Recently, there have been also found associations between hyperhomocysteinemia and oxidative alterations of lipids and proteins (Durand et al., 1996; Ueland et al., 1996). Thus, the current study was undertaken to investigate i) the magnitude of biochemical disturbances in cattle moderately depleted of cobalt ii) the appearance of hyperhomocysteinemia in those animals, and iii) the possible role of homocysteine in the development of oxidative stress.

2. MATERIALS AND METHODS Animals and Diets Twenty-one male cattle of the German Simmental breed with an average body weight of 207 ± 4kg were randomly allocated to two groups of 11 and 10 animals. The Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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groups were fed on a corn silage-based diet which was either cobalt-sufficient supplemented as or cobalt-deficient The duration of the experiment was 43 weeks. The basal diet was supplemented with sufficient amounts of minerals and vitamins according to recommended guidelines (NRC, 1996). The animals were individually fed using electronically controlled feeders and were allowed free access to water and corn silage. Feed refusals were collected and weighed every day. During the whole experiment, the cattle were weighed in two-week-intervals, before feeding. At week 43, 18–20h after the last feeding, all cattle were slaughtered, and blood and liver were excised. The parameters used to assess cobalt deficiency were growth development, vitamin and folate in plasma and liver, and the methylmalonic acid (MMA) and homocysteine levels in plasma. The parameters used to assess oxidative/antioxidative balance were thiobarbituric acid reactive substances (TBARS), as a measure of lipid peroxidation, thiol groups and carbonyl formation as markers for protein oxidation, tocopherol and catalase activity as factors with antioxidative defense potential, and heavy metals as catalysts of oxidative stress.

Analyses Trace element analysis of the diet and livers were done with an atomic absorption spectrophotometer after dry-ashing the samples at 480 °C. Plasma and liver concentrations of vitamin and folate were determined using a SimulTRAC-SNB radioassay kit (ICN Pharmaceuticals, Costa Mesa, CA). Plasma MMA concentration was determined using a capillary gas chromatographic method (McMurray et al., 1986). The other variables measured were done by methods described recently (Stangl et al., 1999). Plasma levels of homocysteine and tocopherol, and the thiobarbituric acid-reactive substances (TBARS) in liver microsomes were determined by high-performance liquid chromatography. The activity of catalase (EC 1.11.1.6), the determination of total thiol groups in plasma (from protein and glutathione), and the measurement of carbonyl formation, an early marker for protein oxidation were done by spectrophotometric methods.

Statistics For evaluation of data analysis of variance was used. The effect of dietary cobalt on vitamin folate, MMA and homocysteine and markers of the oxidative/antioxidative balance was tested for statistical significance (P < 0.05) by the Student’s t-test. Data are represented as means ± standard deviation of the single value (SD).

3. RESULTS Cobalt deficiency in cattle was obvious by a loss of appetite and diminished growth gain. The daily voluntary feed intake of the cattle fed the cobalt-deficient diet was reduced by 18% on average compared to that of the cobalt-sufficient controls (6.24 ± 0.42kg vs. 7.65 ± 0.28kg). At the end of the study at 43 week, the cobalt-sufficient animals were about 3.1-fold of their live weight at week 0 (from 207 ± 17kg to 636 ± 32kg). In contrast, the live weight of the animals fed the cobalt-deficient diet increased more slowly and was at the end of experimental period only 2.6-fold of their live weight at week 0 (from 206 ± 15kg to 535 ± 39kg). Analysis of plasma and liver demonstrate that vitamin concentrations were clearly diminished in prolonged cobalt deficiency, accompanied

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by a slight but statistical significant reduction of liver folate (Table 1). MMA and homocysteine levels in plasma were in excess of 15 and 4.8 times, respectively higher in cobaltdeficient cattle than in controls (Table 1). The antioxidant/prooxidant balance of the cobalt-sufficient and the cobalt-deficient groups was represented in Table 2. A slight change in oxidative status was reflected by the protein carbonyl groups which tended to be higher in tissue vitamin deficiency. The concentration of plasma thiols did not significantly differ between the groups. Liver concentrations of tocopherol, microsomal TBARS and carbonyl groups remained also unaffected by cobalt deficiency induced hyperhomocysteinemia. Catalase activity in the liver of the cobalt-deficient cattle was 11% lower than corresponding values of the cobaltsufficient cattle. However, the concentrations of iron and nickel were considerable elevated in livers of cobalt-deprived cattle. Hepatic iron was increased by 38%, and hepatic nickel was 2.7-fold higher in cobalt-deprived cattle relative to the controls. Copper levels in liver were not influenced by cobalt deprivation.

4. DISCUSSION From the foregoing observations it is evident that, although cobalt-deficient cattle used in this study were only moderately depleted of cobalt because their diet contained

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and are already considered adequate for cattle (NRC, 1996), they developed distinct accumulations of MMA and homocysteine in plasma. The much increased levels of these variables observed with the cobalt-deprived animals emphasizes the use of MMA and homocysteine as extreme sensitive indices for the diagnosis of cobalt deficiency in cattle. The ability of homocysteine to generate hydrogen peroxide has been implicated as potential mechanism leading to endothelial dysfunction which was observed in subjects with hyperhomocysteinemia (Kang et al., 1986), and it has been unequivocally demonstrated that hyperhomocysteinemia induced rapid changes in plasma redox thiol status, probably via thiol-disulphide exchange and redox reactions (Ueland et al., 1996). According to man, also animals with hyperhomocysteinemia have been shown to develop increased lipid peroxidation as measured by elevated levels of cardiac TBARS in rats (Brown and Strain, 1990) and elevated levels of cardiac malondialdehyde in pigs (Young et al., 1997). However, from the foregoing observations it is evident that, although homocysteine in plasma was in excess of 4.8 times higher in cobaltdeprived cattle than in controls, no abnormalities of the antioxidant/prooxidant balance as measured by TBARS, tocopherol, thiol and carbonyl groups, were observed. The slight change in the concentration of plasma carbonyl groups, an early marker for protein oxidation, may indicate incipient protein degradation. The significant change in the activity of the heme-enzyme catalase might probably result from a decreased heme formation observed with vitamin rather than from hyperhomocysteinemia. Although, the present findings are in contrast with some studies, there are also a number of experiments indicating that homocysteine did not act as trigger for changes in oxidative status (e.g. Blom et al., 1995; Mele and Meucci, 1996). It can be concluded from the foregoing observations that hyper-homocysteinemia, along with a slight reduction of hepatic catalase activity and an accretion of iron and nickel do not induce distinct prooxidative situation in cattle.

REFERENCES Blom, H.J., Kleinveld, H.A., Boers, G.H., Demacker, P.N.M., Hak-Lemmers, H.L.M., Te Poele Pothoff, M.T.W.B., and Trijebels, J.M.F., 1995, Lipid peroxidation and susceptibility of low-density lipoprotein to in vitro oxidation hyperhomocysteinaemia, Eur. J. Clin. Invest. 25:149–154. Brown, J.C.W. and Strain, J.J., 1990, Effect of dietary homocysteine on copper status in rats, J. Nutr. 120:1068–1074. Durand, P., Prost, M., and Blache, D., 1996, Pro-thrombotic effects of a folic acid deficient diet in rat platelets and macrophages related to elevated homocysteine and decreased n-3 polyunsaturated fatty acids, Atherosclerosis 121:231–243. Kang, S.S., Wong, P.W., Cook, H.Y., Norusis, M., and Messer, J.V., 1986, Protein-bound homo-cyst(e)ine. A possible risk factor for coronary artery disease, J. Clin. Invest. 77:1482–1486. Kennedy, D.G., Young, P.B., Blanchflower, W.J., Scott, J.M., Weir, D., Molloy, A., and Kennedy, S., 1994, Cobalt-vitamin B12 deficiency causes lipid accumulation, lipid peroxidation and decreased tocopherol concentrations in the liver of sheep, Internat. J. Vit. Nutr. Res. 64:270–276. McMurray, C.H., Blanchflower, W.J., Rice, D.A., and McLoughlin, M., 1986, Sensitive and specific gas chromatographic method for the determination of methylmalonic acid in plasma and urine of ruminants, J. Chromatogr. 378:201–207. Mele, M.C. and Meucci, E., 1996, Homocysteine and oxidative modifications of plasma proteins, Amino Acids (Vienna) 11:99–104. NRC (National Research Council), 1996, Nutrient requirements of beef cattle, National Academy of Sciences, Washington DC. Stangl, G.I., Schwarz, F.J., and Kirchgessner, M., 1999, Cobalt deficiency induced hyperhomocysteinaemia and oxidative status of cattle, Br. J. Nutr., submitted.

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Ueland, P.M., Mansoor, M.A., Guttormsen, A.B., Muller, F., Aukrust, P., Pefsum, H., and Svardal, A.M., 1996, Reduced, oxidized and protein-bound forms of homocysteine and other aminothiols in plasma comprise the redox thiol status—A possible element of the extracellular antioxidant defense system, J. Nutr. 126:1281S–1284S. Van Der Westhuyzen, J., Van Tonder, S.V., Gibson, I.E., Kilroe-Smith, T.A., and Metz, J., 1985, Plasma amino acids and tissue methionine levels in fruit bats (Rousettus aegyptiacus) with nitrous oxide-induced vitamin B12 deficiency, Br. J. Nutr. 53:657–662. Young, P.B., Kennedy, S., Molloy, A.M., Scott, J.M., Weir, D.G., and Kennedy, D.G., 1997, Lipid peroxidation induced in vivo by hyperhomocysteinaemia in pigs, Atherosclerosis 129:67–71.

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ROLE OF DIET RELATED HABITS AND COOKING PRACTICES ON BIOAVAILABILITY OF IRON, COPPER, AND ZINC AND STATUS OF IRON IN VEGETARIANS

V. V. Agte, M. Indumadhavi, V. Kakade, S. Palkar, S. Girigosavi, K. Tarwadi, and S. A. Chiplonkar Agharkar Research Institute G. G. Agarkar Road, Pune 411004 India

Average diet in most of the Asian countries being cereal based, its iron and zinc content has poor bioavailability. Investigations were carried out using in vitro studies and then diet survey to assess the effect of dietary practices like use of acidic foods, green leafy vegetables (GLV), naturally fermented foods for enhancement of biavailability of iron and zinc while use of tannic acid rich foods, frequent drinking of strong tea/coffee in place of meals, chewing of betel leaf and betel nuts, tobacco for their inhibitory action on absorption of these trace metals. When tea, coffee, betelnut or curry powder was added to “idli” (a fermented food using cereal and legume mixture), 65–88% reduction in ionisable iron was observed, while addition of fresh coriander to “idli” increased the ionisable iron by two to three times in 8 out of 9 cereal -legume combinations. The bioavailable densities of iron, zinc and copper for 25 commonly consumed green leafy vegetables based preparations were 0.13 ± 0.01, 0.11 ± 0.01, 0.07 ± 0.01mg/100Kcals, which are higher than the foods based on cereals or legumes. Bioavailability iron density in particular, was three times higher than average value for composite cereal-based meal that is 0.036mg/100Kcal. This indicated GLV as promising fortificants for vegetarians. When hemoglobin values were examined in light of habits, on 324 men and women, groups consuming more condiments (Tamarind), and coffee had higher (p < 0.05) prevalence of anemia than the other two groups. Similar observations were noted on 106 pregnant women where users of tobacco and betelnut had significantly lower levels of hemoglobin (p < 0.05).

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DIALYSABILITY OF CALCIUM, IRON, AND ZINC IN BEANS, CHICK PEAS, AND LENTILS

V. Sebastiá, R. Barberá, R. Farré, and M. J. Lagarda Nutrition and Food Chemistry Faculty of Pharmacy University of Valencia Spain

Legumes can be a dietetic source of minerals, although their bioavailability is considered lower than that of other foods. The aim of our work is to study the effect of different cooking processes on Ca, Fe and Zn bioavailability estimated by an in vitro method.

SAMPLES Chickpeas (Cicer arietinum L.), beans (Phaseoulus vulgaris L.) and lentils (Lens culinaris L.) were provided by a Spanish company. In all cases raw and processed ready-toeat samples were available. The cooking methods applied were: traditional and microwave (l,400w/5 to 25min, depending on the legume).

DIALYSABILITY ASSAYS The method described by Luten et al. (1996) was used. Elements were measured by flame atomic absorption spectroscopy, except in the case of dialysate Fe which was measured using the batophenantroline method. All the assays were carried out in duplicate (in all 12 values for element, legume and cooking treament were obtained).

STATISTICAL ANALYSIS Two-factor legumes and treatment/cooking method ANOVA and Minimal Significant Differences (p < 0.05) were applied. 306

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RESULTS The following % dialysis were obtained.

Ca Traditional and microwave cooking slightly reduce the dialysis percentage with respect to the dry cooked product. Ready-to-eat legumes can be classified according to their calcium dialysis percentage as follows: lentils > beans > chickpeas.

Zn The high % dialysis of Zn from dry uncooked lentils (48.5%) with respect to also dry uncooked beans and chickpeas is noteworthy, although in all cases the high Zn dialysis % were those corresponding to ready-to-eat samples.

Fe The % dialysis of iron were the lowest of the three element studied, except for readyto eat samples. It should to be pointed out that iron % dialysis values are higher (10 to 20 times) in ready-to-eat samples than in dry uncooked legumes. This effect could be due to the ascorbic and citric acids added during processing which increases the bioavailability of this element.

ACKNOWLEDGMENT This study is part of project ALI 97-0890, financed by the CICYT (Spain).

REFERENCE Luten, J., Crews, H., Flynn, A., Van Dael, P., Kastenmayer, P., Hurrell, R., Deelstra, H., Shen, Li-Hua., Fairweather-Tait, S., Hickson, K., Farré, R., Schlemmer, U., and Frohlich, W., 1996, Interlaboratory trial on the determination of the in vitro iron dialysability from food, J. Sci. Food Agric. 72:415–424.

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EFFECT OF ZINC DEPLETION ON IRON TRANSPORT ACROSS ISOLATED DUODENAL SACS

Peter W. F. Fischer and Bartholomeus Belonge Nutrition Research Division Health Protection Branch Health Canada, Ottawa Ontario, K1A 0L2, Canada

Since low dietary zinc has been shown to increase tissue iron stores, as well as increase hemoglobin concentrations, the objective of this study was to determine if zinc depletion results in changes in the mucosal uptake and serosal transfer of iron, using isolated everted duodenal sacs. Changes in mucosal iron binding were also assessed. Male Sprague-Dawley rats (120 weighing 250 ± 5%g) were block randomized into 3 groups, zinc depleted (3mg/kg diet), pair-fed (30mg/kg) and control (30mg/kg). At 2, 4, 6, 8, and 10 weeks, 8 rats per group were sacrificed and a 10 cm segment of duodenum was removed, washed and everted. It was filled with previously dialysed rat serum and suspended in 50mM acetate buffer (pH 5.5), 104mM NaCl and 1 g/L d-glucose. Added to this solution was 35. g as ferrous sulphate labelled with Fe-59. Aliquots of serum were removed at 0, 5, 10, 20, and 30 minutes to determine the amount of iron transferred from the mucosal to serosal side. At the end of the incubation, the iron remaining within the mucosal cell was determined. A mucosal supernatant was prepared and the amount of iron bound to the various protein fractions was determined by gel chromatography. At 6, 8, and 10 weeks, the intestinal segments from rats fed the zinc depleted diet transferred significantly more iron than the segments from the other two groups. There were no significant differences in the total iron bound within the mucosal cells nor in the distribution of iron bound to different protein fractions. These results indicate that zinc depletion increases iron absorption, but that it has no effect on binding of iron within the mucosal cell.

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MINERAL ELEMENT STATUS IN RATS FED DIETS WITH EXTRUDED OR NOT EXTRUDED EVENING PRIMROSE (OENOTHERA PARADOXA)

M. A. Gralak, H. Leontowicz, M. Leontowicz, A. Bogucka-Sciezynska, and G. W. Kulasek Department Animal Physiology Fac. Veterinary Medicine Warsaw Agricultural University

Poland

Evening primrose (Oenothera paradoxa) contains essential polyunsaturated fatty acids especially linoleic acid (C18;3 n-6). However fibre and antinutritional factors (ANFs) contents, e.g. protease inhibitors and lectins, are very high in evening primrose. They can influence bioavailability of nutrients but some of the ANFs are thermolabile. Hence the objective of our study was to determine the effect of evening primrose addition to the semipurified diets (10% of casein) on mineral status in rats. The 28 days experiment was performed on 30 Wistar rats divided into three groups. One diet was control (CTR), the others were supplemented (10%) with evening primrose, raw (REP) or extruded (EEP). The bioavailability of mineral elements (RBV) was estimated as the ratio of the total mineral content in liver of rats fed experimental diets and control diet. For statistical evaluation analysis of variance and Scheffe test were used.

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Generally, 10% of evening primrose in the rat diet decreased bioavailability of the mineral elements. Differences were significant, except calcium because of high variation. Extrusion did not improve bioavailability of mineral elements. Moreover it seems that extrusion had detrimental effect on total zinc and copper content in liver.

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COMBINED EFFECTS OF COMPLEX CARBOHYDRATES AND A RESISTANT PROTEIN ON INTESTINAL ABSORPTION AND STATUS OF IRON AND ZINC IN THE RAT D. Grizard1, C. Coudray2, M. Tahiri2, J. C. Tressol2, Y. Van Doesum3, Y. Rayssiguier2, and C. Barthomeuf1 1

Laboratoire de Pharmacognosie et de Biotechnologies Faculté de Médecine et Pharmacie 28 place H. DUNANT 63001 Clermont-Ferrand France 2 Centre de Recherches en Nutrition Humaine d’Auvergne Unité Maladies Métaboliques et Micronutriments INRA de Theix 63122 St. Genès Champanelle France 3 Gist-Brocades, P.O. Box 01 26000 Massachusetts Delft, Netherlands

The well-known beneficial action of dietary pectins, mainly on lipid metabolism, should not mask their putative unfavorable effects on the digestibility of minerals. In this study, our aim was to limit this potential negative impact of pectins. For this purpose, we evaluated the combined effects of a pre-enzymatic hydrolyzed pectins (MW:100,000, 10% of diet) and a resistant protein, namely human recombinant lactoferrin (1% of diet) on intestinal absorption and tissue retention of iron and zinc in rats. Results were compared to those of 1) control diet containing sucrose 10% of diet), 2) diet containing the same pectins alone (10% of diet). Compared to control diet, rats fed pectins or pectins plus human recombinant lactoferrin had a significant cecal hypertrophy associated with an enhancement of fecal short-chain fatty acids (SCFA) pool. Compared to pectins diet, resistant protein stimulated the pectin fermentation response through a significant increase of ceal SCFA pool. The apparent absorption of Fe was significantly depressed (–27%) in the experimental rats fed pectins diet. Despite an increase of apparent absorption of Zn (+29%), its status was not significantly altered by pectins. The resistant protein 311

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addition was found to limit the negative effects of pectins on the absorption of Fe to level equal to that of the control. However, by decreasing the balance of Zn (–14%), this protein potentialized the unfavorable influences of pectins on Zn bioavalability. We conclude that human recombinant lactoferrin could act as a promoting (Fe) or depressing (Zn) agent on mineral bioavailability in rats. Further studies are necessary to determine the exact mechanisms by which resistant protein acts on mineral uptake.

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NUTRIENT RISK ASSESSMENT Implications for Food Fortification Policy Mary R. L’Abbé1, Kevin A. Cockell1, Sheila Dubois2, and William H. Ross2 1 2

Bureau of Nutritional Sciences, and Bureau of Biostatistics and Computer Applications Food Directorate Health Protection Branch Health Canada, Ottawa Ontario Canada K1A 0L2

The addition of vitamins and minerals to foods can be an effective public health intervention to correct inadequate intakes of nutrients in both the general population and in specific population sub-groups. Canada has a distinguished history of effectively using food fortification to combat nutritional deficiencies and to improve the nutritional quality of the food supply, for example the mandatory fortification of fluid milk with vitamin D or the iodization of salt which have virtually eliminated childhood rickets and goitre respectively. Without appropriate regulation, addition of vitamins and minerals to foods can also pose safety concerns. The addition of vitamins and minerals to foods should not increase the risk of health hazards due to nutrient excesses, deficits or imbalances. Individual nutrients differ in the nature and severity of their adverse effects and in the margin of safety between requirement and the level at which adverse effects are observed. Vitamins and minerals can also impact on the bioavailability and efficacy of other nutrients. For many nutrients, systematic determination of the effects of high intakes over extended periods of time has not been conducted. Governments and other bodies are just beginning to establish the methodology for and to conduct nutrient risk assessments for setting “tolerable upper levels of intake” for nutrients. Nutrient risk assessment provides the framework for using available information to evaluate the addition of nutrients to foods in order to optimize intakes and public health benefits while minimizing risks.

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DESIGN OF PRODUCT AS SOURCE OF VARIANCE IN “IN VITRO” MINERAL AVAILABILITY OF HOMOGENISED WEANING FOODS

Olivares A. B., Martínez C., and Ros G. Area de Nutritión y Bromatología Facultad de Veterinaria Universidad de Murcia Campus Espinardo 30071-Murcia España

Homogenised weaning foods supply the main meal of babies aged from 6 to 12 months and young children from 1 to 3 years old. The ingredients used in the elaboration of baby-foods must be analysed for their mineral content. However, it is necesary to know as exactly as possible the amount of mineral that is available for absorption and utilisation. In addition, some constituents of vegetable foods, such as phytate may decrease the absorption of trace elements. Their content in infant food must be known and reduce to improve trace element status in children. The aim of study was to ascertain the influence of the desing of the product (percentages of raw ingredients included in the formulation process) on the mineral content and on their “in vitro” bioavailability in a wheat-based weaning food named “chicken with rice” to establish the best combination of percentages of raw ingredient which provides. Twenty five samples were studied, all of them designed from the combination of different percentages of four ingredients (carrots, rice, pea and chicken-liver) Fe, Zn, Ca, and Mg analyses were carried out by inductvely coupled plasma and the proportion of available Ca, Zn, and Fe in samples was determined following the method described by Miller et al. (1981). Not only mineral content but also in vitro availability changed significantly (p < 0.05) depending on design of the weaning foods. Percentage of rice included in the formulation process determined the differences in Zn and Fe availability as significant negative pearson correlation was found among this variables. In fact, it has been previous stated that rice flour is the main ingredient which increases phytic acid content in weaning foods.

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IRON DIALYSABILITY IN ENTERAL DIETS

C. H. Azevedo, A. P. Galhardo, and C. Colli Department of Food Science and Nutrition Faculty of Pharmacy University of S. Paulo. Av. Prof. Lineu Prestes, 580 CEP:05508-900 S.Paulo.Brazil

Iron deficiency anemia has been described in patients under prolonged enteral nutrition therapy and commercially available enteral diets have different formulations according to their potential use. In order to evaluate if iron dialysability differs among these diets, samples of one isosmolar standard (IS), two isosmolar with fiber (ISf-A, ISf-B), one formulation blend of natural food (BNF) and one high lipid diet (HLD) for patients with respiratory failure where analyzed by the “in vitro” method. Total iron was determined by (5:1) digestion in triplicate samples. For the dialysis evaluation, samples where acidified (pH 2) with HCl 0.1N and the percentage of dialysable iron (dialysis tubing of 6,000–8,000m.w. cut-off) was determined after pepsin and pancreatinbile (pH 7.5) digestions. Colorimetric determination was made by iron complexation with ferrozine. The percentage of dialysable iron of diets IS, BNF, ISf-A and HLD was 5.5 ± 0.4%; 5.0 ± 0.3%; 4.6 ± 0.8% and 3.3 ± 0.3% respectively, and the diet ISf-B showed the highest value of 9.0 ± 0.5%. The ISf-B diet has the lowest calcium and the highest carbohydrate content of all the five diets and are possible stimulating conditions for iron dialisability. Besides nutrient density, the balance among these and other factors influencing iron bioavialability should be considered when evaluating enteral diets.

E-mail: [email protected]

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NEW INDICES FOR ASSESSMENT OF TRACE ELEMENT STATUS AND REQUIREMENT, WITH A SPECIAL FOCUS ON SELENIUM

Jean Nève Université Libre de Bruxelles Institut de Pharmacie Campus Plaine 205-5 B-1050 Bruxelles Belgium

1. INTRODUCTION Assessment of status is essential to know whether the measurable activity (e.g. the concentration) of a given trace element is sufficient to maintain at an optimal level traceelement dependent biological function(s) or whether there is an excess or a deficiency of this activity that could produce a biochemical defect and lead to a clinical abnormality. A wide range of methods are nowadays available to assess trace element status among which the most popular is the determination of biochemical “indices” or “markers” such as trace element concentrations or trace-element dependent enzymatic activities or protein concentrations in easily accessible body fluids or cells. These procedures can generally be applied to individuals and are therefore useful in the routine clinical diagnosis. Other more sophisticated techniques exist that can only serve scientific purposes, e.g. determination in unusual body fluids or tissues (liver or kidney, platelets, etc.), balance studies, dynamic or tolerance tests, or use of radioisotopes. The latter are precious when trying to define the desirable levels of dietary intake, i.e. the “recommended dietary allowances” (RDA) on which so many aspects of the modern nutritional science rely. Considering there is quasi no ideal method or procedure to assess trace element status and because the choice of parameters and their interpretation may considerably vary from element to element, this contribution will focus on one element, selenium. Its case is actually of great interest owing to the potential beneficial effects on health of dietary intakes higher than the classically admitted RDAs. The review will consecutively

Tel: ++ 32 2 650 51 77; fax: ++ 32 2 650 52 49; email: [email protected] Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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consider the recent evolution of classical parameters and the newly developed biological markers.

2. OLD MARKERS REVISITED Selenium may appear as a nutrient the status of which is rather easy to assess because (i) its concentration can be determined without major difficulty in most biological fluids of interest, and (ii) the activity of the selenium-dependent glutathione peroxidase enzyme (GSH-Px) can be measured in cells such as erythrocytes or platelets and in plasma and is considered to reflect an essential function of the element. It has been shown that it quite well reflects modifications in selenium status (Nève, 1991 and 1995). Moreover, the enzyme activity reaches saturation at certain intake levels that serve for estimation of selenium requirement. However, the recent discovery of isoforms of GSHPx different from the classical enzyme and of the second and the third mammalian selenoenzymes (iodothyronine deiodinase and thioredoxin reductase) offers new perspectives in the study of selenium-dependent biological indicators even if they substantially complicate the assessment of selenium status. Concerning selenium concentrations in biological matrices, it is well known that they substantially vary in healthy subjects with geographical location and age as main determinants of differences. Alfthan and Nève (1996) carefully reviewed a few years ago published reports on selenium concentrations in serum and plasma according to various quality criteria (TRACY) and again concluded that no “universal” reference value could be established for serum selenium. The main serum selenium concentrations of healthy adult subjects in different parts of the world indeed vary from 40 to Therefore, results of such determinations can only be interpreted with reference to carefully matched controls. Other biological fluids such as whole blood or urine do not seem to offer advantages as compared to plasma selenium. Determination of whole blood and urine selenium poses analytical problems and their relationship to selenium intake is rather complex (Nève, 1991 and 1995). Hair or toenail clippings can be convenient indicators for long-term exposure in populations where blood or urine samplings pose problems. Selenium-dependent enzymes are essential in the assessment of selenium status as they reflect one of the biologically active part of the element. Measurements performed in population groups with a large range of individual selenium intakes show that typical hyperbolic curves are obtained when relating GSH-Px activity (measured in red blood cells or plasma) with blood or plasma selenium concentration. Both parameters very significantly correlate for relatively low selenium concentrations reflecting the strong interdependency of both variables. Thereafter, the relationships becomes less significant for increasing selenium concentrations, and even becomes not significant above a threshold value which indicates that selenium requirement for this function is reached. Such curves were unfortunately not often published, so it is difficult to generalise and establish threshold values that could serve as cut-off levels for characterising selenium depletion or sufficient intake. Relevant information can also be derived from dynamic tests where the response of enzyme activities to selenium supplements are analysed. We studied these aspects several years ago (Nève, 1991 and 1995) and showed that most interesting data could be obtained when GSH-Px activity is measured in platelets, which appeared as a very sensitive indice of selenium exposure. When retrospectively examining the results of about 10 selenium

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supplementation trials in population groups with variable selenium intake, we observed that this activity increased in all the examined population groups who consume less than (from 10 to 50) and reached a stable plateau after several weeks supplementation. It therefore seems that selenium requirement of the enzyme in this biological compartment was not met by the usual selenium intake. On the contrary, no increase in GSH-Px activity could be observed in a group consuming about It was possible to estimate that platelet GSH-Px activity reaches a plateau as soon as corresponding plasma selenium levels are in the range of 95 to i.e. at values higher than pre-intervention levels which, in the examined population groups, were in the range of 20 to This therefore implies that an intake in the range 80 to Se/day (values calculated by extrapolation from pre-intervention intakes and blood levels) is adequate for saturation of platelet GSH-Px activity. Several other authorities also established dietary standards for selenium using approaches based on the saturation of GSH-Px activity. The US National Research Council experts based their calculations on data from Chinese searchers who established that is a sufficient dietary intake for adult male residents of Keshan disease areas (a disease linked to selenium-deficiency) to maximise GSH-Px activity in plasma. By correcting this value by a weight factor in order to make it more appropriate for North Americans and by adding a safety factor of 1.3 to convert the physiological requirement to a dietary recommendation, the American experts obtained a RDA of 55 to Se/day, respectively for women and men (National Research Council, 1989). The discrepancy with the previously cited values obtained using maximisation of glutathione peroxidase activity in platelets may be due to the poorer sensitivity of plasma GSH-Px to changing selenium intake, to different physiological requirements of the GSH-Px isoforms, and also to differences in bioavailability of the chemical forms of selenium used for supplementation (Nève, 1991 and 1995). It is unknown why American experts did not consider maximisation of GSH-Px activity in platelets to establish their dietary recommendations. Recommendations from other countries does not seem to be based on experimental data, e.g. (all values for adults): 30 to for Nordic countries, 60 to for Great Britain or 70 to for Australia (Reilly, 1996). The recent recommendations of a joint WHO/FAO/IAEA expert group (1996) were more troublesome. Indeed, this committee proposed as the lower limits of the safe ranges of population mean intake of dietary selenium values from 21 to Se/day, respectively for basal and normative requirement (see definition of these terms in WHO/FAO/IAEA, 1996). The basal requirement was derived from Chinese experiments showing that this intake was sufficient to protect against Keshan disease, while the normative intake was derived from the same Chinese data previously used by the American experts and establishing that is necessary to maximise plasma GSH-Px activity in a 60-kg adult male. However, and contrary to American experts, the joint committee considered for calculation that full expression of GSH-Px activity is not necessary for optimal nutritional status and adopted as desirable an intake corresponding to 2/3 of maximal plasma GSH-Px activity. This demonstrates that the crucial point in establishing nutritional requirements is not the choice of the marker, but the way it is interpreted. Even if the necessity of intake levels causing saturation of enzyme activity can be discussed, one can again question about the choice of results derived from one study performed in Chinese selenium-deficient subjects to establish recommendations valid for other countries without considering the data that can be extracted from similar investigations precisely performed in subjects from these countries.

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Moreover, it seems more and more questionable to base nutritional recommendations for selenium on only one biochemical function. Indeed, after the recent identification of new selenium-dependent proteins including GSH-Px isoforms, it has been demonstrated that they do not have a similar selenium requirement. The cellular (cytosolic) form of GSH-Px (also called cGSHPx or GSH-Px1) is nowadays considered more as a storage form of body selenium to serve a homeostatic function in selenium metabolism than as an antioxidant effective in vivo (Ho et al., 1997). Animal and cell culture experiments showed that some isoforms of GSH-Px like phospholipid hydroperoxide GSH-Px (PHGSH-Px), an intracellular membrane-bound enzyme, or gastrointestinal GSH-Px (GIGSH-Px) are more resistant to selenium depletion than GSH-Px 1. More generally, there are differences in the sensitivity of each selenoprotein to selenium supply within and between tissues. The priority for incorporation of selenium into proteins seems to be in favour of PHGSH-Px, then 5'-desiodinase and finally selenoprotein P (Lei et al., 1995). GIGSH-Px seems also very high in the hierarchy of selenoproteins. Plasma GSHPx is an extracellular enzyme which originates mainly but not exclusively from the kidney proximal tubules. Its activity is influenced by renal function and only more or less 15% of serum selenium is associated with this selenoprotein.

3. NEW MARKERS COME ON STAGE Concerning the “new” selenoproteins distinct from GSH-Px, it is up to now rather difficult to evidence concordant observations that could serve in the assessment of human selenium status. Iodothyronine deiodinase (ID) is an enzyme involved in thyroid hormone metabolism that has 3 isoforms (type I to III) with different tissue distribution. Most recent data suggest that the three types contain selenocysteine in their active site and are selenium-dependent. However, the effects of selenium deprivation in vivo are dependent on both the degree of selenium deprivation and which tissues are examined. Cellular ID activities can be indirectly assessed in humans by measuring circulating T3 and T4 levels. A limited number of authors have reported in different groups of subjects either an association between selenium status and low plasma T3 levels reflecting decreased ID activity (Strain et al., 1997) or a decrease in T4 levels after selenium supplementation (Olivieri et al., 1995; Calomme et al., 1995). Interestingly, high levels of dietary selenium intake (in a seleniferous area in Venezuela) were significantly associated with decreased T3 levels suggesting that activity of ID is depressed in conditions of high intake, i.e. more than (Bratter and Negretti de Bratter, 1996). This level could correspond to the limit of the daily maximal safe intake. Selenoprotein P (SeP) is a potentially interesting new marker that can be quite easily determined in plasma by radioimmunoassay. This protein, that could be a transport form of selenium, but may also exert antioxidant properties, accounts for 60 to 80% of selenium in human plasma and differs from all other known selenoproteins by its high selenium content. In populations with variable selenium intake, its concentration was significantly related to several other parameters reflecting selenium status such as serum selenium (correlation coefficients from 0.35 to 0.93 in 5 studies), plasma GSH-Px (r from 0.69 to 0.92 in 6 studies) or erythrocyte GSH-Px (r = 0.51 in one study) (Akesson et al., 1997; Huang et al., 1995; Persson-Moschos et al., 1995; Rannem et al., 1996). Only few experiments were performed to explore the sensitivity of this protein to changes in selenium status. In Swedish adults, a diet switch leading to a 50% decrease in selenium intake caused a decrease by 11% in serum selenium from 0 to 3 months. Erythrocyte GSH-Px

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was also significantly decreased after that period, but SeP did not change, even after 1 year. A return to normal diet increased serum selenium and E-GSHPx, but did not modify SeP (Persson-Moschos, 1995). In Chinese subjects from Se-deficient and control areas, selenium supplementation (100 to as selenate in salt) increased SeP after 1 week in both groups and after 2 weeks only in Se-deficient subjects (Hill et al., 1996). Data on the newly identified third selenium-dependent enzyme thioredoxin reductase, a NADPH-dependent-flavoenzyme involved in the intracellular reduction of substrates, are extremely limited. The few data from animals or cell lines suggest it is an indicator of selenium intake (Hill et al., 1997; Gallegos et al., 1997) but its localisation limits its use as a clinically-relevant indicator of selenium status. Interestingly, supranutritional quantities of selenium given to rats seem to be able to directly increase the specific activity of the enzyme in some tissues (Bergren et al., 1999). Most recent data about the regulation by selenium of selenoprotein W (Se-W) content of animal tissues are also of great interest in attempts to define selenium requirement. Like other selenoproteins, this one again contains a selenocysteine residue inserted by an in-frame UGA codon. Although originally isolated from muscle, it is now evident that it is also present in other tissues and that its concentration depends on selenium intake (Sun et al., 1998). In some tissues, Se-W increases linearly with increasing selenium intake and a plateau is reached above a certain level (Yeh et al., 1997). Moreover, Se-W and cellular GSH-Px seem to be differently regulated by selenium intake in some organs (Sun et al., 1998).

REFERENCES Akesson, B., Huang, W., Persson-Moschos, M., Marchaluk, E., Jacobson, L., and Lindgarde, F., 1997, Glutathione peroxidase, selenoprotein P and selenium in serum of elderly subjects in relation to other biomarkers of nutritional status and food intake, J. Nutr. Biochem. 8:508–517. Alfthan, G. and Nève, J., 1996, Reference values for serum selenium in various areas—Evaluated according to the TRACY protocol, J. Trace Elements Med. Biol. 10:77–87. Berggren, M., Mangin, J., Gasdaska, J., and Powis, G., 1999, Effect of selenium on rat thioredoxin reductase activity, Biochem. Pharmacol. 57:187–193. Bratter, P. and Negretti de Bratter, V., 1996, Influence of high dietary selenium intake on the thyroid hormone level in human serum, J. Trace Elements Med. Biol. 10:163–166. Calomme, M., Vanderpas, J., François, B., Van Caillie-Bertrand, M., Herchuelz, A., Vanovervelt, N., Van Hoorenbeke, C., and Vanden Berghe, D., 1995, Thyroid function parameters during a selenium repletion/depletion study in phenylketonuric subjects, Experientia 51:1208–1215. Gallegos, A., Berggren, M., Gasdaska, J., and Powis, G., 1997, Mechanisms of the regulation of thioredoxin reductase activity in cancer cells by the chemopreventive agent selenium, Cancer Res. 57:4965–4970. Hill, K., McCollum, G., Boeglin, M., and Burk, R.F., 1997, Thioredoxin reductase activity is decreased by selenium deficiency, Biochem. Biophys. Res, Commun. 234:293–295. Hill, K., Xia, Y., Akesson, B., Boeglin, M., and Burk, R.F., 1996, Selenoprotein P concentration in plasma is an index of selenium status in selenium deficient and selenium-supplemented Chinese subjects, J. Nutr. 126:138–145. Ho, Y., Magnenat, J.L., Bronson, R., Cao, J., Gargano, M., Sugawara, M., and Funk, C., 1997, Mice deficient in cellular glutathione peroxidase develop normally and show no increased sensitivity to hyperoxia, J. Biol. Chem. 272:16644–16651. Huang, W., Akesson, B., Svensson, B., Schutz, A., Burk, R.F., and Skerfving, S., 1995, Selenoprotein P and glutathione peroxidase in plasma as indices of selenium status in relation to the intake of fish, Brit. J. Nutr. 73:455–161. Lei, X.G., Evenson, J., Thompson, K., and Sunde, R., 1995, Glutathione peroxidase and phospholipid hydroperoxide glutathione peroxidase are differentially regulated in rats by dietary selenium, J. Nutr. 125:1438–1446.

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National Research Council, 1989, Recommended dietary allowances, 10th ed., National Academy Press, Washington, D.C. Nève, J., 1995, Human selenium supplementation as assessed by changes in blood selenium concentration and glutathione peroxidase activity, J. Trace Elements Med. Biol. 5:65–73. Nève, J., 1991, Methods in determination of selenium states, J. Trace Elem. Electrolytes Health Dis. 5:1–17. Olivieri, O., Girelli, D., Azzini, M., Stanzial, A.M., Russo, C., Ferroni, M., and Corrocher, R., 1995, Low selenium status in the elderly influences thyroid hormones, Clin. Sci. 89:637–642. Persson-Moschos, M., Huang, W., Skrikumar, T., and Akesson, B., 1995, Selenoprotein P in serum as a biochemical marker of selenium status, Analyst 120:833–836. Rannem, T., Person-Moschos, M., Huang, W., Pharm, B., Staun, M., and Akesson, B., 1996, Selenoprotein P in patients on home parenteral nutrition, J. Parent. Ent. Nutr. 20:287–291. Reilly, C., 1996, Selenium in food and health, Blackie Academic and Professional, London. Strain, J.J., Bokje, E., van’t Veer, P., Coulter, J., Stewart, C., Logan, H., Odling-Smee, W., Spence, R., and Steele, K., 1997, Thyroid hormones and selenium status in breast cancer, Nutr. Canc. 27:48–52. Sun, Y., Ha, P., Butler, J., Ou, B., Yeh, J., and Whanger, P., 1998, Effect of dietary selenium on Selenoprotein W and glutathione peroxidase in 28 tissues of the rat, Nutr. Biochem. 9:23–27. WHO/FAO/IAEA, 1996, Trace elements in human nutrition, World Health Organisation, Geneva, Switzerland. Yeh, J., Vendeland, S., Gu, G., Butler, J., Ou, B., and Whanger, P.D., 1997, Dietary selenium increases selenoprotein W levels in rat tissues, J. Nutr. 127:2165–2172.

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ARE YOUNG WOMEN WITH LOW IRON STORES AT RISK OF ZINC AS WELL AS IRON DEFICIENCY?

R. S. Gibson, A.-L. Heath, N. Prosser, W. Parnell, U. M. Donovan*, T. Green*, K. E. McLaughlin*, D. L. O’Connor*, W. Bettger*, and C. M. Skeaff University of Otago Dunedin, New Zealand and *University of Guelph Guelph, Ontario, Canada

INTRODUCTION The quality of the diets of young women even in developed countries can be poor, resulting in low intakes of poorly available iron that fail to meet their high physiological requirement for growing body tissues, expanding red cell mass, and onset of menarche. As a consequence, low iron stores have frequently been reported in young women, sometimes in association with adverse health consequences (FairweatherTait, 1996). Additional exacerbating non-dietary factors associated with low iron stores in young women may include high menstrual losses, frequent blood donations, nose bleeds, and, sometimes, strenuous exercise. These same etiological factors may also predispose young women to suboptimal zinc status (Yokoi et al., 1994), when there is often a decline in red meat consumption, a readily available source of zinc and iron (Richardson, 1994). Nevertheless, very few studies have examined the zinc status of this age group. In this study, we have compared the biochemical zinc status of Caucasian young women living in Dunedin, New Zealand (NZ) with that of a group of young women from Guelph, Ontario, Canada, a group with lower iron stores, based on lower serum ferritin values. We have also evaluated the impact of certain biological (e.g. infection, use of oral contraceptive agents (OCA), age, body weight, height and body mass index) and dietary (e.g. presence and absence of red meat, flesh food intake, and [phytate]:[zinc] molar ratios) factors on the biochemical zinc indices measured. From these results, we have determined whether young women with low iron stores are at risk of suboptimal zinc status. Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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SUBJECTS AND METHODS A group of 376 Caucasian young women from Dunedin, South Island, NZ were studied. They were subdivided into two age groups: a younger group (n = 104) aged 19–20 years and an older group (n = 272) aged 21–40 years. Of the younger group (mean age ± SD, 19.5 ± 0.7y), 79 were omnivores and 25 avoided red meat whereas in the older group (28.9 ± 6.2y), 213 were omnivores and 59 avoided red meat. The Canadian Caucasian adolescents (n = 72 were omnivores; n = 90 avoided red meat) were 14–19 years (18.2 ± 1.4 y). All groups were recruited from schools, universities, publicity in the media and community groups. Exclusion criteria were presence of chronic disease, pregnancy, lactation, and for the Canadians, use of drugs known to interfere with folate metabolism (Houghton et al., 1997). Informed, written consent was secured from each participant after the nature of the study had been fully explained to them. For those adolescents below 16y, written consent was also obtained from a parent or legal guardian. The study protocols were approved by the University of Otago Human Ethics Committee, Dunedin, New Zealand and the University of Guelph Human Ethics Committee, Guelph, Ontario, Canada. Fasting morning peripheral venipuncture blood samples were taken with the young women in the sitting position using trace-element free evacuated tubes. Scalp hair samples and selected anthropometric measurements were also obtained, and instructions given to the Canadians for completing three-day weighed food records on two weekdays and one week-end day. For the New Zealanders (NZs), a previously validated computeradministered food frequency questionnaire covering the previous month, and designed to assess habitual intakes and dietary modifiers of iron and zinc absorption, was used. Pretested questionnaires on health, socio-eonomic and demographic status, life-style, activity levels, medical and menstrual history, habitual food consumption patterns, and use of alcohol, medications, cigarettes, oral contraceptive agents, and vitamin and mineral supplements, were also administered. Data on blood loss associated with menstruation (via a validated menstrual blood loss questionnaire), blood donations, and nose bleeds over the past year, were also collected for the New Zealanders. Heights and weights of all the adolescents were measured in duplicate using standardized procedures and calibrated equipment (Lohman et al., 1988); Quetelet’s Body Mass Index was calculated. Dietary intakes of energy, selected nutrients, dietary fibre (as non-starch polysaccharide-NSP), phytic acid, and molar ratios of Phy: Zn were calculated using the NZ (Burlingame, Milligan, Quigley, Spriggs, 1995) or Canadian (Sabry, Gibson, and Pen, 1984) computerized nutrient databases. Phytate values were added to the two nutrient databases as described previously (Gibson et al., 1991). Trace element-free techniques were used during the handling and analysis of all the blood samples. Presence of infection was assessed via leucocytosis (i.e. leucocyte count for the Canadians, and by serum C-reactive protein (via turbimetry) for the NZ’s. Zinc in serum was analyzed by flame atomic absorption spectrophotometry (ASS), using a standardized collection, separation and analytical procedure (Smith, Butrinovitz, and Purdy, 1979). The CV (as %) for zinc in pooled serum samples was 9.1% (n = 6) in Canada and 4.3% (n = 20) in NZ. Values for the quality control sera (Bovine Serum Reference Material, National Institute of Standards & Technology, Gaitherburg, MD) (mean ± SD; CV%) were: 13.3 (±0.9; 6.9%; n = 7) umol/L in Canada compared to the certified value of 13.6 ± 0.9umol/L, and 13.6 (±0.9; 1.7%; n = 8) umol/L in New Zealand compared to the certified value of 13.9 ± 1.5umol/L. Alkaline phosphatase in serum was also measured by a colorimetric procedure using p-

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nitrophenol phosphate as the substrate and commercial kits (Sigma Chemical Co., St. Louis, MO). The mean (+SD) (%CV) of the quality control sera (Sigma Enzyme Control 2-N) were 44.2U/L (+1.4; 9.5%; n = 8) in Canada and 70.3 U/L (+1.08, CV, 4.8%; n = 10) in New Zealand compared to the expected ranges of 35.1–55.1 U/L, and 65.13–78.49 U/L, respectively. In both Canada and New Zealand, scalp hair samples were collected, and then washed using the same standardized method (Harrison, Yursachek and Benson, 1969), prior to analysis for zinc by instrumental neutron activation analysis (INAA) in Canada and flame AAS in NZ after acid digestion with ultra pure nitric acid (70%) (Aristair BDH Laboratory Supplies). The CV (as %) for zinc in aliquots of a powdered hair sample were 3.3% (n = 13) by INAA and 2.5% (n = 7) by AAS. Aliquots of human hair (Commission Bureau of Reference, Reference material No. 125) were analyzed; mean (±SD, CV%) values were 3.05umol/g (±0.11; 3.5%; n = 24), compared to the certified value of 3.04 ± 0.08 umol/g. All data were analyzed using the Statistical Package for Social Sciences (SPSS for Windows version 7.5.1). Data were tested for normality using the Kolomogorov-Smirnov or Shapiro-Wilk test, and log-transformed, where appropriate, prior to analyses. Spearman rank correlation coefficients were calculated to examine relationships among anthropometric, biochemical, and dietary variables, where appropriate. Analysis of variance (ANOVA) was also used to investigate the explanatory effect of OCA use, infection, age, blood loss associated with menstruation, nose bleeds, and blood donation, and selected dietary and anthropometric variables on serum zinc and alkaline phosphatase in the NZ non-OCA users. For consistency, all the biochemical and dietary data are presented as medians . All tests were considered significant at p < 0.05.

RESULTS Parents of the Canadian adolescents and younger NZs represented the middle and high income groups. All of the young women studied were at least two years post menarche. In New Zealand, oral contraceptive agents were used by 40% and 36% of the younger and older subjects respectively, and by 25% of the Canadian adolescents. In both countries, less than 6% of the studied subjects regularly consumed mineral supplements containing zinc, whereas 14% of the Canadians and 19% of the NZs used a mineral supplement containing iron. The mean (±SD) BMI (wt/ht2) of the subjects ranged from 21.8 (±2.8) for the younger NZs aged 19–20y, 22.3 (±2.9) for the Canadians, and 23.7 (±4.0) for the NZs aged 21–40y. Table 1 shows the median daily dietary intakes of energy, selected nutrients, and antinutrients for the omnivores from the three groups. Canadians had lower intakes of energy, protein, zinc, dietary fibre (as NSP), phytate, and meat + poultry + fish (MPF), but intakes of calcium and iron that were comparable or slightly higher than for the NZs. Of these, the most notable differences are the low intakes of zinc and MPF in the Canadians. Similar differences distinguished the intakes of the Canadians and New Zealanders who excluded red meat (ERM) from their diet. Table 1 also summarizes the median biochemical zinc indices for the omnivorous subjects. Median hair and serum zinc concentrations were lower for the Canadians compared to the New Zealanders. Twenty-six percent of the Canadian omnivores (non OCA’ users) had serum Zn values <10.71umol/L) compared to 14% & 19% for the younger and older New Zealanders; a comparable trend was noted for serum

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ferritin values. Median serum alkaline phosphatase activity was higher in the younger Canadians compared to the older New Zealanders. ANOVA confirmed that OCA use had a negative effect on both serum zinc and alkaline phosphatase (p < 0.05) (but not hair zinc) in all three groups of subjects. Consequently, OCA users were excluded from any subsequent ANOVA with serum zinc and alkaline phosphatase values. There was no significant effect of infection (based on Creactive protein), recent blood donation, nose bleeds, or menstrual blood loss (based on our validated menstrual blood loss questionnaire) on serum zinc or alkaline phosphatase activity in the non-OCA users of the NZ group. However, in this group, ANOVA results showed that no red meat (p = 0.02) and high [Phy : Zn] molar ratios (p = 0.04) had a negative impact on serum zinc; mean serum zinc for non-red meat eaters (n = 58) was lower than for the omnivores (n = 180)(11.8 vs. 12.2umol/l; p = 0.02). Dietary factors that were negatively correlated with hair zinc were: high phytate intakes (r = –0.194; p = 0.03) and high [Phy: Zn] molar ratios (r = –0.215; p = 0.01) in the Canadians. Serum zinc and serum ferritin values were positively correlated (r = 0.104; p = 0.05) in the non-OCA users of the New Zealand group. Negative correlations were observed between body weight (r = –0.249; p = 0.02) and BMI (r = –0.299; p = 0.004) and hair zinc in the younger but not the older New Zealanders. No other significant relationships were observed between biological or dietary zinc variables and biochemical zinc indices.

DISCUSSION The omnivorous Canadian adolescents had a lower biochemical zinc status compared to the New Zealanders (Table 1), paralleling the trends observed in serum ferritin values. Indeed, 26% of the Canadians omnivores (not using OCA’s) compared to 19% of

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the New Zealander omnivore’s (not using OCA’s) had serum zinc values below the level associated with functional consequences of mild zinc deficiency (Pilch and Senti, 1984). Further, the median serum zinc value for the Canadians omnivores (non-OCA users) fell just above the 25th percentile of the US reference data for white women aged 9–19 years, whereas those for both the younger and older NZ omnivores (non-OCA users) were between the 50 and 75th percentiles (Pilch and Senti, 1984). Our ANOVA results suggested that certain dietary and biological factors impacted significantly on the biochemical zinc indices measured, and hence played some role in the biochemical zinc status of the subjects. Of the dietary factors, high phytate intakes (mg/day), or expressed as [Phy]/[Zn] molar ratios, appeared to have the most impact, as indicated by the negative correlations noted with hair zinc in the Canadians, as well as a negative impact on serum zinc in the NZ non OCA users. Excluding red meat also had a negative effect (p = 0.04) on the serum zinc in this same group. Of the biological factors, OCA use had a major negative influence on serum zinc (and alkaline phosphatase activity), possibly reflecting alterations in the postabsorptive utilization of zinc induced by estrogens rather than poor zinc status per se (King, 1986). Infection (based on an elevated serum C-RP), and blood loss from menstruation (based on our validated menstrual blood loss questionnaire), nose bleeds, and recent blood donations did not impact on serum zinc levels in the NZ non-OCA users. Of the anthropometric indices investigated, body weight and BMI were negatively associated with hair zinc concentrations in the younger NZ women, relationships that have been observed in studies of younger NZ children of both sexes (RSG; pers.comm). The lower zinc nutriture of the Canadian compared to the New Zealand young women noted here may be cause for concern. Physiological requirements for zinc peak during adolescence at the time of the pubertal growth spurt, which generally occurs in girls between 10–15 years. Even when the growth spurt has ceased, adolescents may require additional zinc to replete tissue zinc pools depleted as a result of the increased demands arising from the pubertal growth spurt (King, 1996). Suboptimal zinc nutriture during adolescence could impact on growth and bone maturation at an age when the majority of bone mineralization in girls is taking place (King, 1996). Abnormalities in skeletal growth, maturation and mineralization have been associated with marginal zinc nutriture during childhood and adolescence. In male zinc deficient adolescents from the Middle East, zinc supplements stimulated growth and increased bone age, normalizing skeletal maturation (Prasad et al., 1963). Defects are likely to occur after a period of rapid growth, as occurs during the pubertal growth spurt. Moreover, if adolescents enter pregnancy with a compromised zinc status, and continue to receive intakes of zinc that do not meet their increased needs, their resultant poor maternal zinc status could have serious adverse effects on pregnancy outcome (Tamura and Goldenberg, 1996). In conclusion, the young omnivorous Canadian women were at higher risk of suboptimal zinc status than New Zealand women. Certain dietary variables impacted negatively on the biochemical zinc status in both groups, notably high dietary [Phy :Zn] molar ratios and no intake of red meat in the New Zealanders, and high phytate intakes and high [Phy:Zn] molar ratios in the Canadians. Use of OCA’s had a negative effect on serum zinc and alkaline phosphatase levels whereas infection, and blood loss arising from menstruation, recent blood donations and nose bleeds, had no effect. Of the anthropometric variables, body weight and BMI were negatively associated with hair zinc levels in the younger women. Our results suggest that young women with low iron stores may be especially vulnerable to suboptimal zinc status.

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REFERENCES Burlingame, B.A., Milligan, G.C., Quigley, R.J., and Spriggs T., 1995, FOODfiles Manual. New Zealand Institute for Crop and Food Research Ltd., Wellington, N.Z. Fairweather-Tait, S.J., 1996, Iron requirements and prevalence of iron deficiency in adolescents. An overview, in: Iron Nutrition in Health and Disease (L. Hallberg and B,-G. Asp, eds), pp. 137–148, John Libbey and Company, London. Gibson, R.S., Smit Vanderkooy, P.D., and Thompson, L., 1991, Dietary phytate x calcium/zinc millimolar ratios and zinc nutriture in some Ontario preschool children. Biol. Trace Elem. Res. 30:87–94. Harrison, W.W., Yursachek, J.P., and Benson, C.A., 1969, The determination of trace elements in human hair by atomic absorption spectrophotometry. Clin. Chim. Acta 23:83–91. Houghton, L.A., Green, T.J., Donovan, U.M., Gibson, R.S., Stephen, A.M., and O’Connor, D.L., 1997, Association between dietary fiber intake and the folate status of a group of female adolescents. Am. J. Clin, Nutr. 66:1414–1421. King, J.C., 1986, Do women using oral contraceptive agents require extra zinc. J. Nutr. 117:217–219. King, J.C., 1996, Does poor zinc nutriture retard skeletal growth and mineralization in adolescents? Am. J. Clin. Nutr. 64:375–376. Lohman, T.G., Roche, A.F., and Martorell, R. (eds.), 1988, Anthropometric Standardization Reference Manual. Human Kinetics Books, Champaign, Illinois. Pilch, S.M. and Senti, F.R. (eds), 1984, Assessment of the zinc nutritional status of the US population based on data collected in the second National Health and Nutrition Examination Survey, 1976–1980. Life Sciences Office, Federation of the American Societies for Experimental Biology, Bethesda, Maryland. Prasad, A.S., Miale, A., Farid, Z., Schulert, A., and Sandstead, H.H., 1963. Zinc metabolism in patients with the syndrome of iron deficiency anemia, hypogonadism, and dwarfism. J. Lab. Clin. Med. 61:537–549. Richardson, N.J., 1994, UK consumer perceptions of meat. Proc. Nutr. Soc. 53:281–287. Sabry, J.H., Gibson, R.S., and Pen, C, 1982, Nutrient intake system. User’s guide for the program. Guelph, Canada: University of Guelph. Smith, J.C., Jr., Butrinovitz, G.P., and Purdy, W.C., 1979, Direct measurement of zinc in plasma by atomic absorption spectroscopy. Clin. chem. 25:1487–1491. Tamura, T. and Goldenburg, R.L. (1996). Zinc nutriture and pregnancy outcome. Nutr. Res. 16:139–181. Yokoi, K., Alcock, N.W., and Sandstead, H.H, 1994, Iron and zinc nutriture of premenopausal women: associations of diet with serum ferritin and plasma zinc disappearance. J. Lab. Clin. Med. 124:852–861.

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ASSESSMENT OF FEMURS AND TESTIS AS PARAMETERS FOR ZINC BIOAVAILABILITY FROM DIFFERENT FOOD SOURCES

G. S. Henriques, L. F. C. Pedrosa, E. L. Dantas, J. M. de Moura, C. L. Araujo, and S. M. F. Cozzolino Faculdade de Ciências Farmacêuticas (School of Pharmacy) USP São Paulo Brazil

The intake of metals in different chemical forms may influence its bioavailability. Zinc (Zn) is a transition element essential for animals. From a nutritional point of view, it can behave in different ways, depending on the chemical species, organic or inorganic. The aim of this study was to analyze the differences in zinc content between femurs and testis of young rats, after the intake of different chemical forms of zinc in the feed. Two simultaneous experiments were carried out, both of them with 16 male newly-weaned Wistar rats each, divided into two groups and housed in individual cages. Two groups (DC1 and DC2) were fed with a feed similar to AIN-93G; a third group received a similar AIN-93G modified feed (DO), where zinc source was represented by oysters, and a fourth group in which feed was made up of human diet (DM). After 4 weeks, the animals were killed, femurs and testis were isolated and prepared to zinc determination by flame atomic absorption spectroscopy (FAAS). The results showed that zinc in femurs is a good parameter to evaluate bioavailability,whereas in testis there was no significant variation related to zinc source. The low zinc values in femurs of animals of groups DM and DO and the small variability of zinc values in testis suggest that the metal moves to those tissues of greater metabolic demand and also emphasize that a bioavailability parameter should be chosen taking into account the different ways by which zinc is stored and used by tissues.

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THE EFFECTS OF LOW PROTEIN DIET AND LIGHT DEPRIVATION ON ZINC STATUS AND GONAD FUNCTION IN ADULT MALE RATS

N. Virgona, M. Kamiyama, T. Yano, and T. Esashi Division of Applied Food Research National Institute of Health and Nutrition Toyama 1-23-1 Shinjuku Tokyo 162-8636 Japan

Testicular development can be influenced or inhibited by a number of factors including both light and dietary protein deprivation. Since zinc has also been identified to have an influence on the reproductive process, quantitative examination of Zn status due to the combined effects of restricted dietary protein level and light deprivation might provide better understanding of the mechanism involved. Four groups of six male Fischer-344 rats at 10 weeks of age were placed on two different dietary protein levels, 20% or 6.7% casein, under two photoperiod regimes, normal lighting (LD) or continuous darkness (DD). The experimental diets were made in accordance with AIN93G. The phosphorus level of the 6.7% casein diet was adjusted to be equal to the 20% casein diet and L-cystine level was reduced in proportion to the protein level. After a four weeks feeding period there was no significant (p < 0.05) effect of protein level or photoperiod on growth. The groups kept on the low protein diet (6.7%LD and 6.7%DD) had the highest fecal and urinary Zn output so that by week 4 these two groups had significantly (p < 0.05) lower Zn retention. In addition these two groups showed compromised Zn status evidenced by significantly lower (p < 0.05) femur, serum and testes Zn concentration. Serum dihydrotestosterone (DHT) level was significantly reduced by continuous darkness either alone or in combination with protein restriction (20%DD and 6.7%DD (p < 0.001), and 6.7%LD (p < 0.005)). The results suggested that lower dietary protein intake significantly (p < 0.05) reduced Zn status but not to a deficient level, perhaps due to mitigating effects of maintaining dietary phosphorus : calcium ratio equivalency amongst diets. The reduction in serum DHT levels is not caused by low protein level per se but also by Zn status and that DHT is perhaps more sensitive to Zn availability than other parameters examined here. Address all correspondence to: Dr. Nantiga Virgona; Division of Applied Food Research, National Institute of Health and Nutrition, Toyama 1-23-1 Shinjuku, Tokyo 162-8636 Japan; telephone: 03-3203-5602; fax: 033205-6549; email: [email protected]

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ZINC NUTRITIONAL STATUS IN OBESE CHILDREN AND ADOLESCENTS D. N. Marreiro1, M. Fisberg2, and S. M. F. Cozzolino1 1

Faculdade de Ciências Farmacêuticas Universidade de São Paulo Brazil 2 Pediatria (pediatrics) Unifesp and Cepesn Universidade São Marcos São Paulo, Brazil

Studies carried out in animals and humans have shown several changes in zinc metabolism when obesity is present. However, the information available for children and adolescents is limited and controversial. So, the scope of this study was to determine zinc nutritional status in obese children and adolescents by the evaluation of their diets and biochemical parameters. Therefore, 23 obese children and adolescents, assisted at the Centro de Estudo, Pesquisa, Saúde e Nutrição (CEPESN), da Universidade São MarcosSP (Nutrition Health and Research Center- São Marcos University- São Paulo), were evaluated. Venous blood was collected after a 12 hour fast. Zinc determinations were conducted in plasma, erythrocyte and urine by atomic absorption spectroscopy. The feeds were analyzed by a software developed at Faculdade de Saúde Pública da Universidade de São Paulo, Brazil (School of Public Health-USP). The results showed that 39% of the obese patients had zinc concentrations bellow 75mg/dL in plasma, a value considered as a threshold to establish a diagnosis of zinc deficiency in plasma. In erythrocytes, 100% of these patients showed zinc values bellow The results of zinc in urine showed that 83% of the samples agreed with the average reference values hour),while 17% of them were bellow reference values. The analysis on Zn in the feeds showed an average of 10mg/day, what places 59% of the patients bellow the RDA recommendation of 1989. These biochemical data enable us to conclude that there is a change in Zn nutritional status in the studied group. However, we cannot assert that they are deficient.

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REFERENCE VALUES OF SELECTED TRACE ELEMENTS IN THE SERUM OF TERM NEWBORNS FROM THE URBAN AREA OF ROME Alessandro Alimonti1, Francesco Petrucci1, Francesco Laurenti2,

and Sergio Caroli1 1

Instituto superiore di Sanita Applied Toxicology Department Viale Regina Elena 299 00161 Rome, Italy 2 University “La Sapienza” of Rome Pediatric Department Viale Regina Elena 324, 00161 Rome Italy

Reference values for Al, Cd, Co, Cu, Li, Mn, Mo, Ni, Rb, Se and Zn and indicative intervals for Sb are proposed in the serum from cord blood of 143 term newborns of the urban area of Rome. On the basis of the eligibility criteria adopted, only the babies with gestational age >37 weeks and body weight at the delivery >2,500g, i.e. “normal” term infants, were included in this study. With the exception of Cd, Li, Ni and Sb, all other analytes results provided good approximation to the normal distribution. The experimental references values (in ng/ml) were the following: 1.12–6.79 (Al), 0.10–0.52 (Cd), 0.20–0.43 (Co), 140–691 (Cu), 0.31–2.23 (Li), 0.79–3.26 (Mn), 0.36–1.56 (Mo), 0.20–3.15 (Ni), 196–1,302 (Rb), 20.2–69.7 (Se) and 318–1,405 (Zn). For several elements, the present knowledge does not allow for a thorough comparison. This could be done only for Cu, Sb, Se and Zn. Possible correlations between elements concentrations and weights at birth or gestational ages were also attempted.

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HAPTOGLOBIN POLYMORPHISM ASSOCIATION WITH MAGNESIUM AND LIPID PROFILE IN HEALTHY TEENAGERS M. Bicho1, C. P. Monteiro2, L. Sardinha3, S. Llobet4, P. Marques Vidal4, M. J. Halpern4, and M. J. Laires2 1

Genetics Laboratory, FML, UL, Lisboa Portugal 2 Biochemistry Laboratory FMH, UTL, Lisboa, Portugal 3 Exercise and Health Unit FMH, UTL, Lisboa, Portugal 4 Health Sciences Institute Lisboa, Portugal

In the genesis and progression of most pathological situations, genetic and environmental factors are important, as well as the interactions between them. Haptoglobin (Hp) is an serum glycoprotein, synthesised in the liver, that binds free hemoglobin (Hb) in the vascular system after hemolysis. It is also an acute phase protein and it has a role in the modulation of prostaglandin synthesis. In the human species it presents a genetic polymorphism as consequence of the expression of three codominant alleles. On the other end, in recent years, there has been growing interest in the study of magnesium deficiency and its correlation with various cardivascular diseases. The aim of the present study was to determine, in healthy teenagers, the relationship between Hp phenotypes and magnesium status, as well as with some cardiovascular risk factors such as the lipid profile and plasma peroxidation levels. The study included 171 teenagers from Lisbon, 76 boys and 95 girls with a mean age of 13.0 ± 1.7 and 12.9 ± 1.7 years respectively. A blood sample was collected after a 12h fast to evaluate: plasma magnesium by spectrophotometry, the lipid profile: total cholesterol, HDL and LDL cholesterol and triglycerides by commercial kits and apolopoproteins and B (Apo B) by immunonepholometry; and peroxidation indices: plasma TBARS by a thiobarbituric acid assay and in vitro LDL peroxidation induced by phenilhydrazine delected by the same assay. Hp phenotypes were determined by PAGE. Statistical analysis included ANOVA and linear regression. Results are presented in Table 1. 333

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We can observe that the three groups of individuals presenting different Hp phenotypes show significantly different mean values for plasma magnesium, HDL cholesterol and Apo AI. The group of individuals with Hp2.2 phenotype show a lower plasma magnesium mean value and higher HDL cholesterol and Apo AI mean values. Low magnesium levels are associated with higher probability of development of cardivascular disorders and vith increase of oxidative status. Several authors suggest an association between Hp phenotypes and lipid metabolism which includes the binding of Hp to HDL, as well as that the different Hp phenotypes are associated with different pathologies and the Hp2.2 phenotype is more associated to forms of those pathologies involving higher immune reactivity. Magnesium deficiency is also related to inflammatory situations, which, in turn, are associated with the incrase of oxidative status. So, we might suggest that Hp2.2 proteins may condition a higher oxidative stress with may induce the increase of Apo and the decrease of plasma magnesium.

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URINARY LITHIUM Distribution Shape, Reference Values, and Evaluation of Exposure Based on Inductively Coupled Plasma Argon Emission Spectrometry

K. Usuda, K. Kono, T. Watanabe, T. Dote, H. Nishiura, M. Shimahara*, N. Hashiguchi*, and H. Takeishi* Department of Hygiene and Public Health and *Department of Oral Surgery Osaka Medical College, 2-7, Daigakumachi Takatsuki City, Osaka 569-8686 Japan

Inductively coupled plasma argon emission spectrometry (ICPAES) was used and evaluated for the practical analysis of the lithium content of urine samples without dilution. The calibration curve for lithium in standard solution was shown to have good sensitivity and linearity, and lithium addition to urine sample has shown good reproducibility. The detection limit were defined as the blank signal plus three times the standard deviation of the blank. Detection limits of was obtained for aqueous solutions, using ICPAES. This method can be performed with 1 ml of urine in a single tube, using a routine ICPAES analysis. The ICPAES method could be used rapidly and conveniently in the field of toxicology in lithium exposure cases. Urine samples obtained from workers (n = 86) who have not been engaged in lithium works were determined by ICPAES. The obtained concentrations were corrected using a specific gravity of 1.024. The particular frequency distribution resulted in a log-normal distribution diagram for anatomical spread. Geometric mean value for urinary lithium in the non exposed workers was and the confidence interval (C.I.) from a lognormal distribution, was 11.0 to respectively. Taking into consideration a short biological half-life and massive urine excretion of lithium, urinary lithium was considered to be a useful index for monitoring of exposure. We conclude that the guideline established by the obtained lithium reference values are useful for the early diagnosis or gauging the exposure degree of those who have risk of exposure to lithium in their living or working environments.

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ASSESSMENT OF NONHEME IRON STATUS IN THE WHOLE BLOOD, PLASMA, AND SERUM Healthy Neonates and Patients with Iron Overload

O. M. Mykhaylyk1, N. A. Dudchenko1, T. A. Orlova2, N. M. Pyasetska2, and I. P. Lubyanova3 1

Institute for Applied Problems of Physics and Biophysics NAS of the Ukraine 2 Clinics of Neonatology Hospital “OCHMADET” AMS of the Ukraine 3 Institute for Occupational Health AMS of the Ukraine Kyiv, Ukraine

Sets of instrumental and biochemical methods have been developed for the assessment of nonheme iron including non-invasive liver iron susceptometry and MR imaging. Nevertheless, the concentration of plasma or serum iron and also the iron-binding capacity and plasma ferritin are currently determinated in diagnosing an iron overload. The transferrin saturation index is thought to be the most informative parameter to estimate plasma supply with iron. The specific ESR spectra to transferrin iron (III) paramagnetic ions allow one to develop techniques for specific determination of the indices of iron exchange in transferrin pool. The aim of the work was to propose new procedures for the determination of transferrin iron, transferrin protein and transferrin saturation indices in the whole blood microsamples using the electron spin resonance (ESR) technique. The method was tested on a set of blood, plasma and serum samples taken from neonates and from patients with iron overload as a result of its inhalation entry. The samples were taken from the finger of 2-to-6 day newborns (n = 20) whose gestational age varies from 38 to 40 weeks. The venous blood was taken from patients with professional secondary iron overload (20 men of 40-to-55 years of age). The plasma and serum

Address all correspondance to: Dr O. Mykhaylyk, P.O.Box 355, 252001 Kyiv, Ukraine telephone: +38044-25257-71; e-mail: [email protected]

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Assessment of Nonheme Iron Status in the Whole Blood, Plasma, and Serum

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were preparad immediately after the collection of the blood. The hematocrit indices were determined on a Coulter Counter. The plasma iron was determined by the standard method with Sigma chemicals. Comparison of data on transferrin iron concentration in the whole blood and plasma with data on hematocrite index support the idea that there is iron in the structure of transferrin complexes in the formed blood elements. A new index, transferrin iron concentration in the formed blood elements, was introducted. The transferrin iron concentration value in the formed blood elements of newborns was shown to rise exponentially in the range of 5 to 50mkM with an increase in the transferrin iron concentration in the blood in the range of 14 to 38mkM. Highly saturated transferrin was shown to be lost during the praparation of blood plasma and/or serum samples. At high transferrin saturation, the diagnostic value of iron exchange indices determined in the blood exceeded that of the indices determined in the plasma and/or serum samples. The analysis of the blood microsamples ensures an adequate estimation of iron exchange in the transferrin pool, especially at high transferrin saturation. The iron exchanges indices in the transferrin pool were determined in the blood of newborns, patients with professional secondary hemochromatosis and also in the blood of a set of mammals (dolphins, rats, mice) in the norm.

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LIPID PEROXIDATION AND ZINC AND COPPER STATUS IN HEALTHY ADULTS AFTER GINSENG INGESTION

A. Sánchez-Mayoral, R. de Miguel Romera, and Pérez Gallardo, L. Área de Bioquímica y Biología Molecular E. U. de Fisioterapia 42003-Soria University of Valladolid Spain

We investigated the relations between duration of ginseng ingestion, lipid peroxidation and Cu and Zn status in 17 healthy adult volunteers provided with identical amount of Panax ginseng root. Plasma malondialdehyde and serum Zn and Cu were evaluated in 9 women and 8 men, who received 2g daily of root powder during 4 weeks. Serum malondialdehyde concentrations were measured as the product generated by the reaction between thiobarbituric acid (TBA) and malondialdehyde and were analyzed by spectrophotometry (Pye Unicam SP6-400 UV). Serum Cu and Zn concentrations were determined with an atomic absorption spectrophotometer (Perkin-Elmer 272). These parameters were measured at the beginning of the experiment, in the course of ginseng ingestion (3, 8 and 24 hours and 1, 2, 3 and 4 weeks) and 1 week after the last ginseng ingestion. The Spearman test and stepwise multiple regression analysis were used to examine the relations. Simple correlations between the duration of ginseng ingestion and plasma malondialdehyde showed no association in women (r = –0.05) nor in men (r = –0.316). In all group studied Cu serum concentrations after 24h. (r = 0.512, P < 0.05) or 2 weeks of ginseng ingestion (r = 0.589, P < 0.02) were positively correlated with plasma malondialdehyde, and Zn serum concentrations after 1 week of ginseng ingestion (r = –0.466, P < 0.06) were negatively correlated with plasma malondialdehyde. Stepwise multiple regression analysis confirmed only the association (r = 0.62, P < 0.01) between Cu and plasma malondialdehyde. In conclusion, our results confirm that the level of peroxidation stress in healthy adults after 2 weeks of ginseng ingestion was mainly dependent on Cu status.

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VARIABILITY OF MULTIPLE NUTRITIONAL ELEMENTS IN HAIR OF ONE MAN OVER TWO DECADES

L. M. Klevay, D. M. Christopherson, and T. R. Shuler USDA-ARS Grand Forks Human Nutrition Research Center Grand Forks, ND 58202

Chemical elements often are measured in hair samples with the hope of obtaining useful clinical, epidemiologic, forensic, nutritional or toxicologic information (Klevay, et al. Am J Clin Nutr 46:233, 1987). Analytical methods are sufficiently accurate and precise; medical utility remains largely unproven because of insufficient clinical validation and ignorance of potential variability. The subject of this study used two brands of shampoo low in copper and zinc and collected occiptonuchal hair samples with regularity between February, 1968 and December, 1986. No unusual occupational exposures to chemical elements occurred. Elements in hair were measured by inductively coupled plasma spectroscopy after washing, desiccation and dissolution (Klevay, Am J Clin Nutr 23:284, 1970). Analytical sensitivity was optimized for manganese. Analysis of hair samples in random order revealed X, n = 31 except Cr, 29; K, Se; 30): Calcium Chromium Copper Iron

282 ±153 0.55 ± 0.37 16.5 ±13.2 14.8 ± 18.0

Magnesium Manganese Phosphorus Potassium

39.4 ± 30.7 0.19 ± 0.20 139 ± 22 38 ± 79

Selenium Sodium Vanadium Zinc

0.56 ± 0.11 174 ± 111 0.028 ± 0.058 167 ± 24

Coefficients of variation range from 14 (Zn) to 209 (V). Within person variability decreases the potential usefulness of measurement on a single sample. Neither seasonal effects nor time trends have been found so far. Hair analysis for Ca, Fe, K, Mg, Na, and P probably is based more on analytical ease than on medical utility; other methods of status assessment remain superior. Therapeutic intervention based on hair analysis probably is undesirable without supporting data.

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EFFECT OF MAGNESIUM DEFICIENCY ON ENTEROCYTE Ca, Fe, Cu, AND Zn CONTENT

E. Planells, N. Sanchez-Morito, P. Aranda, and J. Llopis Instuto de Nutrición y Tecnologia de Alimentos and Departamento de Fisiologia Facultad de Farmacia Universidad de Granada E-18071 Granada, Spain

Magnesium deficiency is known to be linked with changes in bioavailability of several elements. In previous studies based on indirect procedures, we reported that Mg deficiency increased the apparent absorption of Ca, Fe, Zn and Cu. We investigated the effect of dietary magnesium deficiency on enterocyte Ca, Fe, Cu, Zn and Se concentrations. A group of six male Wistar rats, (body weighed 100g) were allowed access ad libitum to double-distilled water and a semisynthetic diet deficient in Mg (129mgMg/kg food) for 70 days. The results were compared with findings in a control group that was pair fed for 70 days with an identical diet except that it covered this species’s nutritional requirements for Mg (480mgMg/kg food). A modification of the technique of Weiser (Weiser MM. J Biol Chem, 248, 2536, 1973) was used to isolate the whole enterocyte cells population (upper, mid and lower villus cells) from the upper jejunum. Ca, Fe, Cu and Zn were determined by flame ion atomic absorption spectrophotometry (AAS). Bovine muscle (CRB 184, Community Bureau of Reference, Brussels, Belgium) was used for quality control assays. The results showed that consumption of an Mg-deficient diet significantly increased enterocyte content of Ca (p < 0.01), Fe (p < 0.01) and Zn (p < 0.05). However, a decrease in enterocyte Cu concentration (p < 0.05) was observed. These dates support, in large part, the results obtained by indirect methods.

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SERUM COPPER, ZINC, AND SELENIUM LEVELS WITH REGARD TO PSYCHOLOGICAL STRESS IN MEN

A. Pizent, J. Jurasovic, M. Pavlovic, and S. Telišman Institute for Medical Research and Occupational Health Ksaverska cesta 2, PO Box 291, 10001 Zagreb Croatia

Copper (Cu), zinc (Zn) and selenium (Se) are essential trace elements involved in many metabolic processes and enzyme systems, and have an important role in protection against oxidative stress which is implicated in the pathogenesis of over 100 human diseases. This study considers possible alterations of Cu, Zn and Se status in male subjects under psychological stress. Serum concentrations of copper (S-Cu), zinc (S-Zn) and selenium (S-Se) were measured in 34 apparently healthy male prisoners of war immediately on release from a detention camp, and 85 healthy male subjects of comparable age and body mass index who had not been in a war combat zone. All subjects gave their informed consent prior to inclusion in the study. The S-Cu and S-Zn measurements were performed by using flame AAS and S-Se by electrothermal AAS, and the accuracy was controlled by regular participation in the Trace Elements External Quality Assessment Scheme (Guildford, U.K.). The results expressed as median and range were: 1,138 (877–1,337) of S-Cu, 1,087 (514–1,260) of S-Zn and 52.6 (29.6–99.9) of S-Se in the former prisoners, and 1,149 (869–1,487) of S-Cu, 1,131 (874–1,351) of S-Zn and 64.8 (44.6–109.3) of S-Se in the reference subjects. Significantly lower S-Zn (P < 0.02) and S-Se were found in the former prisoners compared to the reference subjects, whereas no significant difference between the groups was found for S-Cu (P > 0.80). In the group of former prisoners, a significant positive correlation was found between the S-Zn and S-Se levels (r = 0.396, P < 0.05) and inverse correlation between S-Zn and body mass index (r = –0.339, P < 0.05), whereas no significant correlation was found of either S-Cu, S-Zn or S-Se with age (38 (19–54) years) or duration of imprisonment (130 (126–270) days). As the body mass index of 23.4 (19.7–28.1) and the body mass relative deviation from nomogram of 105 (89–125) % in the group of former prisoners showed no indication of malnutrition, lowered S-Zn and S-Se levels 341

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may be ascribed to increased psychological stress induced by conditions during imprisonment. The fact that no significant correlation was found between S-Zn, S-Se or body mass index with respect to imprisonment duration, and an inverse association, rather than positive association, was found between S-Zn and body mass index, tends to corroborate this assumption.

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URINARY IODINE AND THYROID STATUS OF NEW ZEALAND RESIDENTS

C. D. Thomson, S. Woodruffe, A. Colls, and T. D. Doyle Department of Human Nutrition University of Otago PO Box 56, Dunedin New Zealand

Recent studies in New Zealand suggest that the risk of iodine deficiency as assessed by urinary iodine excretion is increasing as our iodine intakes decrease. This may result from dietary recommendations to reduce salt intake and a reduction in the use of iodophors as cleaning agents in the dairy industry. The overall aim of this project was to assess the clinical significance of these low iodine excretions in terms of thyroid hormone status and thyroid volume in an adult population in a low soil iodine area of the South Island of New Zealand. Specific aims were (i) to assess iodine status by measuring 24 hour urinary iodide excretion, and serum thyroglobulin and thyroxine (TSH) levels in a random sample of the adult population living in Otago, and (ii) to assess the prevalence of enlarged thyroid glands in adults with low iodine status, comparing thyroid volume in two groups of adults with low and marginal urinary iodine status with a third group of adults with adequate iodine status. Threee hundred and fifty Otago residents were recruited by random selection from the electoral roo and from the Dunedin Blook Bank. Twenty-four urine collections were made by all these subjects on two separate occasions to screen for urinary iodide excretion. Two hundred and twenty of these subjects were allocated to one of three groups according to average iodide excretion, high , medium low according th the WHO criteria for adequate, low and marginal status (where iodine deficiency disorders might be expected) respectively. The thyroid hormones TSH and thyroglobulin were measured by radioimmunoassay and thyroid volumes have been measured by ultrasonography. Median urinary iodine excretions of the three groups were 46. 76 and respectively for males and 44, 69 and respectively for females. Preliminary analysis of the results show no differences in or TSH among the three groups. Thyroglobulin tended to be higher in female subjects in the two groups with low and moderate iodine excretion in comparison to the high group although the differences were not significant. Similarly, a trend towards a hugher thyroid volume in male and female 343

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subjects classified into the lowest of the three groups in comparison to the medium and high excretion groups, but again not statistically significant. When subjects were reclassified according to urinary iodide concentration rather than total 24 hour excretion, the trends described above were similar, but again differences were not significant. These preliminary results suggest that the fall in iodine status of residents of Otago may be starting to influence thyroid hormone status and thyroid volume. Although the effect is small at present, this situation is likely to worsen should iodine intakes continue to fall.

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SERUM SILICON CONCENTRATION OF HEALTHY PERSONS IN THE ANTWERP REGION (BELGIUM)

K. Van Dyck, H. Robberecht, R. Van Cauwenbergh, and H. Deelstra Laboratory of Food Sciences Department of Pharmaceutical Sciences University of Antwerp (UIA) Universiteitsplein 1, B-2610 Antwerp Belgium

In 1972, Carlisle and Schwartz claimed silicon to be an essential element for the chick and rat. They attributed an important role to the element in bone mineralization and soft tissue development. However, till now, no one was able to prove its essentiality for man. Analytical problems due to the very low concentrations in the human body and absence of biological certified standard reference material hampered the assessment. Evaluation of silicon status and determination of reference values, certainly is a major contribution to future research on deficiency, toxicity or essentiality of silicon for man. Therefore in this study normal values for silicon in human serum are determined, since no such data exist for Belgium. In two hospitals in the Antwerp region blood samples were taken, after informed consent, from healthy children and adults. Patients suffering from chronic kidney diseases, lung fibrosis, senile or Alzheimer dementia, osteoporis or atherosclerosis were excluded. For each subject age, sex, dietary habits, intake of food supplements and reason of visit to the clinic was recorded. At least thirty samples were collected for every age-class: 0–12 months, 1–3, 4–7, 8–0, 10–18, 19–39, 40–60 years and +60. Only in the adults classes (19–60 years) a subdivision for sex was made. Optimal conditions for blood prelevation, serum storage and silicon analysis had been evaluated earlier. Blood samples were drawn with a Monovette® syringe and needle to avoid silicon contamination. Serum samples could be stored at –20 °C prior to analysis. The silicon concentration was determined with an optimized electrothermal atomic absorption spectrometric technique (4,100 ZL, Perkin Elmer, Norwalk). The results of these analyses will be presented.

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ZINC AND HUMAN PREGNANCY

Michael Hambidge, Nancy Krebs, and Laura Caulfield Section of Nutrition Department of Pediatrics and the Center for Human Nutrition University of Colorado Health Sciences Center Center for Human Nutrition Department of International Health The Johns Hopkins School of Hygiene and Public Health

The ubiquitous role of zinc in cellular metabolism is only starting to receive appropriate recognition and there is still a great deal to learn about the biology of this trace element. However, the extent and magnitude of the role of zinc in gene expression, cellular growth and cellular differentiation is now sufficiently clear to provide a strong basic rationale for very thorough examination of the practical importance of zinc nutriture in early human growth and development, both pre- and post-natally. The principal interest relates to the development of the embryo, fetus, infant and young child. However, the partially elucidated and extensive role of zinc in hormonal metabolism, perhaps most notably, in the context of this paper, in modulating the effects of estrogen, especially at the time of delivery, reminds us that maternal health and obstetric outcome cannot be ignored. In 1987, Swanson and King concluded that "the relationship between zinc status and pregnancy outcome remains an open question". This brief review will consider what has occurred since then and the research that merits implementation if this question is to be resolved. One change in the interim has been the broadening and strenthening of scientific concern about the necessity of resolving the unanswered questions with which we are still faced and the recognition that, despite the paucity of knowledge which sometimes appears paradoxical and even contradictory, we may be dealing with a problem of public health dimensions in some populations (Caulfield et al., 1998).

Address all correspondence to: Michael Hambidge MD; Box C225, The University of Colorado Health Sciences Center, 4200 E. 9th Av., Denver, Colorado, 80262; telephone: 303 315 5672; fax: 303 315 3273; email: michael.hambidge@ UCHSC. edu Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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This brief review will, of necessity, be highly selective in content and references. The four aspects of zinc and pregnancy that will be considered are: the periconceptional period; placental transfer of maternal zinc to the fetus; the relationship of maternal zinc nutritional status to fetal development and obstetric outcome; and, finally, the longerterm implications to the infant and child of maternal zinc nutritional status during pregnancy.

PERICONCEPTIONAL A great deal of the respect that we have for the importance of zinc for prenatal development is attributable to the research of nutrition scientists working with animal models. Though numerous centers have been involved with this research, the contributions from U.C. Davis under the leadership of L. Hurley have been quite outstanding. More recently, these have been extended by C. Keen and his colleagues. Their studies, in particular, have served to focus attention on embryogenesis and very early events in pregnancy. In addition to the known brief critical windows of time for differentiation of each organ, later fetal/postnatal development and/or duration of gestation may be impacted by maternal zinc status at critical stages of embryogenesis/ organogenesis. Despite the impressive effects of maternal zinc restriction on embryogenesis in animal, especially rodent, models, there is little evidence at this time to support a recent recommendation for universal periconceptional zinc supplementation in the U.S.A. In fact, current evidence that maternal zinc deficiency has deleterious effects on human embryogenesis is very limited. Suggestive evidence includes a disproportionately large number of congenital malformations/fetal wastage in a very small number of women with Acrodermatitis Enteropathica not receiving zinc therapy. Associations between congenital malformations and low maternal or cord plasma zinc have been inconsistent and inconclusive (Hambidge et al., 1993). In short, definitive human studies are lacking. Zinc accumulation in the first quartile of pregnancy is extremely modest, ie. averaging 0.08mg/day (Swanson and King, 1987). Therefore, barring the possible effects of early pregnancy-associated metabolic changes, including changes in appetite and food intake, zinc nutritional status prior to pregnancy should give a useful indication of zinc status in the periconceptional period. We, therefore, examined zinc homeostasis in young adult nulliparous women in north east China, where the incidence of congenital malformations is relatively high. Net zinc absorption in these women whose habitual diet provides only 5 mg zinc per day (modest phytate: zinc molar ratio) has been found to be indistinguishable from that for control women consuming over 8 mg zinc per day (Lei et al., 1996). Intestinal conservation of endogenous zinc was excellent in these women whose dietary zinc was uniformly low This is consistent with the concept that the human has impressive abilities to adapt to restricted intakes of dietary zinc. However, considerable evidence from animal models and human studies suggests that there is a cost associated with this “adaptation”. This was suggested in this study by the relatively low estimates of “exchangable” zinc, which had a positive correlation with dietary zinc and, especially, with absorbed zinc. These studies were accomplished with the application of stable isotope tecniques which have also been utilized to start to examine zinc homeostasis across gestation (Fung et al., 1997).

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TRANSPORT OF ZINC ACROSS THE HUMAN PLACENTA The placenta is likely to have a key role in determining the effects of changes in maternal zinc status and metabolism on fetal zinc status. Given the anatomy of the human placenta, the only barrier between the maternal and fetal circulation is the single layer of cells of the syncytiotrophoblast, a basement membrame and the endothelium of the fetal circulation (all fetal tissues). The higher plasma levels in the cord versus maternal blood suggest an active transport process though, as in the intestine, the molecular details await identification. An abundance of specific alpha-2 macroglobulin receptors (protease-treated) on the trophoblast which bind this zinc-transporting protein in a specific and saturable manner, suggest receptor-mediated uptake. This protein may be important in the transport of maternal zinc to the placenta. Two selected observations that could be of practical significance with to the transfer of maternal zinc to the fetus are: (a) Metal-metal interactions at the level of the trophoblast that can impact fetal status of these metals (Barone et al., 1998). Until we know a great deal more about such interactions, this should sound a cautionary note with respect to the use of mineral supplements or exposure to metals such as cadmium, (b) Maternal redistribution of zinc secondary to acute phase reactions and other factors can diminish placental transfer (Duffy et al., 1997) indicating that factors other than maternal zinc nutritional status can impact the supply of zinc to the fetus.

MATERNAL ZINC STATUS AND PREGNANCY OUTCOME Two sources of information will be considered: (a). Biochemical Indices of Zinc Status Available indices are limited in their utility for assessing zinc status. Space limitations dictate restricting consideration to plasma/serum zinc. There is a physiological decline in maternal plasma zinc concentration across gestation only in part related to volume expansion. This starts quite noticably in the first trimester and continues until late in gestation, with a rapid return to pre-pregnancy levels postpartum. In middleincome Colorado women whose dietary zinc intake averaged about 11 mg/day, administration of a 15mg zinc supplement per day from 12 weeks gestation did not diminish the decline in maternal plasma zinc concentrations across gestation. This indicated that the decline to the extent that it was observed in these subjects was not indicative of maternal zinc deficiency (Hambidge et al., 1983). Of note, the concentrations at 16 weeks gestation were similar to those recently observed in a low-income Mestizo population in Peru, whose zinc intake was only 7mg per day, and probably only of moderate availability (Caulfield et al., 1999a). The subsequent decline in the latter population was greater, however, and, unlike the Colorado population, modest increases were observed both in maternal and cord blood with zinc supplementation. This population will be discussed further below. It is worth noting that much lower plasma zinc concentratons during pregnancy have been reported in Malawi, where the bioavailability, though not the diet zinc, is very poor (Huddle et al., 1998). As in several other studies, iron supplements were associated with lower plasma zincs. Correlations observed between maternal biochemical indices of zinc status and complications of pregnancy (hypertension); delivery (pre-term, post-term, prolonged

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labor etc); and fetal development have been inconsistent. For example, of 41 studies correlating birthweight with biochemical indices of maternal or infant zinc status, 22 were positive and 19 negative (Tamura et al., 1996). (b). Zinc Supplementation Trials Intervention studies with zinc supplements to detect underlying zinc deficiency and evaluate its effects are especially important for human zinc nutrition research because of the lack of adequate biomarkers of zinc status. Inconsistent improvements reported, for example in pregnancy-induced hypertension; incidence of induced labor and cesarian section; premature delivery; and intra-uterine growth retardation could be attributable in part to limitations in study design. Of note, however, were two well designed studies involving substantial numbers of subjects, one in the U.K. (Mahomed et al., 1989), the other in Denmark (Jonsson et al., 1996) the results of which were entirely negative. In contrast, Goldenberg et al. (1995) have reported greater fetal growth associated with supplementing low-income Afro-Americans from 19 weeks gestation with 25 mg zinc per day. Offspring of the zinc supplemented group had birthweights averaging 126g higher than those of placebo-treated women (significant after adjusting for a significantly longer gestation (+0.5wk)); a greater birth length (+0.6cm); and a greater head circumference (+0.4cm). These results were remarkable in part because of the relatively high initial calculated dietary zinc intake (13mgZn/day). There is, as yet, an unexplained difference between these data and those from a more recent, large scale study of Peruvian women whose baseline diet zinc was only 7mg. (Caulfield et al., 1999b). This Peruvian study was unusually comprehensive and serves as a model for future intervention studies. Notable among the results were: differences in fetal heart rate and activity patterns, assessed using electronic monitoring, suggestive of zinc associated improvements in fetal neurobehavioral development (Merialdi et al., 1998); increased transfer of maternal antibodies to the fetus by the zinc supplemented mothers (Shankar et al., 1998) and a lower incidence of diarrheal morbidity among infants whose mothers took zinc supplements during pregnancy (Shankar S. et al., 1998). Preliminary data from a large scale study of zinc supplementation of intra-uterine growth retarded infants in India (Sazawal S. et al., 1999) starting as early as two weeks post-natal age have indicated a decrease in diarrheal morbidity, a decrease in overall mortality, a greater weight gain and evidence of improved motor development. The beneficial results of such early post-natal zinc supplementation suggest that normal neonatal hepatic zinc stores (Zlotkin, 1988; Krebs et al., unpublished data) may have been compromised by sub-optimal maternal zinc status. Results of primate studies support the hypothesis that maternal dietary zinc restriction can have long-term adverse effects on post-natal development.

RESEARCH PRIORITIES In conclusion, our current understanding of human maternal zinc nutrition during pregnancy as reviewed briefly in this paper is limited. There is, however, enough evidence of its practical importance to obstetric outcome and to development of the embryo, fetus and infant to highlight this issue as a priority area for more intensive research. This

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research requires a strong basic component, directed in part to the mechanism of transfer of zinc across the human placenta. We need to achieve a clearer understanding of zinc homeostasis and of dietary requirements before and during the reproductive cycle, including the effects of dietary and host factors. Above all, however, ther is currently an impelling need for high quality intervention studies to provide more definitive answers to the epidemiology, prevalence and sequelae of human zinc deficiency in pregnancy. Ideally, however difficult and expensive, these studies should commence prior to conception. Moreover, recent human data cited above, together with observations on primate models encourage the long term extension of these studies through infancy and early childhood focussing especially on immunity, morbidity from infections, physical growth and neurobehavioral development.

ACKNOWLEDGMENTS DK02240; DAN-5116-A-00-8051-00; HRN-A-00-97-00015-00.

REFERENCES Barone A., Ebesh O., Harper R.G., and Wapnir R.A., 1998, Placental copper transport in rats: effects of elevated dietary zinc on fetal copper, iron and metallothionein. J. Nutr. 128:1037–1041. Caulfield L.E., Zavaleta N., Shankar A.H., and Merialdi M., 1998, Potential contribution of maternal zinc supplementation during pregnancy to maternal and child survival. Am. J. Clin. Nutr. 68:499S– 508S. Caulfield L.E., Zaveleta N., and Figueroa A., 1999b, Adding zinc to prenatal iron and folate supplements improves maternal and neonatal zinc status in a Peruvian population. Am. J. Clin. Nutr. in press. Caulfield L.E., Zaveleta N., Figueroa A., and Leon Z., 1999b, Adding zinc to prenatal iron and folate supplements does not affect duration of pregnancy or size at birth in Peru. J. Nutr. in press. Duffy J.Y., Baines D., Overman G.J., Keen C.L., and Daston G.P., 1991, Repeated administration of alphahederin results in alternations in maternal zinc status and adverse developmental outcome in the rat. Teratology 56:327–334. Goldenberg R.L., Tamura T., Neggers Y., Copper R.L., Johnston K.E., DuBard M.B., and Hauth J.C., 1995, The effect of zinc supplementation on pregnancy outcome. J. Am. Med. Assoc. 274:463–468. Fung E.B., Ritchie L.D., Woodhouse L.P., Roehl R., and King J.C., 1997, Zinc absorption in women during pregnancy and lactation: a longitudinal study. Am. J. Clin. Nutr. 66:80–88. Hambidge K.M., Krebs N.F., Jacobs M.A., Favier A., Guyette L., and Ikle D.N., 1983, Zinc nutritional status during pregnancy: a longitudinal study. Am. J. Clin. Nutr. 37:429–442. Hambidge K.M., Hackshaw A., and Wald N., 1993, Neural tube defects and serum zinc. British J. Obstet. Gynecol. 100:746–749. Huddle J.M., Gibson R.S., and Cullinan T.R., 1998, Is zinc a limiting nutrient in the diets of rural pregnant Malawian women? Brit. J. Nutr. 79:257–265. Jonsson B., Hauge B., Larsen M.F., and Hald F., 1996, Zinc supplementation during pregnancy: a double blind randomised controlled trial. Acta. Obstet. Gynecol. Scand. 75:725–729. Lei S., Xiang M., Miller L.V., Krebs N.F., Lei T., and Hambidge K.M., 1996, Zinc absorption and intestional losses of endogenous zinc in young Chinese women with a marginal zinc intake. Am. J. Clin. Nutr. 63:348–353. Mahomed K., James D.K., Golding J., and McCabe R., 1989, Zinc supplementation during pregnancy: double blind randomised controlled trial. Brit. Med. J. 299:826–830. Merialdi M., Caulfield L.E., Zavaleta N., Figueroa A., and DiPietro J., 1999, Adding zinc to pernatal iron and folate tablets improves fetal neurobehavioral development. Am. J. Obstet. Gynecol. 180:483–490. Sazawal S., Black R.E., Menon V.P., Dhingra U., Dhingra P., Mazumder S., Caulfield L., and Khosla S., 1999, Effect of Zinc and Mineral Supplementation in Small for Gestational Age Infants On Growth and Mortality. FASEB J. 13:A376.

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Shankar A., Ghujawa A., Caulfield L.E., and Zaveleta N., 1998, The effect of prenatal zinc supplementation on immunological status of the neonate and perinatal morbidity. FASEB J. 12:A18. Swanson C.A. and King J.C., 1987, Zinc and pregnancy outcome. Am. J. Clin. Nutr. 46:763–771. Tamura T. and Goldenberg R.L., 1996, Zinc nutriture and pregnancy outcome. Nutr. Res. 16:139–181. Zlotkin S.H. and Cherian M.G., 1988, Hepatic metallothionein as a source of zinc and cysteine during the first year of life. Pediatr. Res. 24:325–329.

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TRACE ELEMENT NUTRITION DURING PREGNANCY AND LACTATION—EFFECT ON MILK TRACE ELEMENTS

Bo Lönnerdal Department of Nutrition University of California Davis, California 95616, USA

The breast-fed infant is dependent on an adequate supply of trace elements for optimal growth and development. For some elements, e.g. iron, stores at birth provide the element during the early neonatal period, while others need to be provided by the diet, i.e. breast milk. During the period of rapid growth during infancy, requirements for trace elements are high, while the concentrations of these elements in milk are considered to be low in relation to the estimated requirements. Concern has therefore been raised whether maternal trace element deficiency or suboptimal intake can reduce the concentrations of trace elements normally occurring in milk. Thus, even if it is generally considered that the amounts of trace elements provided from breast milk will meet the requirements of the breast-fed infant, but not much more, low concentrations of trace elements in breast milk may cause deficiency or suboptimal trace element status. Recently, there has also been some concern that poor maternal trace element status may impair normal mammary gland function, particularly the metabolic handling and/or secretion of other essential nutrients in milk. Maternal factors affecting breast milk trace element concentrations can be studied by at least three different approaches. First, the trace element status of lactating women can be assessed and the possible correlation with breast milk trace element concentrations can be analyzed. This approach is practically possible for trace elements for which status can be accurately assessed, e.g. iron (hemoglobin, ferritin) and selenium (serum/plasma selenium, glutathione peroxidase), while it is difficult for elements for which status is difficult to assess (e.g. zinc and manganese). Second, the effect of maternal trace element intake on milk trace element concentrations can be analyzed. While

Address all correspondence to: Dr. Bo Lönnerdal, Deptartment of Nutrition, University of California, Davis, California 95616, USA, phone: 530-752-8347, fax: 530-752-3564, e-mail:[email protected] Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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it is often difficult to accurately analyze the trace element intake of pregnant/lactating women, it is possible to follow women with habitually low trace element intake who are given supplements. This approach has been used for iron, zinc and selenium. Third, since the mammary gland acquires trace elements from serum for further export into milk, it is possible to study clinical conditions in women, in which serum trace element concentrations are elevated or decreased. One example of this is infection, where serum copper concentrations increase while iron and zinc concentrations decrease. This paper will examine, for each trace element, results from these approaches and also discuss possible mechanisms involved in the transfer of trace elements from the mother to breast milk.

1.1. Iron The possible effect of low maternal iron status on breast milk iron concentration has been explored in lactating women. Maternal iron status assessed by hemoglobin, serum ferritin and transferrin saturation was not found to be correlated with milk iron in a study on Swiss women (Celada et al., 1982). However, iron status of this group of women did not vary much and none of them were anemic. Similar observations were made in a group of Nigerian women with considerably more variable iron status (Murray et al., 1978). The women were divided into three groups: “iron deficient” “normal” and “iron overloaded” (Hb > 120g/L), but no difference in milk iron was found between groups. Transferrin saturation varied widely between groups; 6%, 27% and 64%, respectively. A study on Malaysian women indicated that ethnicity affected milk iron, but there was no correlation between maternal iron status, as assessed by hemoglobin, serum iron or total iron-binding capacity, and milk iron (Loh and Sinnathury, 1971). In contrast to these studies, a study in India suggested a positive effect of maternal iron deficiency on milk iron (Fransson et al., 1985). Severely anemic women (Hb < 80 g/L) had significantly higher milk iron concentrations than non-anemic women (Hb > 110 g/L). Concentrations of lactoferrin, the major iron-binding protein in human milk, were also higher in the anemic women. The number of subjects was very low, however, and samples were taken very soon after delivery (<2 weeks post partum), a period when milk iron changes dramatically, depending on milk volume produced. It is not known whether the anemic women were given iron supplements when diagnosed, which may have increased circulating levels of iron even if it was too early to affect hemoglobin concentration. In a study on Peruvian women, anemic women (Hb < 110 g/L) were found to have similar concentrations of iron and lactoferrin in colostrum (day 2 of lactation) to those of nonanemic women (Zavaleta et al., 1994). The anemic women were given iron (100mg daily) from day 2 to day 30 post partum, which significantly increased their hemoglobin concentrations (from 92 to 105 g/L), but no effect on milk iron or lactoferrin was found. Similarly, iron supplementation during pregnancy (60 mg daily) did not affect colostrum iron concentrations (Chang et al., 1999). Several studies on maternal iron intake and milk iron concentration have found no correlation between these parameters (Lönnerdal et al., 1981). While studies have investigated the potential correlation between hemoglobin or iron stores (ferritin) and milk iron, it is more likely that iron taken up by the mammary gland is supplied by circulating iron, i.e. serum iron. We explored this possibility in women with acute infection during the early post partum period or during established lactation (2–3 mo post partum) (Zavaleta et al., 1995; Lönnerdal et al., 1996). Serum iron decreased during infection, as an acute phase response, but no effect on colostrum or

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mature milk iron concentrations was found. Thus, it appears that breast milk iron is not affected by maternal iron intake or status or infection, suggesting that the mammary gland has a mechanism that regulates milk iron concentrations.

1.2. Zinc Several studies have investigated the correlation between maternal dietary intake of zinc and milk zinc but found no significant correlation (Lönnerdal et al., 1981). Since the concentration of zinc in breast milk decreases precipitously during lactation, it was believed that low maternal zinc status had a negative effect on milk zinc concentrations. However, lactating well-nourished women given daily supplements of zinc had similar milk zinc levels to unsupplemented women (Moser-Veillon and Reynolds, 1990). In a recent study on Peruvian women given zinc (Caulfield et al., 1999; Chang et al., 1999) daily during pregnancy and the first week of lactation, no effect was found on colostrum zinc or milk zinc at 1 or 3 months of lactation. The unsupplemented women had low plasma zinc concentrations and even if no signs of zinc deficiency were observed, it is likely that their zinc status was low. This suggests that marginal maternal zinc status does not affect milk zinc concentrations. The mammary gland is likely to acquire zinc from the plasma pool and low plasma zinc may therefore affect milk zinc. In our study on Peruvian women with acute infection during early or established lactation (Zavaleta et al., 1995; Lönnerdal et al., 1996) serum zinc concentrations decreased significantly as an acute phase response, but no effect on milk zinc concentration was found. Thus, similar to iron, there appears to be a mechanism that regulates breast milk zinc concentrations.

1.3. Copper and Manganese There are few studies on the effect of maternal copper or manganese intake or status on milk concentrations of these elements. No effect of dietary intake of copper on milk copper has been observed (Lönnerdal et al., 1981). In one study, maternal dietary manganese intake was found to be correlated with breast milk manganese (Vuori et al., 1980). It should be noted, however, that human milk manganese concentrations are very low (4–8 g/L) and difficult to analyze accurately and the data base for the manganese content of various food items is limited. Further studies are needed on this potential correlation. There have been no studies on the effect of maternal copper or manganese supplementation on breast milk copper or manganese. In our study on Peruvian women with acute infection during lactation (Zavaleta et al., 1995; Lönnerdal et al., 1996), serum copper increased significantly, but there was no effect on milk copper. Thus, it appears that circulating copper concentrations do not affect milk copper and that a regulatory mechanism exists.

1.4. Selenium Maternal selenium status has been shown to be closely correlated with milk selenium. Studies in areas with low selenium intake (e.g. Finland, New Zealand and China) have shown that women in these regions have lower than normal concentrations of serum and milk selenium (Kumpulainen et al., 1985). It is well known that soil selenium affects selenium concentrations in plants and animals raised on this soil and that selenium status of subjects will be strongly affected by dietary selenium. Selenium supplementation of

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women in low selenium has been shown to increase breast milk concentrations considerably (Kumpulainen et al., 1985). Both inorganic (selenite) and organic (yeast) forms of selenium were effective, with the latter form possibly being better utilized. Since milk selenium concentrations are closely correlated with circulating levels of selenium, it is unlikely there is a mechanism regulating mammary gland selenium uptake and its export into milk.

2. MECHANISMS REGULATING MILK TRACE ELEMENT CONCENTRATIONS It is evident that mechanism exist in the mammary gland that can regulate concentrations of iron and zinc in milk. This regulation may occur at two different sites; either at the uptake of trace elements from serum by the mammary epithelial cell or at the synthesis and secretion of milk from the gland. Control at the uptake phase appears more likely as the cell otherwise may increase/deplete its trace element content. 2.1. Iron Since most cell types utilize transferrin receptors (TfRs) to regulate cellular iron concentrations, we explored the involvement of mammary gland by TfRs in milk iron regulation in an animal model. We found a correlation between the declining trend in milk iron concentration during lactation and mammary gland TfR concentrations (Sigman and Lönnerdal, 1990a), suggesting an involvement of TfR in normal physiological changes during lactation. We also studied the effect of maternal iron intake and status on milk iron and TfRs (Sigman and Lönnerdal, 1990b). The results from this study are more complicated to interpret. Iron-deficient rats had lower milk iron concentrations as compared to controls, which was accompanied by higher mammary TfR concentrations, suggesting up-regulation to compensate for the low iron content. However, rats fed high iron diets had even higher mammary TfR concentrations as compared to controls, while milk iron concentrations were similar.

2.2. Zinc The mechanism by which cells acquire zinc has not yet been established. Zinc in serum is transported by and serum albumin, while some (1–2%) zinc is loosely attached to amino acids. Serum albumin binds zinc non-specifically and to date there has been no evidence of this protein delivering zinc to the cell. Since binds 4 atoms of zinc with high affinity, we hypothesized that this protein is involved in the delivery of zinc to the cell. We found that the binding of to human mammary epithelial cells in culture is specific and saturable which is indicative of a receptormediated mechanism (Beshgetoor and Lönnerdal, 1999). We also found that the mRNA for the is expressed in the mammary cells, suggesting that this is a mechanism to deliver zinc to this cell type. The quantitative significance of this pathway for mammary cell acquisition of zinc is not yet known, nor do we have conclusive data yet on the effect of media or cellular zinc concentration on expression, which is important for an understanding of regulatory mechanisms. We have recently explored the regulation of milk zinc in a rat model. Rats were fed a low zinc diet through lactation only

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or pregnancy and lactation or a control diet (Beshgetoor and Lönnerdal, 1997). Although liver zinc concentration was significantly lower in dams fed the low zinc diet, mammary gland zinc and milk zinc were not affected, suggesting that under conditions of maternal zinc deficiency/marginal zinc intake, milk zinc concentrations are maintained.

3. DYSFUNCTION OF MECHANISMS REGULATING MILK TRACE ELEMENT CONCENTRATIONS Very little is known about cases in which concentrations of trace elements in milk are abnormally low. We have, however, studied several cases of women who had significantly lower concentrations of breast milk zinc as compared to normal women at the same stage of lactation. Most of these cases were women delivering infants prematurely; however, cases have also been found in women delivering at term (Atkinson et al., 1989). We analyzed the concentrations of protein, lipid and carbohydrate, as well as other trace elements, such as iron and copper, and found them all to be within the normal range. Further, concentrations of ligands binding zinc in milk were found to be normal. It is not yet known why with zinc specifically was low in these women, but it is possible that a zinc transporter responsible for the secretion of zinc into milk may be defective. It is possible that similar defects may exist for other trace elements, but in the case of zinc the infants suffering the consequences of zinc deficiency are easier to find and diagnose than those possibly suffering from iron or copper deficiency in which the signs are less specific and take a longer time to manifest themselves.

4. EFFECT OF MATERNAL TRACE ELEMENT DEFICIENCY OR EXCESS ON NORMAL MAMMARY GLAND FUNCTION Although most of the concern about lactating women being suboptimal in trace element status has been related to the concentration of the particular element in milk, the possibility of an effect on mammary gland function in general needs to be considered (Latulippe et al., 1999) have shown that iron deficient women produce milk which is low in folate. The mechanism behind this lesion is not yet known, but it is possible that one of the steps in mammary folate uptake and/or secretion is dependent upon normal iron status. (Brätter et al., 1997) have shown that women with high selenium intake secrete milk which is low in zinc, suggesting that selenium intake above a certain level may impair normal zinc uptake or secretion by the mammary gland.

5. CONCLUSIONS It is evident that mechanisms exist that regulate milk concentrations of trace elements such as iron and zinc, while the transfer of elements like selenium appears unregulated and may be explained by passive diffusion. Increased knowledge of these mechanisms may help us understand the transfer of trace elements into milk in normal conditions, but also help us investigate the lesions behind abnormal transfer of trace elements as well as defects in mammary gland function induced by low trace element status.

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REFERENCES Atkinson, S., Whelan, D., Whyte, R., and Lönnerdal, B., 1989, Abnormal zinc content in human milk: risk for development of nutritional zinc deficiency in infants, Am. J. Dis. Child. 143:608–611. Beshgetoor, D. and Lönnerdal, B., 1997, Effect of marginal maternal zinc deficiency in rats on mammary gland zinc metabolism, J. Nutr. Biochem. 8:573–578. Beshgetoor, D. and Lönnerdal, B., 1999, Identification of an . 2-macroglobulin receptor in human mammary epithelial cells, J. Nutr. 129:152–157. Brätter, P., Brätter, V.E., Recknagel, S., and Brunetto, R., 1997, Maternal selenium status influences the concentration and binding pattern of zinc in human milk, J. Trace Elem. Med. Biol 11:203–209. Chang, S.-C, O’Brien, K.O., Zavaleta, N., Caulfield, L.E., Abrams, S.A., and Bannon, D., 1999, The impact of Fe & Zn supplementation on the mineral content of colostrum, FASEB J. 13:A255 (abstr). Caulfield, L., Yamini, S., Zavaleta, N., and Shankar, A., 1999, Breastmilk (BM) zinc concentrations at one and three months in Peruvian women supplemented with zinc during pregnancy and lactation, FASEB J. 13:A250 (abstr.). Celada, A., Brusset, R., Gutierrez, J., and Herreros, V., 1982, No correlation between iron concentration in breast milk and maternal iron stores, Helv. Paediatr. Acta 37:11–16. Fransson, G.-B., Agarwal, K.N., Gebre-Medhin, M., and Hambraeus, L., 1985, Increased breast milk iron in severe maternal anemia: physiological “trapping” or leakage?, Acta Paediatr. Scand. 74:290–291. Kumpulainen, J., Salmenperä, L., Siimes, M.A., Koivistoinen, P., and Perheentupa, P., 1985, Selenium status of exclusively breast-fed infants as influenced by maternal organic or inorganic selenium supplementation, Am. J. Clin. Nutr. 42:829–835. Latulippe, M.E., Irurita, M.J., Villalpando, S., and Picciano, M.F., 1999, Lactating women with iron deficiency secrete milk low in folate, FASEB J. 13:A696. Loh, T.T. and Sinnathury, T.A., 1971, Haematological data and milk iron in Malaysian women, Aust. J. Obstet. Gynaecol. 11:254–58. Lönnerdal, B., Keen, C.L., and Hurley, L.S., 1981, Iron, copper, zinc and manganese in milk, Annu. Rev. Nutr. 1:149–174. Lönnerdal, B., Zavaleta, N., Kusunoki, L., Lanata, C.F., Peerson, J.M., and Brown, K.H., 1996, Effect of post-partum infection on proteins and trace elements in colostrum and early milk, Acta Paediatr 85:537–542. Moser-Veillon, P.B. and Reynolds, R.D., 1990, A longitudinal study of pyridoxine and zinc supplementation of lactating women, Am. J. Clin. Nutr. 52:135–141. Murray, M.J., Murray, A.B., Murray, N.J., and Murray, M.B., 1978, The effect of iron status of Nigerian mothers on that of their infants at birth and 6 months, and on the composition of Fe in breast milk, Br. J. Nutr. 39:627–630. Sigman, M. and Lönnerdal, B., 1990a, Relationship of milk iron and the changing concentration of mammary tissue transferrin receptors during the course of lactation, J. Nutr. Biochem. 1:572–576. Sigman, M. and Lönnerdal, B., 1990b, Response of rat mammary gland transferrin receptors to maternal dietary iron during pregnancy and lactation, Am. J. Clin. Nutr. 52:446–450. Vuori, E., Mäkinen, S.M., Kara, R., and Kuitunen, P., 1980, The effects of dietary intakes of copper, iron, manganese, and zinc on the trace element content of human milk, Am. J. Clin. Nutr. 33:227–231. Zavaleta, N., Lanata, C., Butron, B., Peerson, J.M., Brown, K.H., and Lönnerdal, B., 1995, Effect of acute maternal infection on quantity and composition of breast milk, Am. J. Clin. Nutr. 62:559–563. Zavaleta, N., Nombera, J., Rojas, R., Hambraeus, L., Gislason, J., and Lönnerdal, B., 1995, Iron and lactoferrin in milk of anemic mothers given iron supplements, Nutr. Res. 15:681–690.

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GLUTATHIONE PEROXIDASE IS NOT A FUNCTIONAL MARKER OF SELENIUM STATUS IN THE NEONATAL PERIOD 1

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L. A. Daniels , R. A. Gibson , and K. Simmer 1

Departments of Public Health Flinders University of South Australia Bedford Park, South Australia Australia 2 Department Paediatrics and Child Health Flinders Medical Centre Bedford Park, South Australia Australia

1. INTRODUCTION The selenoprotein glutathione peroxidase (GSHPx) is a central component of the cellular antioxidant defense system (Daniels, 1996). The high oxygen concentrations associated with respiratory support in preterm infants increase oxidant stress, a primary aetiological factor for chronic lung disease (CLD) (Kelly, 1993). GSHPx requires selenium (Se) for synthesis and Se depletion results in reduced tissue, blood and plasma GSHPx activity (Daniels, 1996). There is evidence that preterm infants are at risk of Se depletion (Daniels et al., 1997), which may, through the antioxidant role of GSHPx, impact on clinical outcome, specifically CLD (Darlow et al., 1995). In adults, with low Se intakes and blood Se levels, there is a strong linear relationship between Se concentration and GSHPx activity. However above blood Se levels of around further increases in GSHPx activity are not found (Thomson et al., 1993). This plateau is thought to represent Se sufficiency (Levander, 1985; Thomson et al., 1993) and a correlation between Se and GSHPx activity implies depletion. In Sedepleted adults, GSHPx activity in various blood components has been shown to reflect cardiac and hepatic Se and enzyme levels (Batist et al., 1985; Diplock, 1993). Thus, GSHPx activity has been widely used as a functional indicator of status in adults with low Se intakes, but is of limited value in assessing Se status in Se-sufficient individuals or populations (Diplock, 1993). Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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Evaluation of GSHPx as an indicator of Se status in infants has been limited. Valid methods for assessing Se status in preterm infants are required, particularly given the risk and potential clinical importance of Se depletion (Daniels et al., 1997; Darlow et al., 1995). There have been reports of weak or non-existent relationships between GSHPx and Se levels in preterm infants (Sluis et al., 1992), even though blood Se concentrations are well within the range where a strong linear relationship would be expected in adults (Thomson et al., 1993). There is both animal (Cross et al., 1977) and in vitro (Jornot and Junod, 1995) evidence to suggest that GSHPx activity and synthesis may also be regulated by oxygen exposure. The purpose of this paper was to examine the hypothesis that GSHPx activity in preterm infants is influenced by oxygen exposure and hence does not reflect preterm Se status in the neonatal period.

2. METHODS AND SUBJECTS Erythrocyte GSHPx activity and plasma and erythrocyte Se were measured between days 2–5 (Week 0) and then weekly until discharge in 63 preterm infants (40 males) with mean ± SE birth weight and gestation of 1,572 ± 60 (range 654–2,930) g and 30.7 ± 0.3 (25–36) weeks. Most of the preterm infants (95%) received respiratory support, primarily for respiratory distress syndrome (49%). Seventy-eight percent were ventilated, 28% developed CLD and 44% were transfused. Preterm infants received parenteral nutrition with negligible Se within 72 hours of birth and then were graded to standard preterm formula (FF) ([Se] or own mothers’ breast milk (BM) ([Se] 13 µg/l (Daniels et al., 1997). The net daily Se intake from all sources was averaged over each week and the first four and six weeks of life. The average daily Se intake of the preterm infants over the first six weeks of life was A healthy term reference group (n = 46, 25 males, birth weight 3,473 ± 81 g) was assessed at day 5 and six weeks and received breast milk or formulae.

3. RESULTS The indicators of Se status at week 0 and week 6 are compared in Table 1. Between weeks 0 and 6 plasma Se declined in preterm and increased in term infants (median –9.9 vs p < 0.001) and erythrocyte Se also dropped in the preterms infants (–136 vs –15, p = 0.009). However, the opposite pattern was seen with GSHPx activity, with an increase in preterm and no change in the term infants evident (median +0.23 vs –0.01 IU/g Hb, p = 0.034). However, when method of feeding and hence Se intake were considered only those preterm infants who received BM showed an increase in GSHPx (Table 1). In term infants, plasma Se increased (p < 0.001) but only in those who were breast-fed and there was no change in erythrocyte Se or GSHPx, regardless of feeding. In both preterm and term infants there were no associations between GSHPx activity and Se intake or plasma Se. There were weak and irregular correlations between erythrocyte GSHPx and Se over the first three months. The relationships at weeks 4 and 6 between GSHPx activity and parameters of respiratory support which quantify oxygen exposure are shown in Tables 2 and 3. Preterm infants who developed CLD and hence were still receiving oxygen at four weeks had higher GSHPx activity at 6 weeks than infants without CLD (1.49 ± 0.53 vs 1.12 ± 0.30 IU/g Hb, n = 16, 18; p = 0.017)

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4. DISCUSSION These data show no evidence of the linear relationship between GSHPx activity and blood Se levels seen in Se depleted adults. Indeed, in the preterm infants there appears to be an anomalous increase in GSHPx activity in the face of declining plasma Se levels, while in the term breast-fed infants, despite a 30% increase in plasma Se, there was no change in GSHPx. This is consistent with New Zealand (a low Se area) data, showing associations between GSHPx and Se levels in adults and term infants that suggest Se depletion. However, no such relationships were evident in preterm infants (Dolamore

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et al., 1992) (Sluis et al., 1992), who are relatively more depleted that adults and term infants in the same region (Daniels et al., 1997). These results report consistent associations between GSHPx levels and oxygen concentration and pressure and hence support the hypothesis that GSHPx activity in preterm infants is confounded by oxygen. It is interesting to note that, while the BM-fed preterm infants had a Se higher intake over the first six weeks than the FF fed group (Daniels et al., 1997), both groups showed a decline in plasma Se, but only the BM-fed preterm infants produced an increase in GSHPx activity. This suggests that, consistent with animal studies (Cross et al., 1977), preterm infants may be able to increase GSHPx activity in response to the oxidant stress of respiratory support, but only if they have adequate Se intake. Data from our previous studies (Daniels et al., 1997; Daniels, Gibson and Simmer, 1996) further support this contention. An increase in GSHPx activity was seen in very sick preterm infants who had substantial PN and therefore extremely low Se intakes and plasma levels, but very high oxygen requirements. In contrast, relatively healthy preterms who had high BM and Se intakes and low oxygen requirements showed no change in GSHPx activity over the first six weeks. We conclude that erythrocyte GSHPx activity is influenced by supplemental oxygen and hence the traditional role as a functional marker Se status is questionable in preterm infants receiving respiratory support. This does not imply that GSHPx activity and Se status are not important for antioxidant protection in preterm infants. It is possible that for preterm infants the capacity to increase GSHPx activity in response to oxygen exposure may indicate adequate Se status. If this is the case then, although the traditional role of GSHPx as a functional marker may not be valid, further studies are required to explore the potential significance and interpretation of GSHPx activity in preterm infants in relation to Se status and risk of oxygen toxicity.

ACKNOWLEDGMENTS A more detailed report of the data presented here has been published under the same title and authors in Journal Pediatric Gastroenterology and Nutrition 1998; 26:263–268.

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REFERENCES Batist, G., Norton, J., Katki, A.G., Wagman, L., Ferrans, V.J., Maher, M., and Myers, C.E., 1985, Cardiac and red blood cell glutathione peroxidase: results of a prospective randomized trial in patients on total parenteral nutrition, Cancer Res. 45:5900–5903. Cross, C.E., Hasegawa, G., Reddy, K.A., and Omaye, S.T., 1977, Enhanced lung toxicity of O2 in seleniumdeficient rats, Res. Commun. Chem. Pathol. Pharmacol. 16:695–706. Daniels, L.A., 1996, Selenium metabolism and bioavailability, Biol Trace Elem. Res. 54:185–199. Daniels, L., Gibson, R., and Simmer, K., 1996, Randomised clinical trial of parenteral selenium supplementation in preterm infants, Archives of Disease in Childhood: Fetal and Neonatal Edition. 74:F158–F164. Daniels, L., Gibson, R., and Simmer, K., 1997, Selenium status of preterm infants: The effect of postnatal age and method of feeding, Acta Paediatrica. 86:281–288. Darlow, B.A., Inder, T.E., Graham, P.J., Sluis, K.B., Malpas, T.J., Taylor, B.J., and Winterbourn, C.C., 1995, The relationship of selenium status to respiratory outcome in the very low birth weight infant, Paediatrics. 96:314–319. Diplock, A.T., 1993, Indexes of selenium status in human populations, American Journal of Clinical Nutrition. 57:256S–258S. Dolamore, B.A., Brown, J., Darlow, B.A., George, P.M., Sluis, K.B., and Winterbourn, C.C., 1992, Selenium status of Christchurch infants and the effect of diet, New Zealand Medical Journal. 105:139–142. Jornot, L. and Junod, A.F., 1995, Differential regulation of glutathione peroxidase by selenomethionine and hyperoxia in endothelial cells, Biochemical Journal. 306:581–587. Kelly, F.J., 1993, Free radical disorders of preterm infants, British Medical Bulletin. 49:668–678. Levander, O.A., 1985, Considerations on the assessment of selenium status, Federation Proceedings. 44:2579–2583. Sluis, K.B., Darlow, B.A., George, P.M., Mogridge, N., Dolamore, B.A., and Winterbourn, C.C., 1992, Selenium and glutathione peroxidase levels in premature infants in a low selenium community (Christchurch, New Zealand), Pediatric Research. 32:189–194. Thomson, C.D., Robinson, M.F., Butler, J.A., and Whanger, P.D., 1993, Long-terra supplementation with selenate and selenomethionine: selenium and glutathione peroxidase (EC 1.11.1.9) in blood components of New Zealand women, British Journal of Nutrition. 69:577–588.

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TRACE ELEMENT TRANSFER FROM THE MOTHER TO THE NEWBORN— INVESTIGATIONS ON TRIPLETS OF COLOSTRUM, MATERNAL, AND UMBILICAL CORD SERA Michael Krachler1,*, Erich Rossipal2, and Dusanka Micetic-Turk3 1

Research Centre Juelich Institute of Applied Physical Chemistry Germany and 2 Department of Pediatrics Karl-Franzens-Universität Graz Austria 3 Department of Pediatrics Teaching Hospital, Maribor Slovenia

The purpose of this study is to elucidate the transfer of trace elements from the maternal organism to the newborn and to clarify the function and permeability of the placenta for selected trace elements. The concentrations of the eight essential elements calcium (Ca), cobalt (Co), copper (Cu), magnesium (Mg), manganese (Mn), molybdenum (Mo), tin (Sn), and zinc (Zn) and of the not essential and toxic elements barium (Ba), beryllium (Be), bismuth (Bi), cadmium (Cd), cesium (Cs), lanthanum (La), lithium (Li), lead (Pb), rubidium (Rb), antimony (Sb), strontium (Sr), and thallium (Tl) were determined in umbilical cord (N = 29) and corresponding maternal sera (N = 29) as well as in colostrum (N = 27). Umbilical cord serum concentrations of Ca, Mn, and Zn were 120%, 150%, and 148% of the maternal value, respectively. Maternal sera had twice the Cu concentrations found in healthy adults and five-times higher Cu than umbilical cord sera. Concentration ratios colostrum/maternal serum and colostrum/umbilical cord serum were approximately 1 for Co, 1.4 for Mg, 2 for Ca, Mn, and Sn, 5 for Cu (maternal serum), 8 for Mo, and 10 for Zn. Concentrations of the toxic elements Cd and Pb decreased in the order colostrum (Pb 2.6µg/L; Cd 0.6µg/L), maternal sera (0.8µg/L; 0.3µg/L), umbilical cord sera (0.4µg/L; 0.2µg/L). Maternal serum Ba and Rb was 182% and 66% of the umbilical cord Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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value. For Sr and Li, an almost perfect correlation between umbilical cord and maternal sera was found. For Ba, Co, Cu, Mn, Zn there was no correlation, and for Ca, Cs, Mn, Mo, Rb only weak positive correlations between these two compartments could be estab– lished. The results of this study indicate that an active transport mechanism for the transport of Ca, Mn, Rb, and Zn from the mother to the newborn exists, whereas Cs, Li, and Sr follow concentration gradients. As regards Cu, the placenta showed to have a blocking effect on the transfer from the mother to the baby.

1. MATERIALS AND METHODS Subjects Umbilical cord blood from 29 newborns (f/m = 17/12) was sampled into uncontaminated polyethylene tubes by manual expression. Venous blood from the 29 mothers (27.6 ± 5.2yrs), from whom blood samples for medical reasons had to be taken, was simultaneously drawn with polyethylene syringes within 20min before delivery and immediately transferred to polyethylene tubes. Serum was obtained by immediate centrifugation of blood for 10 minutes at l,800g at 4°C. The serum samples were transferred into polyethylene tubes and kept at –20 °C, until they were mineralized. Only sera of newborns of healthy mothers who had successfully given a natural birth to a mature baby at the University Maternity Clinic in Maribor, Slovenia in 1996 and 1997 after an uneventful pregnancy were considered. No mother was smoking during pregnacy, although five mothers smoked before pregnancy. Twenty-seven colostrum samples of the mothers (from 2 mothers no colostrum was available) were collected by manual expression of the breast during the first to the third day after delivery. The breast from which the milk was collected, was cleaned with doubly-distilled water and was air-dried. Mothers of the newborns had been fully informed about the aim of the investigation and gave their consent to the study.

Mineralization of Serum, Colostrum, and Milk Powder A closed-pressurized, high-performance microwave digestion unit (MLS 1200 MEGA, MLS GmbH, Leutkirch, Germany) equipped with a rotor for ten Teflon vessels designed for pressures up to 30 bar was used for mineralizations. Aliquots of serum, weighed to 0.1 mg were poured into the colostrum or milk powders digestions vessels and mixed with 1.5 mL concentrated HNO3 purified by sub-boiling distillation in an all-quartz distillation unit and 0.5 mL high purity hydrogen peroxide (30%, Suprapur®, Merck) and mineralized under conditions described previously (Domej et al., 1998; Krachler et al., 1998b). Completely clear, colorless, homogenous digests were obtained and subsequently diluted to 10 mL with high-purity water.

Quality Assurance To guarantee the accuracy and precision of the applied technique (microwave digestion and ICP-MS) two whole blood reference materials (Seronorm™ Trace Elements Whole Blood I+II, Batch No. 203052 + 203056, Nycomed, Oslo, Norway), one serum reference material (Seronorm™ Trace Elements Serum, Batch No. 010017, Nycomed),

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and two certified skim milk powder reference materials (BCR 063—natural level, and BCR 150—spiked, BCR, Brussels, Belgium) were analyzed. Experimental concentrations were in good agreement with certified values (Krachler et al., 1997b; Krachler et al., 1998b+c).

Determination of Trace Elements An inductively coupled plasma mass spectrometer (VG PlasmaQuad Turbo Plus, VG Elemental Ltd., Winford, UK) equipped with a Meinhard concentric glass nebulizer, a double-pass, Scott-type spray chamber (water cooled, 0°C), and a Gilson Minipuls-3 peristaltic pump was applied to the determination of trace elements. Operating conditions of the ICP-MS instrument and details of the analytical procedure are described elsewhere (Krachler et al., 1996; Krachler et al., 1998b+c).

2. RESULTS AND CONCLUDING REMARKS Concentrations of the essential elements Ca, Mn, and Zn in umbilical cord sera are significantly higher than in maternal sera. Therefore, it is most likely that for Ca, Mn, and Zn an active transport mechanism for the transfer of these elements from the mother to the fetus exists. The same holds true for Rb. The mobilization of Cu in mothers during pregnancy leads to 2-fold higher maternal serum Cu concentrations compared to healthy adults. Umbilical cord sera values amount to about only 20% of that of the mothers, providing body weight daily to be accumulated by the human fetus. The high correlation coefficients for concentrations of the alkaline elements Li and Cs in umbilical cord and maternal sera, which have Na and K as neighbors in the periodic table of the elements, suggest that Li and Cs follow concentration gradients, so does Sr. More detailed information regarding the trace element transfer from the mother to the newborn can be derived from the simultaneous quantification of trace elements in arterial and venous umbilical cord sera. By splitting umbilical cord sera into its arterial and venous components, no models for the estimation of the element transfer are nec-; essary. Based on the well-known flow rates of blood within the placenta and the differences of the concentrations of a particular element in arterial and venous umbilical cord sera enable mass balances for each element. A part of these investigations have already been performed (Krachler et al., 1999a), shedding more light on the transfer of trace elements from the maternal organism to the fetus perinataly. A detailed description of this current research work has been published recently in the European Journal of Clinical Nutrition (Krachler et al., 1999b).

ACKNOWLEDGMENT The authors would like to acknowledge the financial support of the “Gesellschaft zur Förderung der Gesundheit des Kleinkindes” Graz, Austria to perform this study.

REFERENCES Domej W., Krachler M., Schlagenhaufen C., Trinker M., Krejs G.J., and Irgolic K.J., 1997: Trace elements in pleural effusions. J. Trace Elements Med. Biol. 11, 232–238.

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Krachler M., Radner H., and Irgolic K.J., 1996: Microwave digestion methods for the determination of trace elements in brain and liver samples by inductively coupled plasma mass spectrometry. Fresenius J. Anal. Chem. 355, 120-128. Krachler M., Wirnsberger G., and Irgolic K.J., 1997a: Trace element status of hemodialyzed patients. Biol. Trace Elem. Res. 58, 209–221. Krachler M., Lindschinger M., Eber B., Watzinger N., and Wallner S., 1997b: Trace elements in coronary heart disease: Impact of intensified lifestyle modification. Biol. Trace Elem. Res. 60, 175–185. Krachler M., Rossipal E., and Micetic-Turk D., 1998a: Concentrations of trace elements in sera of newborns, young infants, and adults. Biol. Trace Elem. Res., in press. Krachler M., Li F.S., Rossipal E., and Irgolic K.J., 1998b: Changes in the concentrations of trace elements in human milk during lactation. J. Trace Elements Med. Biol. 12, 159–176. Krachler M., Rossipal E., and Irgolic K.J., 1998c: Trace elements in infant formulae based on cow and soy milk and in Austrian cow milk determined by inductively coupled plasma mass spectrometry. Biol. Trace Elem. Res. 65, 53–74. Krachler M., Alimonti A., Petrucci F., Irgolic K.J., Forastiere F., and Caroli S., 1998d: Analytical problems in the determination of platinum-group metals in urine by quadrupole and magnetic sector field inductively coupled plasma mass spectrometry, Anal. Chim. Acta 363, 1–10. Krachler M., Alimonti A., Petrucci F., Forastiere F., and Caroli S., 1998e: Influence of sample pre-treatment on the determination of trace elements in urine by inductively coupled plasma mass spectrometry. J. Anal. At. Spectrom. 13, 701–706. Krachler M., Rossipal E., and Micetic-Turk D., 1999a: Concentrations of trace elements in arterial and venous umbilical cord sera. Trace Elem. Electrolytes 16, 46–52. Krachler M., Rossipal E., and Micetic-Turk D., 1999b: Trace element transfer from the mother to the newborn—Investigations on triplets of colostrum, maternal and umbilical cord sera. Em J. Clin. Nutr., in press. Rossipal E. and Krachler M., 1998a: Pattern of trace elements in human milk during the course of lactation. Nutr. Res. 18/1, 11–24. Rossipal E., Krachler M., and Micetic-Turk D., 1998b: Concentrations of trace elements in sera of young infants fed breast milk or infant formula. In: Metal Ions in Biology and Medicine, Collery Ph, Brätter P., Negretti de Bratter V., Khassanova L., Etienne J.C., eds. Paris: John Libbey Eurotext, Vol. 5, pp. 511–515.

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RELATIONSHIP BETWEEN TRACE ELEMENTS, ACTIVITIES OF ANTIOXIDANT ENZYMES IN MATERNAL AND UMBILICAL CORD BLOOD IN POLAND 1

1

2

1

W. Wasowicz , J. Gromadzinska , K. Szram , K. Rydzynski , 3 2 4 P. Wolkanin , Zb. Pietrzak , and J. Tomczak 1

Department Toxicol. Cancerogenesis Inst. Occupat. Med. 8 Teresy St. 90-950 Lodz Poland 2 Department Obstet. Gynaecol. Polish Mother Memorial Hospital Lodz 3 Military Medical Academy, Lodz 4 St. Peter’s Clinic, Duren Germany

1. INTRODUCTION Delivery and the period directly after birth are characterized in the neonate by considerably enhanced reactive oxygen species (ROS) attack, caused, among others, by rapid exposure to oxygen at atmospheric pressure and by the lack of sufficient protection which is provided by the placenta and maternal organism during pregnancy. It may be assumed that the course of pregnancy has an influence on the concentration of microelements and low molecular antioxidants, as well as on the activity of protective enzymes such as glutathione peroxidase (GPx), superoxide dismutase (SOD) and ceruloplasmin in the neonate. Deficiencies of microelements, particularly selenium (Se), are considered to be of special importance. Some authors observe a relationship between premature birth and intrauterine fetal dystrophy and low concentration of the microelement in the newborn (Lockitch et al., 1989), while this was not true in another studies (Wasowicz et al., 1993). Experiments on animals prove that deficiencies of either zinc (Zn) and copper (Cu) during pregnancy are associated with abnormal labor, intrauterine growth retardation, congenital malformations, and developmental disturbances in surviving offspring (Hurley, 1981). Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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Since there are a number of various factors which may cause increase of ROS concentration and intensification of the enzymatic and non-enzymatic systems of their removal, determination of a single parameter will not result in an explicit assessment of antioxidant capacity of the organism. The aim of the present work is to determine the parameters of oxidative stress in women at delivery and to correlate the results with the parameters observed in the neonates.

2. MATERIALS AND METHODS Women with normal pregnancies and normal delivery time (over 37 weeks of pregnancy) were qualified for examination. This study included 117 neonates (65 girls) and 117 women (average age 24; from 19 to 37yrs.). Sixty-three women had no previous deliveries, 36 one previous delivery, and 18 had two or more deliveries. None of the women suffered from metabolic diseases or had undergone a surgery. No developmental defects were found in the neonates. The group of neonates and their mothers was divided according to the neonate’s birth weight (<2,500g and 2,500g) and gender, number of mother’s pregnancies and gestation age. Gestation age of the newborns was determined by calculating from the reported last menstrual period or ultrasonographic examination. Smoking during pregnancy was also taken into consideration. While collecting information, attention was also paid to microelement supplementation during pregnancy. If the answer was positive, mothers were excluded from the study. Blood samples for the investigation were placed in heparinized test tubes free from trace elements. Samples were centrifuged and plasma stored at –20 °C until trace elements analysis. The Ethical Committee of the Medical Academy of Lodz approved the protocol of the study. The data were expressed as means SD and were subjected to statistical analysis by non-parametric Mann-Whitney test, analysis of variance and calculation of correlation coefficient. Differences at p < 0.05 were considered significant.

3. RESULTS It was found that Se concentration in plasma of women at delivery was 40.3 ± 10.5 and was significantly higher (p < 0.001) than in neonates (33.3 ± 10.2µg/l). The concentration of Cu in maternal plasma was significantly higher (p < 0.001), while that of Zn was significantly lower (p < 0.001) than in neonates and was found to be 1.99 ± 0.83 mg/1 vs. 0.56 ± 0.28mg/1 and 0.74 ± 0.21 mg/1 vs. 0.98 ± 0.32mg/l, respectively. GPx activity in both erythrocytes (15.4 ± 2.8 u/g Hb) and plasma, (188 ± 36 u/l) of women at delivery was significantly higher than in umbilical plasma (11.0 ± 4.0 u/g Hb; p < 0.001 and 113 ± 31u/l; p < 0.001, respectively). A similar relation was observed between activities of SOD in erythrocytes and ceruloplasmin in plasma of women at delivery and neonates. Statistically significant differences depending on the birth weight of the neonate were found between the parameters under investigation in the plasma and erythrocytes, except for Se concentration and GPx activity in the plasma of the women and their neonates with birth weight below 2,500g and thiobarbituric acid reactive substances (TBARS) concentration in both the whole examined group and in the group where the birth weight of the neonate was over 2,500g. A weak, but statistically significant linear correlation was found between Zn concentration in the plasma of the mother (r = 0.213; p < 0.05) and the neonate (r = 0.268; p < 0.05; Table 1). Significant relationships between variables

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determined in neonates and the determined parameters are presented in Table 1. Significant linear correlations were also discovered between the following parameters determined simultaneously in the maternal and umbilical cord blood: Zn (r = 0.474; p < 0.001), Cu (r = 0.588; p < 0.001), GPx RBC (r = 0.503; p < 0.001); plasma GPx (r = 0.658; p < 0.001); SOD (r = 0.557; p < 0.001); TBARS (r = 0.404; p < 0.001). No such relations were found in the case of ceruloplasmin activity and Se concentration. No differences were found between the parameters under study, related to the number of pregnancies or deliveries, the week of pregnancy or smoking habit.

4. DISCUSSION During normal pregnancy, a demand for and use of oxygen by woman’s organism and the developing fetes increases. The higher use of oxygen may result in intensification of free radical processes, caused, among others, by increased ROS synthesis or lowered antioxidative activity of the organism. Enhanced ROS attack is a characteristic feature during delivery and the period directly following birth (Jain, 1986), hence the crucial importance of appropriate antioxidative potential of the neonate organism. It is now believed that the antioxidative potential of the fetus is affected by the course of pregnancy and, it seems, the deposit of macro- and microelements in the mother’s organism as proper amounts of these components can be supplied only through the placenta (Hurley and Keen, 1988). In this study we proved the significantly higher concentration of Zn in umbilical than maternal plasma, and higher level of Se and Cu in maternal than cord plasma. This observation is in agreement with the pattern observed in our previous study (Wasowicz et al., 1993), and by other authors (Bro et al., 1988a,b). The results of the activity of antioxidative enzymes seem to confirm the hypothesis that the protection from the intensified lipid peroxidation process provided by low erythrocyte SOD and GPx activity, accompanied by simultaneous increased TBARS concentration is not efficient enough. As demonstrated by the analysis of our results, there are statistically significant linear correlations between the determined parameters in the group of women at delivery and in the neonates. It was found that the child’s birth weight is positively correlated with the concentration of Zn in the plasma of the mother, and inversely correlated TBARS concentration (Table 1). Thus, the higher plasma Zn concentration in the mother, the higher the neonate’s birth weight. TBARS concentration, on the other hand, is lower in neonates with higher birth weight, which may indicate that peroxidation processes are intensified in newborns with lower birth weight. The data concerning the relation between maternal blood Zn concentration and the child’s birth weight are not explicit. The analysis of 41 studies on this subject, carried out by Tamura and Goldenberg (1996), did not result in formulation of clear conclusions (22 studies confirm the relation and in 19 the

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relation is not observed). The distribution of maternal plasma Se, Zn and Cu concentrations, as well as antioxidant enzymes activities according to the number of pregnancies or deliveries, the week of pregnancy and smoking habit suggested no statistical association with these variables. Mbofung and co-workers. (Mbofung et al., 1986), however, found changes in the level of microelements related to increasing number of parity. In conclusion it should be observed that concentrations/activities of the parameters under study in umbilical blood differ significantly from those found in maternal blood, and that changes in the parameters in which occur in maternal and cord blood exhibit dependence on the birth weight of the neonate. There are statistically significant linear correlations between individual parameters in maternal and neonatal blood.

ACKNOWLEDGMENTS This work was supported by the State Committee for Scientific Research (KBN); grant No 0627/P05/98/14.

REFERENCES Bro S., Berendtsen H., Norgaard J., Host A., and Jorgensen P., 1988, Serum selenium concentration in maternal and umbilical cord blood: relation to course and outcome of pregnancy, J. Trace Elem. Electrolytes Health Dis. 2:165–169. Bro S., Berendtsen H., Norgaard J., Host A., and Jorgensen P., 1988, Serum zinc and copper concentrations in maternal and umbilical cord blood. Relation to course and outcome of pregnancy, Scand. J. Lab. Invest. 48:805–811. Hurley L.S., 1981, Teratogenic acpects of manganese, zinc, and copper nutrition, Physiol. Rev. 61:249–295. Hurley L.S. and Keen C.L., 1988, Fetal and neonatal development in relation to maternal trace element nutrition: manganese, zinc, and copper, in: Vitamins and Minerals in Pregnancy and Lactation, Volume 16 (H. Berger, Nestle Nutrition Workshop Series), pp. 215–230, Nestle Ltd. Vevey/Raven press Ltd. New York. Jain S., 1986, Membrane lipid peroxidation in erythrocytes of the newborn, Clin. Chim Acta 161:301–306. Lockitch G., Jacobson B.E., Quigley G., Dison P., and Pendray M.R., 1989, Selenium deficiency in low birthweight neonates: an unrecognized problem, J. Pediatr. 114:865–870. Mbofung C.M.F., Antimo T., and Omololu A., 1986, Neonatal, maternal, and intrapartum factors and their relationship to cord- and maternal-plasma trace-element concentration, Biol. Trace Elem. Res. 9:209–219. Tamura T. and Goldenberg R.L., 1996, Zinc nutriture and pregnancy outcome, Nutr. Res. 16:139–181. Wasowicz W., Wolkanin P., Bednarski M., Gromadzinska J., Sklodowska M., and Grzybowska K., 1993, Plasma trace element (Se, Zn, Cu) concentrations in maternal and umbilical cord blood in Poland. Relation with birth weight, gestational age and parity, Biol. Trace Elem. Res. 38:205–215.

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COMPENSATORY CHANGES IN SELENOENZYMES IN FULL TERM HUMAN PLACENTAE INDUCED BY ENVIRONMENTAL CONDITIONS P. Zagrodzki1,2, L. Zamorska3, and M. Zadrozna3 1

Department of Food Chemistry and Nutrition Jagiellonian University School of Medicine, Medyczna 9 30-688 Kraków 2 Institute of Nuclear Physics Radzikowskiego 152, 31-342 Kraków 3 Department of Cytobiology and Histochemistry Jagiellonian University School of Medicine Medyczna 9, 30-688 Kraków

There is a growing interest in considering human placenta as an indicator in environmental studies based on changes in histological, biochemical and morphological features of placenta. The possible differences in selenoenzymes activities were investigated in full term human placentae collected in low-polluted (Bieszczady) and high-polluted (Cracow) regions. This was accompanied with histochemical measurements of oxidative chain enzymes activities in villous syncytiotrophoblast. The placentae examined consisted of 24 cases, 14 of which originated from Bieszczady region, and 10 from Cracow. The parturients were in good health, nonsmokers and permanent residents of studied areas. All neonates were alive and free from evident developmental defects. The following parameters were measured: plasma glutathione peroxidase (plGSHPx) and Se (in plasma of maternal and umbilical cord blood), cellular glutathione peroxidase (cGSHPx), thioredoxin reductase (TR), NADH-dehydrogenase (NADHD), cytochrome c oxidase (CCO) (in placentae). The apparent dissimilarity of populations being compared was revealed due to statistically significant differences in plGSHPx (only in umbilical cord plasma, p < 0.05), cGSHPx (p < 0.005), TR (p < 0.005), NADHD (p < 0.001), and CCO (p < 0.001) 373

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activities (Bieszczady: plGSHPx: 205.8 ± 94.8U/L; cGSHPx: 0.94 ± 0.41 U/mg; TR: 1.66 ±0.57mU/mg;NADHD 184.4 ± 6.1 (arbitrary units); Cracow: plGSHPx: 293.0 ± 86.3 U/L; cGSHPx: 1.74 ± 0.62U/mg; TR: 2.84 ± 1.21mU/mg; NADHD The deterioration of the activities of OCC and NADHD and the induction of selenoenzymes in placentae from Cracow are likely to be a simultaneous response to the enhanced pollution of the environment.

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MATERNAL ANEMIA AT DELIVERY, OUTCOME OF PREGNANCY AND INFANT BIRTH WEIGHT, IN A GROUP OF LOW SOCIOECONOMIC LEVEL, IN GREATER BUENOS AIRES

M. L. de Portela, L. López, S. H. Langini, S. Fleischman, A. Weisstaub, M. García, O. Moreno, and C. R. Ortega Soler Department Nutr. & Food Sciences & D. Paroissien Hospital (La Matanza) School of Pharmacy & Biochemistry University of Buenos Aires Junín 956,2p.-(1113) Argentina

Iron deficiency anemia is prevalent among pregnant women, but its influence in maternal and neonatal health is still uncertain. Latin America shows a high prevalence of maternal and child morbidity, mortality and anemia, reflecting nutritional problems and socio-cultural behavioural factors. However, Argentina is an exception within Latin America due to its high meat consumption, even in the low socio-economic population. A cross-sectional study was carried out in 157 pregnant women assisted at delivery in a suburban hospital, between July and September/1998. Most of the women did not have a follow-up at the Hospital and they had not taken iron or vitamin supplements during pregnancy. Maternal venous fasting blood samples were collected with EDTA and Hematocrit (Htc), Red Blood Cell (RBC), Hemoglobin (Hb) and Mean Cell Volume (MCV) were determined by an electronic counter. Infant birth weight (IBW) and delivery gestational age (DGE) were recorded. The mean values, standard deviation and ranges (between brackets) were: RBC (X 106/µl): 3.75 + 0.53; Htc(%): 34.1 + 5.1 (52–21); MCV(fL): 92 + 7 (115–71); Hb (g/dL): 11.1 + 1.8 (16.9–7.1); (DGA) (weeks): 38.8 + 2.4 (26–41). Distribution of the population according to Hb (g/dL) was: <7.0: 1.9%; 7.1–8.9: 10.5%; 9.0–10.9: 30.5%; 11.0–13.2: 45%; >13.2: 12%. MCV(fL) was <80 in 4.5% of the women and >100 in 5%. The percentage of preterm birth (<38 weeks of DGA) was 5.9% in the whole population, but there was no correlation with the haematologic parameters. Two women (1.3%) had dead infants. IBW (g) of the other alive born infants was: 3252 + 618 (5350–810). These results show that, in spite of an important percentage of women with Hb values <11.0 g/dL at delivery, there was a high percentage with Hb values >12.0 375

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g/dL. Therefore, the mandatory follow-up would be advisable as a Public Health policy, taking into account not only the low Hb levels but also the high ones. Moreover, special prenatal care and follow-up should be intensified to study the factors that influence maternal health and nutritional status on outcome of pregnancy. On the other hand, it would be mandatory to promote the implementation of public health guidelines, enhancing laboratory and nutritional practices in clinical management. Supported by University of Buenos Aires, Grant FA 060.

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TRACE ELEMENT AND VITAMIN DEFICIENCIES IN FRENCH PREGNANT WOMEN

I. Hininger*, M. Favier**, H. Faure*, J. Arnaud*, F. Khatir†, J. Thoulon†, E. Hariveau††, A. Favier*, and A. M. Roussel* *LBSO,

Université Joseph Fourier Domaine de la Merci, 38700 La Tronche France ** Service de gynécologie obstétrique Hôpital Sud. CHU de Grenoble France †Service de gynécologie obstétrique Hôpital E. Herriot, Lyon France ††Laboratoire Boiron Sainte Foy Les Lyon France

The prevalence of vitamin (A,E, beta-carotène, Bl, B6, B9) and mineral (Zn, Fe, Mg) deficiency in normal antenatal women, was studied. One hundred women at 5 to 16 weeks of gestional age, aged 20–38 years old were recruited in the maternal department of the Lyon and Grenoble University Hospitals. At baseline 6% of the women had low concentrations of serum vitamin No subjects had values in the range of major vitamin A deficiency but low values corresponding to a moderate risk of vitamin deficiency were observed in 10% of pregnant women. High risk of vitamin B6 deficiency was observed to 15% of the study subjects. For vitamin B2, a high risk of deficiency (>1.52) is observed in 8% of the pregnant women. The higher risk for vitamin deficiencies was observed for beta carotene and for vitamin Bl (1.18), respectively 40% dan 63%. None of our subjects had serum alpha-tocopherol or folate concentrations below the limit cut-off point respectively). Concerning trace elements low serum zinc concentration were found in 76% of the pregnant women. Plasma magnesium (<0.76 µmol/l) were low for 76% of pregnant women, but none had a low erythrocyte magnesium. Iron level was low in only 3% of the pregnant women and iron-deficient anemia were found in 4% of these women, which is the prevalence in French women. Indeed, at this time of 377

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pregnancy concentration were minimally affected by hemodilution since we verified that none had a protein concentration below the cut-off value.

CONCLUSION Until now the major trace element which was considered during pregnancy was iron and iron supplementation was quite systemic among clinical pratician. Our findings indicate that the proportion of pregnant women at risk of Zn deficiency is around 20 times more important than iron. Considering the role of zinc in the replication protein a particular attention to this trace element must be considered during pregnancy.

SPONSRING Bioron Laboratory (Ste Foy les Lyon). France.

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VINEGAR DRINK TO IMPROVE IRON STATUS DURING PREGNANCY

U. Heins, C. Koebnick, and C. Leitzmann Institute of Nutritional Science Wilhelmstr. 20, 35392 Giessen Germany

INTRODUCTION Iron-deficiency anemia is common among young women. Many pregnancies start with diminished iron stores. If the increased need cannot be met by the usual diet, iron supplementation is given to prevent anemia. For women refusing iron supplements because of its side effects, some midwives recommend a vinegar drink to improve iron status during pregnancy.

OBJECTIVES To investigate the influence of vinegar on iron status of iron deficient pregnant and non-pregnant women.

METHOD 27 pregnant (24th week of pregnancy ±5) and 6 non-pregnant women with low iron status took a vinegar drink three times a day with their meals over 4 weeks instead of iron supplements. The drink consists of 200ml water, 1 tablespoon vinegar (acidity 5%), and 1 teaspoon honey (optional). The nutritional iron intake was calculated from two 4day-estimated food records. Blood was taken before and after the intervention. Besides the usual parameters like Hb, MCV, serum iron, ferritin and transferrin, zinc protoporphyrin (ZPP) was analysed. 379

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RESULTS AND DISCUSSION ZPP decreases significantly in all participants (pregnant 31 to 28µmol/mol heme p < 0.001; non-pregnant 38 to 32 µmol/mol heme p = 0.046). ZPP is a minor metabolic byproduct of heme synthesis; in iron deficiency it is produced instead of heme. Its concentration is measured in erythrocytes and is therefore independent of plasma dilution due to pregnancy. Changes of the other parameters also give hints for an improvement of iron status even though they do not reach statistical significance. According to these data there is a positive effect of the above-mentioned vinegar drink on iron status in pregnant as well as in non-pregnant women. This might be due to the reducing potency of the acid which improves the absorption of iron. The effect is more obvious for non-pregnant than for pregnant women. If pregnant women are already iron deficient, vinegar drink alone is not sufficient to improve the iron status. An intake before or at an earlier stage of pregnancy might prevent iron deficiency.

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EVOLUTION OF COPPER CONTENTS FROM COLOSTRUM TO TRANSITIONAL HUMAN MILK

*M. D. Silvestre, M. J. Lagarda, R. Farré, **C. Martinez-Costa, **J. Brines, and ***G. Clemente Area de Nutrición y Bromatologia Universidad de Valencia Burjassot *Area de Veterinaria CEU-San Pablo, Moncada (Valencia) **Dpto. de Pediatría, Obstetricia y Ginecología Facultad de Medicina Universidad de Valencia ***Dpto. de Estadística Universidad Politécnica de Valencia España

The composition of human milk undergoes important variations during the first post-partum days. Knowing how trace elements elements contents evolve during this period is of interest in setting up nutritional requirements and understanding the physiology of secretion. In order to study the copper content evolution from colostrum to transitional human milk a longitudinal study has been carried out so as to eliminate interindividual variations.

SUBJECTS AND SAMPLES 39 healthy women agreed to participate in the study. Milk samples were collected the third and fifteenth days postpartum; similar milk volumes from both breast were taken before the infants’ feeding, always between 11 a.m. and 4 p.m.

METHODS Copper was measured by FAAS after a microwave digestion (2ml milk + 1.5ml 381

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RESULTS No significant differences (p < 0.05) were detected when a one-way ANOVA was applied to Copper contents of milk. From the individual study of the 39 considered cases it can be concluded that: a) Copper contents of colostrum milk are in a wide range whereas the range is narrow (from 0.31 to 0.50 µg/ml) for values corresponding to transitional milk; b) in 30 of 39 cases there is a large difference between the copper contents of colostrum and transitional milk, not always in the same sense. Hypothesis: the copper content of human milk from the third to the fifteenth day postpartum could evolve in two different ways depending on the particular woman a) high copper content in colostrum decreasing in transitional milk and b) low copper content in colostrum increasing in transitional milk. To confirm this hypothesis a two factors ANOVA (stage and Copper content of colostrum higher or lower than was applied and then the Minimal Significant Difference (MSD) was estimated. The results show that: The copper content of human milk during the first 15 days of lactation shows two divergent types of behaviour depending on the particular woman: in one case the original contents are very low (with a mean value lower than 0.20 mg/ml) and then increase in transitional milk (to a mean value of 0.42mg/ml). in the other case, the original contents are high (mean value >0.50 mg/ml) and decrease in the following stage (transitional milk) to a mean value of 0.45 mg/ml. The variations observed in each group considered alone are highly significant, this could be observed because it was a longitudinal study. While, when both groups are considered together these results were masked. In the reviewed literature there is no mention of this dual copper evolution. This kind of behaviour was not observed in the evolution of iron and zinc in the study carried out with the same samples.

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CERULOPLASMIN OXIDASE EXPRESSION IN THE ROWETT HOODED RAT AND HUMAN PLACENTA DURING PREGNANCY

L. Gambling, W. Bruce-Johnson, R. G. Lea, M. J. Bingham, and H. J. McArdle Rowett Research Institute Greenburn Road, Aberdeen, Scotland AB21 9SB

During pregnancy, Fe is transferred from mother to fetus across the placenta. Uptake is through transferrin-receptor mediated endocytosis. After this little is known. Iron is carried across the placenta by unknown carriers and released into the fetal circulation subsequently being bound to fetal transferrin. In the accompanying abstract (Danzeisen & McArdle), we presented evidence that the placenta synthesises an endogenous ceruloplasmin, located ont he plasma membrane. We hypothesise that this protein oxidises iron on the fetal face of the placenta prior to incorporation into the fetal transferrin. Here, we examine the expression of the protein during pregnancy in the human and the Rowett hooded Lister rat. Firstly, we demonstrate that both rat and human placental membranes express oxidase activity. We verify that the protein shows at least partial homology to serum ceruloplasmin, since antibody to serun protein significantly decreases placenta ceruloplasmin oxidase activity. Serum ceruloplasmin levels in the rat plasma increase slightly to a maximum at day 15, falling thereafter to term, as has been recorded for other strains of rat. There is no change in ceruloplasmin activity or expression in the hooded rat placenta during pregnancy. In the human, a different pattern is seen. It is well established that levels of ceruplasmin in the maternal serum increase and, in parallel, levels in the placenta are also much higher in the third trimester than in the firs. As is also the case in the rat, the ceruloplasmin in the human placenta is located primarily in the syncytiotrophoblast layer, but presently we cannot determine whether it is predominantly on the apical or basolateral membrane. The data presented in this abstract provide preliminary evidence for a membrane-attached ceruloplasmin which may have a role in iron efflux to the fetal circulation. The placenta expresses RNA for ceruloplasmin, but using this model we cannot definitively say whether the protein 383

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associated with the membrane is synthesised by the cell or is attached to the cel by the ceruloplasmin receptor. On balance, we consider the former to be the more likely hypothesis, but conclusive evidence can only be found using cell models for placenta function. This work was supported by SOAEFD.

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ANEMIA AND ITS THERAPY WITH PERORAL ANTIANEMICS IN PREGNANCY AND PUERPERIUM 1

1

1

2

2

M. Mara , V. Eretova , J. Zivny , J. Kvasnicka , A. Umlaufova , 3 and E. Marova 1

Department of Obstetrics and Gynecology Department of Clinical Haematology 3 Department of Clinical Biochemistry st 1 Medical Faculty of Charles University and General Faculty Hospital in Prague Czech Republic 2

OBJECTIVE To examine changes of blood count, iron metabolism, serum erythropoetin and acute phase reactants at puerperal women. The dynamics of these parameters after spontaneous delivery and their influence by oral iron and folic were studied.

STUDY DESIGN 80 patients were divided into three groups. One group (T) was treated with 80 mg Fe++ daily (Tardyferon) during the first two months after delivery, the second group (F) with 80mg Fe++ plus 0.35mg of folic acid a day (Tardyferon-fol). The control group (C) did not get any medication. Each woman was examined in the third trimester and further four times during the first three months after delivery.

RESULTS In our study all women after the delivery suffered from anemia (combined influence of sideropenia and peripartal blood loss). Hematocrit and hemoglobin concentrations were increased in both treated groups compared with the control group in the first two months after delivery. Three months after labor there were no significant changes in 385

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hematocrit, hemoglobin concentration and red blood cells count among the groups. Decrease of transferin was significantly faster in the groups T and F than in the group C. One month after delivery the count of reticulocytes remained slightly elevated in the groups T and F while subnormal in the group C. There were significantly higher values of serum transferin receptor in all groups in the first two postpartal months than three months after delivery.

CONCLUSIONS In the base of our results we recommend the iron supplementation in all women during the first two months after delivery. Longer than two months therapy, or addition of the folic acid, seems to be useless.

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REQUIREMENTS FOR THE ESSENTIAL TRACE ELEMENTS IRON AND ZINC DURING PREGNANCY

Shahla M. Wunderlich Department of Human Ecology Montclair State University Upper Montclair, New Jersey USA

The requirements for the essential trace elements iron and zinc are increased during pregnancy in response to fetal and maternal physiological needs. At the same time, the rates of absorption and metabolism of these elements are changing during the gestational period. Low-income pregnant women are often consuming diets that are low in these critical essential elements including iron and zinc. The low intake of these two trace elements is reportedly associated with adverse outcomes including preterm delivery, low birth weight, and maternal morbidity such as vulnerability to infection. The issues of supplementation of these nutrients during pregnancy are not resolved among scientists. Therefore, the purpose of this paper is to investigate and analyze the current research findings regarding the levels of these nutrients in the diets of pregnant women and to offer guidelines to provide optimal levels these trace elements through foods. Several studies done by the author indicated that the dietary level of iron and zinc are low. In the most recent study with 249 women, the mean dietary level for iron was 15.6mg ± 7.8 (Standard Deviation) and zinc was 10.8mg ± 6.3 (SD). These levels meet 52% and 72% of the US Recommended Dietary Allowances for pregnant women for iron and zinc respectively. Assessment of dietary trace element status must be done in very controlled conditions because the possibility of under or over reporting of serving sizes is a common challenge for nutritionists. Another important factor in this assessment is bioavailabity and cellular metabolism of these nutrients. The dietary intake can only provide the amount of these elements in foods, however, how much of these is actually utilized and metabolized remains uncertain. Therefore, effective educational measures are necessary to ensure the intake of these nutrients through foods until more is certain about the dietary supplementation of these nutrients. Furthermore, more studies are needed to provide the knowledge of dietary trace element bioavailabity and metabolism during pregnancy from different foods. 387

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URINARY SELENIUM IN PRETERM INFANTS Effect of Inorganic Versus Organic Selenium Intake

L. A. Daniels, R. A. Gibson, and K. Simmer Flinders University of South Australia Adelaide, South Australia Australia

In adults urinary Se has been used to assess Se status as there is a strong correlation between Se intake and urinary excretion. However, supplementation with inorganic selenite results in higher urinary excretion than organic Se, which is the form in infant formulae and breast milk. There is recent evidence that urinary Se excretion in preterm infants is not directly related to renal function infants and hence may also be a useful indicator of Se status in these infants. However, there is very little data available on urinary Se in infants. The aim of this paper is to assess the effect of organic versus inorganic exogenous Se on Se excretion and evaluate the utility of urinary Se as an indicator of Se status in preterm infants. Plasma and urine Se (single void sample) in preterm infants were measured between days 1–5 and then weekly until discharge. Standard parenteral nutrition (negligible Se) was used for 19 infants (PN + 0) (15 males, mean ± SD gestational age 28 ± 2 weeks). Additionally, PN supplemented with selenite (inorganic Se) at 3 (PN + 3) and 6 (PN + 6) µg/kg/day was used for 19 (8 males; 29 ± 2 weeks;) and 8 (5 males; 29 ± 2 weeks;) infants respectively. All infants were graded from PN to standard preterm formulae and/or breast milk (13µg/l). Average daily Se intake over each week was determined. For the PN + 0 group urine Se declined (p < 0.01) over the first 6 weeks despite increasing organic Se intake (0.2 to 1.5µg/kg/day). There was also a net decline (p < 0.05) in urine Se over the same period in both the supplemented groups. At each of the first five weeks urinary Se losses in the PN + 6 group were greater that those for the PN + 3 group which in turn were greater than in the PN + 0 group. There was a significant increase in plasma Se (p < 0.0001) for the PN + 6 group over the first 6 weeks compared with no change in the PN + 3 group and a decline (p < 0.001) in the PN + 0 group. At week 6, when Se intakes were the organic form as PN use was minimal, there was no difference in urine Se between the three groups despite significant differences in intake (PN + 0 = 1.6 ± 0.6, PN + 3 = 2.0 ± 0.6 and PN + 6 = 3.0 ± 1.3µg/kg/day; p < 0.05). 388

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Organic Se intake showed a weak relationship (r = 0.3 to 0.5) with plasma Se but none with urine Se and there was no relationship between the latter parameters. These data indicate that urinary Se is substantially influenced by the form of Se intake and the intake of inorganic but not organic Se. In conclusion, the relationship between urinary, dietary and plasma Se in preterm infants appears to be different to that in adults and requires further study before urinary Se can be recommended as a useful indicator of Se status in preterm infants.

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TRACE ELEMENT BALANCE STUDY IN VERY LOW BIRTHWEIGHT INFANTS Andrea Raab1, Andrea Loui2, Peter Brätter1, and Michael Obladen2 Hahn-Meitner-Institut, Berlin Germany1 and Charité Champus Virchow Klinikum, Klinik für Neonatologie Berlin (Germany)2

Very low birthweight (VLBW) infants are in danger of developing trace element deficiency, because of their lower stores at birth, their incomplete digestive system and their high rate of synthesis of tissue cells. In this early stage of life several organs (e.g. brain), which are sensitive to trace element imbalances, are differentiating. It is under discussion, whether a deficiency in trace elements is a factor which contribute to typical preterm diseases, as retinopathy of prematury or bronchopulmonary dysplasia. Accordingly any imbalance concerning the trace elements (iron, zinc, selenium and copper) might well influence the antioxidant defence system. Less is known about the trace element needs of the rapid grown VLBW infants during their first three months. To evaluate the adequate supplementation of the trace elements cobalt, copper, iodine, iron, molybdenum, selenium and zinc balance studies were performed in 10 VLBW infants with birthweights less than 1000g. All infants were fed enteral with breast milk plus fortifier. To estimate the trace element uptake from the breast milk urine and faeces were collected during a 72 hour period on week 5 and 10 after birth, respectively. The trace element content in breast milk, serum, urine and faeces was determined by means of ICP- mass spectrometry, ICP-atomic emission spectrometry and neutron activation analysis. Nine infants showed negative zinc balance during both collection periods. Two infants developed significant clinical deficiency symptoms. After oral zinc supplementation the clinical symptoms disappeared, but the serum levels of zinc remained below the controls. Of the 10 infants six had negative iron balance despite of iron supplementation. The copper retention of four infants during the second balance period were negative, probably due to low copper intake and inadequate copper reabsortion in the gut. No infant had problems with iodine, molybdenum, selenium and cobalt absorption.

Address all correspondence to: Dr. Peter Brätter, Hahn-Meitner-Institut Dept. NG, Glienicker Strasse 100, D-14109 Berlin; telephone: 030-8062-2785; fax: 030-8062-2781; email: [email protected]

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NUTRITIONAL STATUS OF IRON OF PRE-SCHOOL CHILDREN FROM THE CITY OF JUIZ DE FORA, MG, BRAZIL E. L. Chicourel1, A. F. Pinto1, A. E. C. Lassance1, M. Fisberg2, and S. M. F. Cozzolino3 1

Faculdade de Farmácia e Bioquímica UFJF, Juiz de Fora/MG-Brazil 2 Pediatria UNIFESP and CEPESN Universidade São Marcos-São Paulo/SP 3 Faculdade de Ciências Farmacêuticas (School of Pharmacy) USP São Paulo/SP-Brazil

Iron deficiency is a nutritional disorder of high prevalence throughout the world, especially in children and women, considering iron deficiency anemia as the responsible for about half of all different types of anemias. Children with iron deficiency anemia can show physical and mental impairment, as well as a reduced immune response against diseases. However, iron deficiency, in terms of iron body level, can exist without any clinical anemia evidence. Based on these premises, the present work had the following aims: (1) evaluate iron related biochemical parameters, through a transverse study, in 111 pre-school children, aged between 47 and 85 months, from three nurseries of Municipal Education System, from the city of Juiz de Fora, MG, Brazil; (2) evaluate the children nutritional status by an anthropometric evaluation. The following variables were selected: hemoglobin, hematocrit, ferritin, serum iron, total iron-binding capacity and transferrin saturation; weight, height, sex and age. The anthropometric data were verified by a sofware developed by the Computer Center Applied to Health of the Medicine School of São Paulo—Pediatrics Nutritional Status Evaluation System, which uses reference standards from NCHS, recommended by OMS. Regarding iron related biochemical parameters, 18.3% of the children showed hemoglobin content less than l l g%; 34.9% of ferritin content less than 20ng/mL; 43.9% of serum iron content less than 4.6% hematocrit less than 33%, 64.9% of total iron-binding capacity more than and 54.6% of transferrin saturation less than 16%. The results of the anthropometric evaluation revealed that most of the children were eutrophics (70%); 12.7% were overweight or obese and 17.3% showed present or previous chronic malnutrition. The results obtained allow us to conclude that anemia occurs in a significant number of children, even if eutrophia was founded in 70% of them. 391

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EXERCISE EFFECTS ON TRACE ELEMENT METABOLISM

Richard A. Anderson Nutrient Requirements and Functions Laboratory Beltsville Human Nutrition Research Center U.S. Department of Agriculture ARS, Beltsville, Maryland 20705 USA

The trace elements, chromium, copper and zinc, are not only involved in energy production and utilization but also in the control of free radicals generated during both aerobic and anaerobic exercise. The healthy act of exercise can increase oxygen consumption and glucose utilization 10–20-fold leading to significant increases in potentially damaging free radicals. The effects of exercise on the trace elements may be paradoxical in that exercise leads to increased trace element losses but trace elements are also needed to counteract the effects of stress related to the generation of free radicals. It was established during the eighties that strenuous exercise leads to increased losses of Cr, Cu, and Zn. This becomes a potentially important human health problem since the dietary intake of these nutrients is often suboptimal.

CHROMIUM Chromium is an essential nutrient required for normal carbohydrate and lipid metabolism. Marginal signs of Cr deficiency that are reversed by improved Cr nutrition are widespread including elevated blood glucose, insulin, cholesterol, triglycerides and decreased HDL cholesterol (Anderson, 1998a,b). More severe signs of Cr deficiency including neuropathy and brain disorders have been observed in patients on total parenteral nutrition. Normal dietary Cr intake is suboptimal and is 50 to 60% of the minimum suggested daily intake of

Address all correspondence to: Dr. Richard A. Anderson; USDA, ARS, BHNRC, NRFL; Bldg. 307, Rm. 224, BARC-East; Beltsville, MD 20705-2350 USA; telephone: 301-504-8091; fax: 301-504-9062; email: [email protected] Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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Coupled with low dietary intake, exercise poses a potential health problem since it increases Cr losses. Chromium losses were increased nearly fivefold two hours following a strenuous 6-mile run and daily Cr losses were almost 2-fold higher on the exercise day compared with sedentary days (Anderson et al., 1982). The intensity and duration of the exercise are related to the amount of Cr lost. The degree of stress, as determined by the stress hormone, cortisol, is proportional to the amount of Cr lost (Anderson et al., 1991). While acute exercise leads to definite increases in Cr losses, there is no evidence that well-trained individuals are displaying more signs of Cr deficiency than control sedentary subjects. In fact, exercise training leads to improvements in glucose uptake, glycogen synthesis and insulin sensitivity that are also improved by supplemental Cr. Exercise training alters Cr metabolism and trained subjects have lower basal Cr excretion than untrained subjects. This could be due to a depletion of Cr stores and therefore lower Cr excretion but also an adaptive mechanism to preserve Cr stores. Exercise and training have also been shown to increase Cr absorption (Rubin et al., 1998). By utilizing a stable isotope of Cr, the effects of exercise on basal Cr losses can be differentiated from changes in Cr absorption. Changes in Cr absorption can be separated from mobilization of Cr stores since none of the stable isotope would be present in the endogenous stores and any increase in excretion of the stable isotope would be derived from newly administered Cr. Therefore, the combination of studies showing reduced basal Cr excretion (Anderson et al., 1991) and the increased absorption of Cr given as a stable isotope (Rubin et al., 1998) demonstrate that exercise leads to increased efficiency in the metabolism of Cr. Chromium has also been reported to increase lean body mass in people who exercise, i.e., football players, which has been substantiated in animal studies. However, some follow-up studies have not supported these observations. These results have been reviewed (Anderson, 1998b) and the effects of Cr on lean body mass appear to be related to a combination of Cr status of the subjects and duration and amount of supplemental Cr. Studies to demonstrate an effect of supplemental Cr on lean body mass and body weight of subjects who exercise should be 24 weeks or longer and involve or more daily of supplemental Cr in a form that is readily absorbed (Anderson, 1998b). Additional studies involving of supplemental Cr or less per day and/or studies shorter than 24 weeks will be of minimal benefit and will serve mainly to cloud the issue of whether supplemental Cr has an effect on body composition and weight loss in humans who exercise strenuously. An effect of Cr on weight loss and body composition of sedentary subjects has also been reported (Kaats et al., 1996). The response to Cr has also been shown to have a genetic component and people with a specific allele, A2 DRD2, displayed much greater effects on loss of fat and body weight due to supplemental Cr than remaining subjects (Blum et al., 1998). In addition to its role in glucose and lipid metabolism primarily via its effect on increased insulin sensitivity, Cr also functions as an antioxidant. Chromium, like the antioxidant vitamin E, protects rats from oxidant stresses associated with exposure to carbon tetrachloride. Chromium also protects against lipid peroxidation in isolated rat hepatocytes. Chromium has recently been shown to lead to a decrease in the effects of free radicals as determined by a decrease in lipid peroxidation based on a reduction of thiobarbituric acid reactive substances in people with type 2 diabetes. Similar effects were observed with zinc and the combination of zinc and Cr (Roussel et al., 1998).

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COPPER Copper is an essential nutrient involved in carbohydrate and lipid metabolism, immune function, collagen and elastin formation, amino acid metabolism, hematopoiesis and protection against free radical damage. Clinical signs of copper deficiency have been associated with elevated cholesterol, abnormal electrocardiogram, glucose intolerance and ischemic heart disease. Copper deficiency in animals is characterized by arterial damage, elevated cholesterol, cardiac hypertrophy, anemia, inflammation and fibrotic changes of the heart and sudden death (Klevay, 1994). While Cu plays a key role in free radical protection and is a component of superoxide dismutase, an enzyme involved in destruction of free radicals, free Cu is linked to the production of free radicals. Free Cu release is augmented by exercise. For example, transient hypoxia which occurs with intense weight lifting can lead to an increase in hydrogen ions which react with superoxide anions to produce additional reactive oxygen species. Tissue hypoxia can also lead to the release of free Cu which can function in vivo to catalyze free radical reactions similar to its effects in vitro (Kanter, 1998). Dietary intake of Cu, like that of Cr, is suboptimal and most diets contain less than the minimum suggested intake of 1.5mg and approximately one-third of the diets provide less than 1mg/d (Klevay, 1994). The Cu intake of athletes is likely to be similar to the rest of the population. If intakes for individuals consuming normal diets are marginal, the intakes for those on weight limiting diets such as gymnasts, wrestlers and others, are likely to be well below the suggested intakes. Athletes also often consume high amounts of simple sugars which have been shown to exacerbate the effects of low Cu intakes. Exercise training has been reported to lead to an increase in serum Cu but other researchers reported no relationship between training mileage and serum Cu levels (see review, Anderson, 1991). Serum Cu and ceruloplasmin have been reported to decrease or remain constant during training for competitive swimmers (Lukaski et al., 1990). Activity of Cu/Zn superoxide dismutase has been reported to be higher for female swimmers than non swimmers and also increases with training (Lukaski et al., 1990). Effects of acute exercise on serum Cu are also varied with reports of increases, decreases, and no change (Buchman et al., 1998). The majority of the Cu is excreted via the bile with only small losses, 1–2% via the urine. However, the amount of Cu lost via the sweat appears to be substantial. It has been estimated than in strenuously exercising individuals, Cu lost via the sweat could be in the region of 1 mg which is very substantial since this approximates normal Cu intake of 1 to 1.5 mg/d (Anderson, 1991).

ZINC Zinc is a nearly ubiquitous trace element and is the most abundant trace element in tissues, other than the blood, and is widely distributed in the nuclear, mitochondrial and supernatant fraction in every tissue in the body. Zinc functions in numerous and diverse reactions including carbohydrate, fat and lipid metabolism and regulation of numerous hormones which impact on energy production and utilization. Thousands of zinc binding proteins from different sources, have been isolated and characterized. Zinc

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nutriture is of importance to the athlete since exercise and other forms of stress enhance zinc losses, and dietary intake of zinc may be suboptimal or in individuals who consume unbalanced supplements containing Zn, may create nutrient imbalances. Nutrient imbalances due to supplemental Zn are associated with impaired Cu nutrition and may lead to decreased HDL-cholesterol and increased risks of cardiovascular disease. Suboptimal intakes of Zn in humans are associated with slow wound healing, skin lesions, anorexia, oligospermia, loss of taste and smell acuity and decreased immune responses (Prasad, 1995). Hypozincemia in distance runners is well documented but not universal and likely relates to dietary Zn as well as other components of the diet (see review, Lukaski, 1997). In the initial study of Dressendorfer and Sockolov (1980) serum Zn decreased as the weekly training distance increased. Female marathon runners were also shown to have low plasma Zn values with 22% of the values below value of (Singh et al., 1990). Acute exercise leads to increased urinary losses of Zn with a 50% increase in urinary Zn excretion on the day of a 6-mile run compared with nonexercise days (Anderson et al., 1984). Similar increases in Zn excretion were also observed following a 10-mile run in trained men and in untrained men following stair climbing to exhaustion. Brief bouts of high intensity (90% peak work capacity) treadmill exercise was of insufficient duration to induce increased Zn losses (Anderson et al., 1991). There is a redistribution of Zn in the blood during exercise with increases in blood Zn in shorter duration exercise but unchanged following a marathon run and intense skiing (see reviews, Anderson, 1991; Lukaski, 1997) The effects of supplemental Zn on antioxidant variables of distance runners with hypozincemia have not been completed. However, Zn supplementation leads to a reduction of thiobarbituric reactive substances and an increase in the antioxidant enzyme glutathione peroxidase in sedentary subjects (Faure et al., 1995; Roussel et al., 1998) suggesting that compromised Zn status, prevalent in some people who train intensely for long duration, may be of benefit in trained individuals with low Zn status. In summary, exercise increases trace metal losses. Many athletes, especially those on weight limiting diets, are likely consuming suboptimal amounts of the trace elements, Cr, Cu and Zn. Additionally studies are needed to ascertain the role of suboptimal intake of trace elements, especially in individuals who exercise strenuously.

REFERENCES Anderson, R.A., 1991, New Insights on the trace elements, chromium, copper and zinc, and exercise, in: Advances in Nutrition and Top Sport, Med Sport Science, (Hebbelinck M. and Sheppard, R.J. eds.), pp. 38–58, S. Karger, Basel, Switzerland. Anderson, R.A., 1998a, Chromium, glucose intolerance and diabetes, J. Am. Coll. Nutr. 17:548–555. Anderson, R.A., 1998b, Effects of chromium on body composition and weight loss, Nutr. Rev. 56:266–270. Anderson, R.A., Polansky, M.M., Bryden, N.A., Roginski, E.E., Patterson, K.Y., and Reamer, D.C., 1982, Effect of exercise (running) on serum glucose, insulin, glucagon and chromium excretion, Diabetes 31:212–216. Anderson, R.A., Polansky, M.M., and Bryden, N.A., 1984, Strenuous running: acute effects on chromium, copper, zinc and selected clinical variables in urine and serum of male runners, Biol. Trace Elem. Res. 6:327–336. Anderson, R.A., Bryden, N.A., Polansky, M.M., and Thorp, J.W., 1991, Effect of carbohydrate loading and underwater exercise on circulating cortisol, insulin and urinary losses of chromium and zinc, Eur. J. Appl. Physiol 63:146–150.

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Blum, K., Kaats, G, Davis, K., Eisenberg, M., Sherman, M., Cull, J.G., Woods, R., Bucci, L., Chen, T.H.J., and Braverman, E., 1998, Chromium picolinate induces changes in body composition as a function of TAQ 1 dopamine D2 receptor A2 alleles, J. Am. Coll, Nutr. 17:504. Buchman, A.L., Keen, C., Commisso, J., Killip, D., Ou, C-N., Rognerud, C.L., Dennis, K., and Dunn, J.K., 1998, The effect of a marathon run on plasma and urine mineral and metal concentrations. J. Amer. Coll. Nutr. 17:124–127. Dressendorfer, R.H. and Sockolov, R., 1980, Hypozincemia in runners, Phys. Sports Med. 8:97–100. Faure, P., Benhamou, P.Y., Perard, A., Halimi, S., and Roussel, A.M., 1995, Lipid peroxidation in insulin-dependent diabetic patients with early retina degenerative lesions: effects of an oral zinc supplementation, Eur. J. Clin. Nutr. 49:282–288. Kaats, G.R., Blum, K., Fisher, J., and Adelman, J.A., 1996, Effects of chromium picolinate supplementation on body composition: a randomized double-masked placebo-controlled study, Curr. Ther. Res. 57:747–756. Kanter, M., 1998, Free radicals, exercise and antioxidant supplementation, Proc. Nutr. Soc. 57:9–13. Klevay, L.M., 1994, Ischemic heart disease: nutrition or pharmacotherapy, J. Trace Elem. Electrolytes Health Dis. 7:63–68. Lukaski, H.C., Hoverson, B.S., Gallagher, S.K., and Bolonchuk, W.W., 1990, Physical training and copper, iron and zinc status of swimmers. Am. J. Clin. Nutr. 51:1093. Lukaski, H.C., 1997, Zinc, in: Sports Nutrition: Vitamins and Trace Elements, (Wolinsky, I. and Driskell, J.A., eds.), pp. 157–174. Prasad, A.S., 1995, Zinc: an overview, Nutrition 11:93–99. Roussel, A.M., Zouari, N., and Anderson, R.A., 1998, Antioxidant effects of zinc and chromium in people with type 2 diabetes mellitus, J. Am. Coll. Nutr. 17:504. Rubin, M.A., Miller, J.P., Ryan, A.S., Treuth, M.S., Patterson, K.Y., Pratley, R.E., Hurley, B.F., Veillon, C., Moser-Veillon, P., and Anderson, R.A., 1998, Acute and chronic resistive exercise increase urinary chromium excretion in men as measured with an enriched chromium stable isotope, J. Nutr. 128:73–78. Singh, A., Deuster, P.A., and Moser, P.B., 1990, Zinc and copper status of women by physical activity and menstrual status, J. Sports Med. Phys. Fitness 30:29–36.

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ALTITUDE RELATED CHANGES IN RED BLOOD CELL MEMBRANE LIPIDS AND PROTEINS. POSSIBLE LINKS WITH REDOX EQUILIBRIUM, ACID BASE STATUS AND CELL CALCIUM Claus Behn1, Manuel Ivan Estrada1, Eliseo Hibert Dávila1, Oscar Araneda1, Max González1, Alejandro Carrasco1, Rudy Soria2, Mauricio Araos2, Mercedes Villena2, Wilma Téllez2, Hilde Spielvogel2, Enrique Vargas2, Jorge Cajigal3, Gloria Celedón4, and Gustavo González5 1

Programa de Fisiología y Biofísica ICBM, Facultad de Medicina Universidad de Chile Santiago, Chile 2 Instituto Boliviano de Biología de la Altura (IBBA) La Paz, Bolivia 3 Centro de Alto Rendimiento DIGEDER, Santiago 4 Departamento de Fisiología Universidad de Valparaíso 5 Instituto de Química Universidad Católica de Valparaíso Valparaíso, Chile

1. INTRODUCTION Survival in extreme environments may be limited by some life inherent paradoxes such as the lack of oxygen presumably exacerbating toxicity of the latter gas. Hypoxia generates electron surplus promoting free radical reactions (Degroot and Littauer, 1989), increases lipid peroxidation in rats (Yoshikawa et al., 1982) and enhances cell susceptibility to oxidative injury (Degroot and Littauer, 1989). Adjustments to cope with oxygen, on the other hand, may alter homeostasis (Samaja et al., 1997). Hyperventilation

Telephone: 56-2-678-6215; fax: 56-2-777-6916; e-mail: [email protected] Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers. New York, 2000.

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increases supply, but reduces alveolar Respiratory alkalosis at 5,050m is not compensated within a week staying at high altitude (Grassi et al., 1996). Uncompensated respiratory alkalosis, in turn, associates with lack of tolerance to hypoxia in climbing Caucasians (Samaja et al., 1997). Dehydration may be a main reason for excessive bicarbonate reabsorption at high altitude (West, 1996). At least, metabolic alkalosis can be corrected by supplying chloride, despite persisting plasma volume depletion. Failure in adequately compensating respiratory alkalosis at high altitude, thus, may also rely on a defect in chloride/bicarbonate exchange. Recent work shows short term hypobaric hypoxia to affect band 3 protein, an anion exchanger in the red cell membrane (Celedón et al., 1998). This finding is presented here in relation with results from current field investigations on the role of acid base balance in acclimatisation to high altitude (Cajigal, 1999). The possibility of oxidative stress playing a role in high altitude effects is discussed.

2. MATERIALS AND METHODS 2.1. Barochamber Studies Three healthy male volunteers (19–23 yr) were subjected for 20 min to simulated altitude (4,500m) in a low barometric pressure chamber (433mmHg). De- and recompression were, respectively, completed within 15 min. Venous blood samples were obtained immediately prior to and after exposure to hypobaric hypoxia, red blood cell membranes (RBCM) being isolated according to Dodge, Mitchell, and Hanahan (1963). RBCM were examined for a) short range lateral diffusion status in the membrane bilayer and b) susceptibility of membrane proteins to degradation. 2.1.1. Lipid Lateral Diffusion. RBCM were probed with 12-(1-pyrene)dodecanoic acid (PDA) to assess short-range lateral mobility of lipids in the membrane plane on the basis of diffusion-controlled intermolecularly excited dimer (excimer) formation (Celedón et al., 1992). Spectra of probe loaded membrane suspensions were recorded with a Fluorolog photon-counting spectrofluorometer (Spex). Excitation was realized both directly (344 nm) and through resonance energy transfer from membrane proteins (289 nm). Energy transfer efficiency rapidly decreasing with distance implicates excitation at 289 nm reaching only PDA molecules located very close to proteins. Maximum monomer (Imonom) and excimer emission (Iexcim) was evaluated at 374 nm and 480 nm, respectively. 2.1.2. Membrane Protein Degradation. Densitometry was applied to electrophoretic profiles obtained by SDS-PAGE in the Laemmli discontinuous buffer system, prior to and after incubation of RBCM resuspended in phosphate buffered saline (pH 7.4; 1.67mg protein/ml) for 6h at 37°C (Celedón et al., 1997). Incubation effects were examined in membrane proteins from RBCM obtained before and after exposure to hypobaric hypoxia.

2.2. Field Studies Arterial blood gases, plasma electrolyte concentrations and blood lactate concentrations were studied in lowlanders (LL) and highlanders (HL) climbing together the

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Huayna Potosí mountain (6,060m) in the Bolivian Andes. LL were physically well trained, healthy males (n = 5, 26 to 31 y) living at Santiago, Chile (640m). After arriving by plane at La Paz, Bolivia (3,600m), joint climbing with HL was undertaken two days later. HL were subjects comparable to LL, but normally living in La Paz (n = 7, 22 to 28 y). Blood samples for gas analysis were obtained by arterial puncture at La Paz, prior to, and at the base camp (4,800m) after climbing. Arterial blood obtained at the base camp was collected into glass tubes which were immediately sealed and transported to La Paz for gas analysis being done about one hour later. Electrolyte concentrations were determined in plasma of venous blood obtained at the base camp, both before and after climbing. Lactate concentration was measured in whole venous blood samples by the Acusport device. Venous blood samples also rendered red cells to determine free amino groups in the cytosol and thiols in RBCM, standard methods respectively being applied.

3. RESULTS AND DISCUSSION 3.1. Barochamber Studies Exposure of volunteers to 4,500m for 20 min increased PDA Iexcim/Imonom in RBCM by about 30% (Table 1). As an indicator of lateral diffusivity of membrane lipids, PDA Iexcim/Imonom comparably augments in RBCM when incubation temperature is elevated from 37° to 43 °C (Galla and Luisetti, 1980). The increase of PDA Iexcim/Imonom by simulated altitude was noticed, both when the probe was excited directly at 344 nm or indirectly at 289 nm, via resonance energy transfer (Table 1). Hypobaric hypoxia, thus, appears to increase lipid lateral diffusivity both in the bulk of the bilayer matrix, as well as, in the lipid core of membrane proteins in RBCM. Membrane protein bands obtained by SDS-PAGE reveal simulated altitude to decrease band 3 protein in relation to all other membrane proteins in RBCM, the latter being incubated for 6h at 37°C prior to analysis (Fig. 1). The latter effect could be blocked by protease inhibition. Acute hypobaric hypoxia, thus appears to increase usceptibility of band 3 protein to endogeneous proteolytic degradation.

3.2. Field Studies Climbing increased the difference between plasma and from 32.5 ± 13.0 to 53.1 ± 7.8mmol/l in LL (p < 0.01).

concentration on the other hand,

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did not change by climbing in HL (33.5 ± 9.1 vs. 40.3 ± 6.6mmol/l). Arterial blood gas analysis of samples obtained at 4,800 m, after climbing, revealed plasma bicarbonate concentration being higher in LL than in HL (Table 2). Blood lactate concentration increased by climbing, both in LL and in HL. The climbing related increase of blood lactate concentration correlates with the concomitant increase of (r = 0.74, p < 0.05). Alkalinization at high altitude, thus, seems to be inversely related to physical performance capacity, bicarbonate handling appearing to be more adequate in HL than in LL. Free amino groups in the cytosol of red blood cells were less prevalent in HL than in LL (Fig. 2A). SH group content in RBCM, on the contrary, was higher in HL than in LL (Fig. 2B). Thiol oxidation and/or limited proteolysis may be involved in the conversion of xanthine dehydrogenase into xanthine oxidase, a major source of oxygen free radicals during hypoxia/reoxygenation (Greene and Paller, 1994). Proteases can be activated by an increase of cytosolic free concentration (Mellgren, 1987). Cytosolic free concentration plays a pathogenic role in ischemic or hypoxic acute organ failure (Schrier et al., 1987). By cholesterol modification (Neyses et al., 1985), and/or by favouring intracellular alkalinization (Nitschke et al., 1996; Jiang and Steinberg, 1997), oxidative stress may increase cytosolic free concentration at the expense of membrane associated calcium. Oxidative stress mediated proteolytic degradation of band 3 protein may affect anion exchange as a possible basis for respiratory alkalosis not adequately being compensated at high altitude in LL. In HL, on the contrary, membrane shielding by antioxidants may contribute to improve anion handling and, thus, physical performance at high altitude.

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ACKNOWLEDGMENTS We thank Professor Marc Nigon (IBBA) for logistic support of field studies and Dr. Mario Sandoval (ACHS) for providing hypobaric chamber facilities. This work was financed by FONDECYT (1950454).

REFERENCES Cajigal, J., 1999, Plasma bicarbonate concentration at very high altitude (above 6,000m), Thesis in preparation. Celedón, G., Behn, C., Montalar, Y., Bagnara, M., and Sotomayor, C.P., 1992, Transbilayer asymmetry of pyrene mobility in human spherocytic red cell membranes, Bio chim. Biophys. Acta 1104:243-249. Celedón, G., Lips, V., Alvarado, C., Cortes, M., Lissi, E.A., and Gonzalez, G., 1997, Protein degradation in red cells exposed to 2,2´–azo-bis(amidinopropane) derived radicals, Biochem. Mol. Biol. Int. 43:1121–1127. Celedón, G., Gonzalez, G, Sotomayor, C.P., and Behn, C., 1998, Membrane lipid diffusion and band 3 protein changes in human erythrocytes due to acute hypobaric hypoxia, Am. J. Physiol. 275:C1429–C1431. Degroot, H. and Littauer, A., 1989, Hypoxia, reactive oxygen and cell injury, Free Radic. Biol. Med. 6:541–551. Dodge, J.T., Mitchell, C., and Hanahan, D.J., 1963, The preparation and chemical characterization of hemoglobin-free ghosts of human erythrocytes, Arch. Biochem. Biophys. 100:119–130. Galla, H.J. and Luisetti, J., Lateral and transversal diffusion and phase transitions in erythrocyte membranes. An excimer fluorescence study, Biochim. Biophys. Acta 596:108–117. Greene, E.L. and Paller, M.S., 1992, Xanthine oxidase produces in posthypoxic injury of renal epithelial cells. Am. J. Physiol. 263:F251–F255. Grassi, B., Marzorati, M., Kayser, B., Bordini, M., Colombini, A., Conti, M., Marconi, C., and Cerretelli, P., 1996, Peak blood lactate and blood lactate vs. workload during acclimatization to 5,050m and in deacclimatization, J. Appl. Physiol. 80:685–692. Jiang, T. and Steinberg, S.F., 1997, receptors enhance contractility by stimulating -dependent intracellular alkalinization. Am. J. Physiol. 273:H1044–H1047. Mellgren, R.N., 1987, Calcium-dependent proteases: an enzyme system active at cellular membranes. FASEB J. 1:110–115. Neyses, L., Locher, R., Stimpel, M., Streuli, R., and Vetter, W., 1985, Stereospecific modulation of the calcium channel in human erythrocytes by cholesterol and its oxidized derivatives. Biochem. J. 227:105–112. Nitschke, R., Riedel, A., Ricken, S., Leipziger, J., Benning, N., Fischer, K.-G, and Greger, R., 1996, The effect of intracellular pH on cytosolic in cells. Pflügers Archiv- Eur. J. Physiol. 433:98–108. Samaja, M., Mariani, C., Prestini, A., and Cerretelli, P., 1997, Acid-base balance and transport at high altitude, Acta Physiol. Scand. 159:249–256. Schrier, R.W., Arnold, P.A., Van Putten, V.J., and Burke, T.J., 1987, Cellular calcium in ischemic acute renal failure: role of calcium entry blockers. Kidney Int. 32:312–321. West, J.B., 1996, Physiology of extreme altitude. Handbook of Physiology—Environmental Physiology. 57:1307–1325. Yoshikawa, T., Furukawa, Y., Wakamatsu, S., Takemura, H., Tanaka, H., and Kondo, M., 1982, Experimental hypoxia and lipid peroxide in rats, Biochem. Med. 27:207–213.

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EFFECT OF SUPPLEMENTATION WITH BRAZIL NUTS (CP, BERTHOLLETIA EXCELSA H. B. R.), IN CAPOEIRA PLAYERS ON SELENIUM (SE) CONCENTRATION AND GLUTATIONE PEROXIDASE’S ACTIVITY (GSH-PX, E.C.1.11.1.9)

V. F. Coutinho, V. B. Bittencourt, and S. M. F. Cozzolino Faculdade de Ciências Farmacêuticas (School of Pharmacy) USP, SP, Brazil

Brazil nuts (CP) have a high content of Se. The purpose of this study was to establish a correlation between the effect of supplementation with CP and both nutritional status of Se and GSH-Px activity in young adult capoeira players The GSH-Px selenodependent enzyme is the main enzyme of the antioxidant system. CP samples were brought from Manaus (BR) and the population under study consumed only one Brazil nut per day during 70 days. 34 capoeira players resident in São Paulo, with a mean age of 20.1 ± 2.52 composed the population for women and 21.6 ± 2.92 for men. The average training period per week was 3.51 ± 0.84 and the duration of each training was 109.46 ± 20.27 minutes. The collections of venous blood after a 12-hour fast, were done previous to supplementation (T1) and after 70 days of supplementation (T2), and the collected material was fractionated into plasma and erythrocyte. Se concentrations were determined by a modified fluorimetric method (Watkinson, 1966).

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The Brazil nuts showed concentrations of Se of 1985, or 59.57 ± per nut, weighting 3 g each. The results suggest a positive correlation between the effect of supplementation with CP, and Se concentrations and the activity of the GSH-Px enzyme.

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EFFECTS OF WEIGHT LOSS AND WEIGHT-BEARING EXERCISE ON BLOOD AND ORGAN CONCENTRATIONS OF LEAD AND SOME ESSENTIAL METALS

Francis W Kemp, Shenggao Han, Wenjie Li, David Tiber, Chenzang Wang, David Sepulveda, Justin Holmes, and John D Bogden Department of Preventive Medicine and Community Health New Jersey Medical School UMDNJ, Newark NJ 07103-2714, USA

Thousands of years of human evolution have resulted in the selection of genes that enhance the ability to survive during extended periods of starvation or reduced energy intake. However, this has occurred in the absence of extensive exposure to environmental pollutants. Adults cur-rently living in industrialized countries have body lead burdens about 500 times greater than those who lived prior to widespread lead dissemination in the environment. Most (>95%) of the lead burden is stored in the skeleton. During rapid weight loss, there is a decline in bone mass in addition to soft tissue mass. This bone mass loss may mobilize skeletal lead. However, weight-bearing exercise during weight loss may prevent or reduce bone lead mobilization and redistribu-tion to other organs. The objective was to determine the effects of weight loss, with and without weight-bearing exercise, on lead and essential metal stores in rats with prior lead exposure. Fifty-five, 12-week-old, female, Sprague-Dawley rats were exposed to 400 ppm lead as the acetate in drinking water for 4 weeks, followed by a one-week period without lead exposure. The rats were randomly assigned to seven groups before the start of food restriction. The seven groups were weight maintenance (WM) with ad lib feeding, moderate weight loss (MWL) with 25% food restriction and substantial weight loss (SWL) with 50% food restriction, with or without treadmill running, and one background group euthanized before food restriction. Tread-mill running speed was gradually increased to

Adrress all correspondence to: Dr. John Bogden; Department of Preventive Medicine and Community Health, UMDNJ-New Jersey Medical School, Newark, NJ 07103-2714; telephone: 973-972-5432; fax: 973-972-7625; email: [email protected]

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25m/min with a surface inclination of for 2 hours per day. After a four-week period of food restriction, the rats were killed and their blood and organs (liver, kidneys, brain, lumbar spinal column, quadriceps muscle, fat tissue, femurs), were obtained for analysis for Pb, Ca, Cu, Fe, Mg, and Zn. Food restriction substantially reduced body, liver, and kidney weights. Rats fed restricted diets had consistently higher blood lead concentrations than their (WM) controls with significant differences among groups beginning one week after the start of food restriction. Liver lead con-centrations of food restricted rats were 51% (SWL) and 44% (MWL) higher than those of (WM) controls (ANOVA, P < 0.02). Spinal column bone lead concentrations were 6% (MWL) to 19% (SWL) lower than those of (WM) rats, and were higher for running rats than non-running rats. Weight bearing exercise did not significantly influence most organ metal concentrations. Iron concentrations of food restricted rats (MWL, SWL) were significantly higher in all organs except brain, for which no differences were found for any metals. Concentrations of other essential divalent metals (Ca, Cu, Mg, Zn) were increased in some organs (liver, fat), and decreased in others (spinal column, kidney and muscle). However, the organ contents of all these metals were substantially decreased by weight loss. In contrast, the contents of iron and lead in the liver and other organs were not reduced by weight loss. The conservation of Pb suggests that lead toxicity is possible during rapid weight loss in individuals previously exposed to excessive lead. (Supported by NIH# HL 56581).

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TRACE ELEMENT DEFICIENCIES AND SUPPLEMENTATIONS IN THE ELDERLY

Anne-Marie Roussel LBSO UFR de Pharmacie, UJF Domaine de la Merci F 38700 La Tronche

INTRODUCTION Considering the rapid increase in the number of “baby-boomers” and other aging people in the general population, the maintenance of health with age is one of the main public health challenges of the next millenium. Trace element status influences immune functions (Zn, Se), oxidative stress (Zn, Se, Cu, Fe, Mn), lean body mass (Cr), bone density (Cu, B, F, Sr) and insulin sensitivity (Cr). The risks of deficiencies appear to be especially important for Se, Zn, Mn and Cr, and in postmenopausal women possibly Cu and B. Trace element deficiencies result in altered immune functions, increased oxidative stress, impaired cognitive functions, glucose intolerance and osteoporosis. Several trials of supplementation have reported interesting data, particularly in improving immunity, decreasing lipoperoxidation, preventing loss of bone density and improving glucose tolerance. These data are important to be considered to prevent the increased incidence of infections, cardiovascular diseases, osteoporosis, and diabetes in the elderly.

1. RISK FACTORS OF TRACE ELEMENT DEFICIENCIES Numerous factors tend to limit the trace element nutriture in elderly (Mertz, 1990). Functional modifications of the gastrointestinal tract lead to decreased trace element bioavailability. Even if efficient adaptative mechanisms to these changes exist in healthy elderly people, chronic diseases affect absorption and bioavailability of trace elements,

Address all correspondence to: Pr Roussel, LBSO, UFR de Pharmacie, F38700 La Tronche Phone 0033476637131, Fax 0033476637180, email [email protected] Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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especially when subjects consume drugs. The lesions caused by gastritis may also be responsible frequently for a loss of appetite and a modification of dietary habits with selected food intakes. Pancreatic function is impaired with age and a delay in the peak of insulin secretion which is associated with decreased chromium intake causes decreased glucose tolerance and peripheral insulin resistance. Moreover, the pancreas being highly susceptible to oxidative stress, antioxidant intakes of selenium and zinc should be considered, in addition to increased chromium requirements. In parallel, economic, social, environmental or mental problems also may contribute to nutritional disorders in aging. Trace element intakes and status are significantly influence by loneliness, low socioeconomic levels, depression, drug, and functional or mental impotence. Another agerelated change with respect to trace element nutriture to be considered is the decline in the lean body mass where trace elements are mostly distributed. In free living people, the consequences of marginal dietary intakes are limited, as recently observed in EVA (Coudray, 1997) and SUVIMAX studies (Preziosi, 1998) in pre-aging population and aging free living subjects (EURONUT SENECA, 1991). In contrast, in hospitalized or house-bound older people, protein energy malnutrition and micronutrient acute deficiencies are frequently observed. Thus, low caloric intakes, associated with changes in gastrointestinal and endocrine functions and environmental factors may result in increased risks of trace element deficiencies since it is known that caloric intake below 1,500kcal/day cannot fulfil the biological needs for micronutrients.

2. TRACE ELEMENTS IN THE AGING PROCESS Aging and related diseases may be described as a process which results from impaired immunological, genetic, neurological, endocrinological or antioxidant functions. The free radical theory of aging (Knight, 1995) suggests an age-related imbalance between pro-oxidant and antioxidant equilibrium associated with a potential decrease in antioxidant defenses. It has been shown, in vitro, that oxidative stress led to genomic alterations and accelerated cellular aging (Toussaint, 1998). Data concerning the relationship between age and DNA oxidation are not yet available from large scale epidemiological studies, but in limited groups of elderly subjects it has been observed that a higher level of oxidized bases are present than in younger adults (Piperakis, 1998). The immunological theory of aging is also well documented; aging alters both humoral and cell-mediated immunity; direct consequences of alterations in immune defenses will be an increased susceptibility to many illness and infections. Some trace elements are intimately involved in the maintenance of the integrity of the immune function, particularly Zn and Se.

Selenium Several lines of evidence have shown that Se plays a key role in protecting cells against free radical attack; this property is mainly due its antioxidant effect as the constitutive trace element of the active site of GSH peroxidase and as a limiting factor for the synthesis of this enzyme (Neve, 1992). Se is also part of other antioxidant compounds (selenoprotein P, thioredoxin reductase) and can also modulate the immune system (Mac Kenzie, 1998). It also plays a role in detoxifying heavy metals (Hg, Cd, Pb) and xenobiotics. This function offers an interesting field of action in elderly patients consuming multiple drugs.

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Zinc The role of Zn as an antioxidant is complex (Bray and Bettger, 1990) and its influence on aging process is not totally understood. It is a component of antioxidant Cu-Zn SOD, and metallothionein. Zn, by competition with other transition metals, protects SH groups from oxidation, stabilizes SH groups and might influence transcription factors as p53 (Verhaegh, 1998). Zn also functions as a cofactor for enzymes of nucleic acid catabolism (endonuclease and caspase) (Perry, 1997). Low levels of Zn result in increased cellular death and apoptosis (Parat, 1998). It is also essential for host defense mechanisms, lymphocyte maturation and responses in cell mediated immunity and the functional capacity of phagocytes.

Copper The role of Cu in oxidative stress remains controversial, since Cu could act both as an antioxidant and as a pro-oxidant. Nevertheless, a revised standpoint should be considered with respect to the possible cardiovascular and osteoporosis risks associated with Cu deficiency (Strain, 1988).

Chromium This element attracts a great attention in consideration of its relationships with the aging process and diabetes (Anderson, 1998). Chromium deficiency results in glucose intolerance, fasting hyperglycemia, glycosuria, hypoglycemia, elevated circulating insulin, decreased insulin receptor number and binding, modifications of lipid metabolism and neurological disorders (Anderson, 1997). Most of this manifestations are precisely those frequently encountered during aging.

Iron In free living healthy elderly individuals Fe deficiencies do not seem to be major problem (Johnson, 1994) In contrast, an increase in iron stores was documented, following the observation of increased serum ferritin levels; the pro-oxidant effects of an excess of Fe are now currently accepted in relation with cardiovascular diseases (Ascherio, 1994), cancers (Fairbanks, 1994) and Alzeihmer’s diseases (Smith, 1997)

3. TRACE ELEMENT DEFICIENCIES IN ELDERLY Numerous studies involving hospitalized or institutionalized elderly subjects reported deficient intakes and crucial alterations of trace element status.

Zinc and Selenium In the MINVITAOX study (Monget, 1996), the frequency of abnormally low zinc status reached 47% in institutionalized people, and 53% for low selenium status. Only 5.9% of the patients presented no deficiencies. In the NOVE study, selenium status assessed by plasma selenium concentration and gluthathione peroxidase activity was systematically lower than in younger adults (Olivieri, 1994). This situation causes increased

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oxidative stress, such as recently documented by a higher susceptibility of erythrocytes to hemolysis (Girodon, 1997 a) and by parameters of lipid peroxidation (Congy, 1995). In long stay hospitalized elderly women (Schmuck, 1996), 83 to 100% of the patients had intakes, assessed by the duplicate diet technique, below two-thirds the recommendations (Zn, Se, Mn) or below the lower limit of the ESSADI (Cu). 38% of the patients exhibited Zn plasma concentrations <10.7umol/l. Selenium intakes were as low as 23 ug/d, 71% of them had Se plasma concentrations below 0.76umol/l. Moreover, red cell gluthathione activity which is described as age-related (Samiec, 1998) in healthy elderly, was dramatically low and reached only 50% of that in healthy adults 35 to 60 years old. In parallel, determination of exchangeable pools of Zn and Se (Ducros, 1998) using stable isotopes, showed that the total pool sizes were decreased in institutionalized subjects compared with free living subjects. A low Se status has also been associated with an enhanced risk of cancer and cardiovascular disease. Recently the decline of cognitive functions has been correlated with the decrease of plasma Se level (Berr, 1999).

Chromium Davis (1997)demonstrated a continuous decline in tissue chromium concentrations with age and confirmed previous reports. Poor nutrition, especially excessive consumption of refined carbohydrates might explained this decline and could be consistent with several studies of chromium metabolism (Anderson, 1990). However, it is still unanswered whether the decline is a normal physiological development or due to poor nutrition. The consequences of chromium deficient intakes and status in aging lead to increased glucose intolerance and risks of diabetes (Anderson, 1994). Other trace elements are also at risk of deficiencies in elderly. For Copper, further large-scale epidemiological studies and complementary intervention studies are needed to demonstrate functional deficiencies in the elderly. Measured copper intakes of the elders are lower than the recommendations. In the French Val de Marne study (Hercberg, 1991), more than 50% of women over 60 years exhibited copper intakes below 2/3 ESSADI. Boron is essential for bone metabolism, lipid metabolism, and immune functions (Beattie, 1993). In postmenopausal women, increased osteoporosis incidence may be related to low dietary boron (Saltman, 1993). Recently, it has been considered possible relationships between boron nutriture and brain and psychological functions. B deprivation results in poorer performance (Penland, 1998).

4. TRACE ELEMENT SUPPLEMENTATIONS IN ELDERLY Selenium Several supplementation trials were conducted in elderly subjects. Both plasma selenium and glutathione peroxidase activity could be improved in all the published studies using different selenium forms, doses to or association with antioxidant vitamins (Monget, 1996). A decrease of lipoperoxidation, assessed by plasma TBARs is reported in some trials (Bortoli, 1991; Simonoff, 1992) and a stimulation of parameters reflecting an improvement of immune response as lymphocyte proliferative response to mitogens (Peretz, 1991a, 1991b) is reported in two important Belgium studies. A significant and spectacular effect of high doses of Se on cancer incidence

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has been recently reported by Clark and Combs (1996); A decreased incidence of 69% on prostate cancer was observed in a double blind study (2 to 7 years of supplementation versus placebo).

Zinc Many studies reported also the benefit of zinc supplementation (15 to 100 mg/d) on the immune functions. A lot of various protocol (type of subjects, doses and forms) have quite constantly documented pronounced effects of zinc supplementation on parameters of immunity (number of circulating T cells, delayed skin response to purified protein derivatives, lymphocyte proliferative response to mitogens, etc). Most of these studies (Boukaiba, 1993; Prasad, 1993) utilized supplements containing zinc as the sole active substance. Other reported effects of combined supplementation (Bogden, 1994; Galan, 1997; Fortes, 1998). Some studies used high zinc doses, over 50mg/day, but such doses are generally not justified, causing possibly side-effects, (gastric intolerance, decreased Chol-HDL, modified Zn/Cu ratio). Antioxidant effects were reported in the elderly after Zn supplementation (25 mg/d). The supplementation resulted in decreased plasma lipoperoxidation (Fortes, 1997).

Chromium In elderly, variable responses to supplemental chromium have been reviewed depending on age and degree of glucose intolerance (Anderson, 1994). This may be related to the decreased ability to convert chromium to a usable form that appears to decline with age and degree of impairment of glucose tolerance. Subjects with marginally impaired glucose tolerance respond within 3 weeks to of inorganic chromium, but older subjects with more severe glucose intolerance may require higher amounts of chromium. Improved chromium nutrition, by supplementation in elderly subjects, mean age 78, also leads to decreased total cholesterol and triglycerides and improved total cholesterol HDL. This is particularly important for age associated cardiovascular diseases.

Copper The consequences of a possible moderate deficit is still uncertain and in spite of the cardiovascular risks associated with a low copper status, copper supplementation in elderly is not yet accepted. A recent study (Jones, 1997) showed a significant increased Cu-Zn SOD activity and resistance of lipoproteins to oxidative stress, at nutritional doses (2mg/day for 4 weeks). However, since Cu acts both as antioxidant (Cu-Zn SOD, Ceruloplasmin, Ferroxidase) and as also pro-oxidant, the role of copper in oxidative stress remains controversial. Increased iron stores associated with copper deficient status might lead to increased oxidative stress and cardiovascular risk.

Combination of Trace Elements and/or Vitamins Several long-term studies were recently published with interesting results. Girodon (1997b) showed a significant decrease in the mean number of infectious events associated with a multimicronutrient supplementation, including Zn as 20mg/d and Se as 100 ug/d.

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CONCLUSIONS Trace element intakes and status are frequently marginal in elderly, resulting in nutrition-related diseases. Considering the essential protective roles of zinc and selenium against oxidative damage and decline of immune functions, or the beneficial effect of chromium on glucose tolerance, there is now increasing evidence that appropriate supplementations, restoring marginal trace element status, should reduce the risk of several common age-related degenerative diseases. Institutionalized people should also be monitored with respect to their antioxidant trace element status. Surprisingly, little information is available concerning the real mineral requirements of elderly people. Additional research also needs to be done to evaluate the efficiency of preventive supplementations associating several complementary micronutrients. In any case, many authors agree that more attention should be devoted to elderly people who represent the best target of preventive intervention, since the challenge is to maintain in a better state the growing older population.

REFERENCES Anderson, R.A., Bryden, N.A., Polansky, M.M., and Reiser, S., 1990, Urinary chromium excretion and insulinogenic properties of carbohydrates, Am. J. Clin, Nutr., 51:864–868. Anderson, R.A., 1994, Chromium nutrition in the elderly, Handbook of Nutrition in the Aged, R.R. Watson, Ed, CRC Press, Boca Raton, FL, 385–392. Anderson, R.A., 1997a, Chromium as an essential nutrient for humans, Regul. Toxicol. Pharmacol, 26:S35–S41. Anderson, R.A., 1997b, Nutritional factors influencing the glucose/insulin system: Chromium, J. Am. Coll. Nutr., 16:404–410. Anderson, R.A., 1998, Chromium, glucose intolerance and diabetes, J. Am. Coll. Nutr., 17(6):548–555. Ascherio, A., Willett, W.C., Rimm, E.B., Giovannucci, E.L., and Stampfer, M., 1994, Dietary iron intake and risk of coronary disease among men, Circulation, 89:969–974. Beattie, J.H. and Peace, H.S., 1993, The influence of a low-boron diet and boron supplementation on bone, major mineral and sex metabolism in postmenopausal women, Br. J. Nutr., 69(3):871–884. Berr, C., Richard, M.J., Roussel, A.M., and Bonithon-Kopp, C., 1998, Systemic oxidative stress and cognitive performance in the population-based EVA study, Free Radic. Biol. Med, 24(7/8):1202–1208. Bogden, J.D., Bendich, A., Kemp, F.W., Bruening, K.S., Skumick, J.H., Denny, T., Baker, H., and Louria, D.B., 1994, Daily micronutriment supplements enhance delayed hypersensibility skin test responses in older people, Am. J. Clin. Nutr., 60:437-442. Bortoli, A., Fazzin, G., Marchiori, M., Mello, F., Brugiolo, R., and Martelli, F, 1991, Selenium status and effect of selenium supplementation in a group of elderly women, J. Trace Elem. Electrolytes Health Dis. 5:19–21. Boukaiba, N., Flament, C., Acher, S., et al., 1993, A physiological amount of zinc supplementation effects on nutritional, lipid, and thymic status in an elderly population, Am. J. Clin. Nutr., 57:566–572. Bray, T.M. and Bettger W.J., 1990, The physiological role of zinc as an antioxidant, Free Rad. Biol. Med., 8:281–291. Clark, L. and Combs, J., 1996, Effects of Selenium supplementation for cancer prevention in patients with carcinoma of the skin, JAMA, 25:1957–1963. Congy, F, Bonnefont-Rousselot, D., Dever, S., Delattre, J., and Emerit, I., 1995, Oxidative stress in the elderly, Press. Med., 24:115–118. Coudray, C., Roussel, A.M., Arnaud, J., and Favier, A., 1997, Selenium and antioxidant vitamin and lipoperoxidation levels in preaging French population. EVA Study group, Biol. Trace. Elem. Res., 57(2): 183–190. Davis, S., Mc Laren-Howard, J., Hunnisett, A., and Howard, M., 1997, Age-related decreases in chromium levels in 51,665 hair, sweat, and serum samples from 40,872 patients: implications for the prevention of cardiovascular disease and type II diabetes mellitus, Metabolism, 46:469–473.

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Ducros, V., Faure, P., Ferry, M., Couzy, F., Biajoux I., and Favier, A., 1998, The sizes of the exchangeable pools of selenium in elderly women and their relation to institutionalization, Br. J. Nutr., 3:379–396. Euronut Seneca Investigators, 1991a, Intake of vitamins and minerals, Eur. J. Clin. Nutr., 45(suppl.3):121–138. Fairbanks V.F., 1994, Iron in Medicine and Nutrition, in “Modern nutrition in health and diseases,” vol. 1, 185–213, 8th Ed, Shils ME, Olson JA, ShikeM, edts., Philadelphia: Lea and Febiger. Fortes, C, Agabiti, N., Fano, V., Paciflci, R., Forastiere F., Virgili, F., Zuccaro, P., Perruci, C.A., and Ebrahim, S., 1997, Zinc supplementation and plasma lipid peroxides in an elderly population, Eur. J. Clin. Nutr., 51:97–101. Fortes, C., Forastiere, F., Fano, V., Agabiti, N., Paciflci, R., Virgili, F., Piras, G., Bartoloni, C., Tricerri, A., Zuccaro, P., Ebrahim, S., and Perruci, C.A., 1998, The effect of Zinc and Vitamin A supplementation on immune response in an older population, JAGS, 46:19–26. Galan, P., Preziosi, P., Monget, A.L., Richard, M.J., Arnaud J, Lesourd B, Girodon P, Bourgeois C.F., Keller H., Favier, A., and Hercberg S., 1997, Effects of trace element and/or vitamin supplementation on vitamin and mineral status, free radical metabolism and immunological markers in elderly long-term hospitalized subjects, Internat. J. Vit. Nutr. Res., 67:45–460. Girodon, F., Blache, D., Monget, A.L., Lombard, M., Brunet, P., Arnaud, J., Richard, M.J., and Galan, P., 1997a, Effect of a two year supplementation with low doses of antioxidant vitamins and/or minerals in elderly subjects on levels of nutrients and antioxidant defense parameters, J. Am. Coll. Nutr., 16:357– 365. Girodon, F., Lombard, M., Galan, P., Brunet-Lecomte, P., monget, A.L., Arnaud, J., prezioci, P., and Hercberg, S., 1997b, Effect of micronutrient supplementation on infections in institutionalized elderly subjects: a controlled trial, Ann. Nutr. Met., 41:98–107. Hercberg, S., Preziosi, P., Galan, P., Deheeger, M., Papoz, L., and Dupin, H., 1991, Apports nutritionnels d’un échantillon représentatif de la population du Val de Marne: III Les apports en minéraux et vitamines, Rev. Epid. Santé publique, 39:245–261. Johnson, M.A., Fisher, J.G., Bowman, B.A., and Gunter, E.W., 1994, Iron nutriture in elderly individuals, FASEB J., 8:609–621. Jones, A., DiSilvestro, R., Coleman, M., and Wagner, T., 1997, Copper supplementation of adult men: Effects on blood copper enzyme activities and indicators of cardiovascular disease risk, Metabolism, 12:1380–1383. Knight, J.A., 1995, The process and theory of aging, Ann. Clin. Lab. Sci., 25:11–12. Mac Kenzie, R., Rafferty, T., and Beckett, G., 1998, Selenium: An essential element for immune function, Immunology Today, 19:342–345. Mertz, W., 1990, The role of trace elements in the aging process, Prog. Clin. Biol. Res., 326:229–240. Monget, A.L., Galan, P., Preziosi, P., Keller, H., Bourgeois, C., Arnaud, J., Favier, A., and Hercberg, S., 1996, Micronutrient status in elderly people. Geriatrie / Min. VitAOX network, Int. J. Vitam. Nutr. Res., 66(l):71–76. Neve, J., 1992, Prevention du vieillissement par les oligoéléments essentiels. De Natura Rera, 5:100–115. Olivieri, O., Stanziali, A.M., and Girelli, D. et al., 1994, Selenium status, fatty acids, vitamin A and E, and aging: the Nove Study, Am. J. Clin Nutr., 1994, 60:510–517. Parat, M.O., Richard, M.J., Pollet, S., Hadjur, C., Favier, A., and Beani, J.C., 1997, Zinc and DNA fragmentation in keratinocyte apoptosis: its inhibitory effect in UVB irradiated cells, Photochem. Photobiol, 37:101–106. Penland, J.G., 1998, The importance of Boron nutrition for brain and psychological function, Biol. Trace Elem. Res., 66(l–3):299–317. Peretz, A., Neve, J., Desmedt, J., Duchateau, J., Dramaix, M., and Famaey, J.P. 1991a, Lymphocyte response is enhanced by supplementation of elderly subjects with selenium enriched yeast, Am. J. Clin. Nutr., 53:1323–1328. Peretz, A., Neve, J., Duchateau, J., Siderova, V., Huygen, K.M., Famaey, J.P., and Carpentier, Y.A., 1991b, Effects of selenium supplementation on immune parameters in gut failure patients on total parenteral nutrition, Nutrition, 7:215–221. Perry, D., Smyth, M.J., Stennicke, H.R., Salvesen, G.S., Durier, P., Poirier, G., and Hannun, Y., 1997, Zinc is a potent inhibitor of the apoptotic protease, caspase-3: a novel target for zinc in the inhibition of apoptosis, J. Biol. Chem., 271:18530–18533. Piperakis, S.M., Visvardis, E.E., Sagnou, M., and Tassiou A.M., 1998, Effects of smoking and aging on oxidative DNA drainage of human lymphocytes, Carcinogenesis, 19:695–698. Prasad, A., Fitzgerald, J.T., Hess, J.W., Kaplan, J., Pelen, F., and Dardenne, M., 1993, Zinc deficiency in elderly patients, Nutrition, 9:218–224.

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Preziosi, P., Galan, P., Herberth, B., Valeix, P., Roussel, A.M., Malvy, D., Paul-Dauphin, A., Arnaud, J., Richard, M.J., Briancon, S., Favier, A., and Hercberg, S., 1998, Effects of supplementation with a combination of antioxidant vitamins and trace elements at nutritional doses, on biochemical indicators and markers of the antioxidant system in adult subjects, J. Am. Coll. Nutr., 17(3):244–249. Saltman, P.D. and Strause, L.G., 1993, The role of trace minerals in osteoporosis, J. Am. Coll. Nutr., 12(4):384–389. Samiec, P.S., Drews-Botsch, C, Flagg, E.W., Kurtz, J.C., Sternberg, P., Reed, R.L., and Jones, D.P., 1998, Glutathione in human plasma: decline in association with aging, age-related macular degeneration and diabetes, Free Radic. Biol. Med., 24(5):699–704. Schmuck, A., Roussel, A.M., Arnaud, J., Ducros, V., Favier, A., and Franco, A., 1996, Analyzed dietary intakes, plasma concentrations of zinc, copper, and selenium and related metalloenzyme activities in hospitalized elderly women, J. Am. Coll. Nutr., 15:462–470. Smith, M., Harris, P., Sayre, L., and Perry, G., 1997, Iron accumulation in Alzeihmer disease is a source of redox-generated free radicals, Proc. Natl. Acad. Sci. USA, 94:9866–9868. Strain, J.J., 1988, A reassessment of diet and osteoporosis-possible role for copper, Med. Hypothesis, 27(4):333–338. Toussaint, O., Raes, M., Michiels, C., and Remacle, J., 1998, The response of cells to stress: relationship to the aging process and the pathology, Med. Science, 14:622–635. Verhaegh, G., Parta, M.O., Richard, M.J., and Hainaut, P., 1998, Modulation of p53 protein conformation and DNA-binding activity by intracellular chelation of zinc, Mol. Carcin., 21:205–214.

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SERUM AND URINE SELENIUM CHANGES IN A GROUP OF ELDERLY DURING ONE YEAR OF SELENIUM SUPPLEMENTATION J. Kvícala1, V. Zamrazil1, and V. Jiránek2 1

Institute of Endocrinology Národní 8, 116 94 Praha 1 Czech Republic 2 Immunotech, a.s., Radiová 1, 102 27 Praha 10 Czech Republic

1. INTRODUCTION Essential role of selenium in organism has been proved in recent years by the elucidation of several regulatory and protective functions of its compounds. Se-cysteine was found in the active centre of so important enzymes like Deiodinases (Berry et al., 1991), which control metabolism and regulatory function of thyroid hormones. Se-cysteine was detected also as a component of active centre in Se-dependent GSHPeroxidases (Flohe et al., 1973), which are one of the most powerful parts of antioxidative defence system of the body. Increase of oxidative diseases (cardiovascular diseases, malignity, autoimmune disorders, neural diseases) in elderly is well known as well as worsening of thyroid function. On the other hand, decrease of body selenium content in elderly and decrease of its intake in this period of life has been also repeatedly reported (Bortoli et al., 1991; Ferry and Roussel, 1996; Gratzlova et al., 1992), especially in the countries with mild or even marginal deficiency of selenium. The aim of the presented work was to measure Se indexes (serum Se and urine Se) of the population of seniors in the region of East-North Bohemia and increase them by supplementation of selenium in the form of selenite.

2. RESULTS AND DISCUSSION Methods used for analyses were INAA (instrumental neutron activation analysis) and fluorimetry as stated elsewhere (Kvicala and Havelka, 1987; Kvicala et al., 1995). Quality control of analyses was performed by coanalyses of appropriate standard Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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reference materials (IInd. generation human serum and H-4 animal muscle for INAA; Lyphocheck Urine Metals Control and Urine SRM 2670 for fluorimetry). Serious Se deficiency has been detected for population between 6 and 65 years in several regions of the Czech Republic, average Se in the range of serum and of urine (Kvicala et al., 1993; Kvicala et al., 1996a; Kvicala et al., 1996b; Kvicala et al., 1997). Very low values of of serum and of urine ( creatinine in urine) were detected for the group of 108 elderly in the age between 65 and 91 from East Bohemia. Median and geometric mean were close to arithmetic mean. Slight but significant correlation was found between serum and urine selenium (r = 0.2700; n = 88; p = 0.0110). Assess of average selenium intake on the basis of daily urine excretion was in this group of inhabitants. All these numbers proved low selenium status of the searched subpopulation as compared with both normal and old populations of the West Europe (Neve 1991; Robberecht and Deelstra, 1984). Because of such a low selenium status, group of 89 relatively healthy seniors of the region was selected and 46 of them obtained selenium supplementation in the form of selenite tablets (together with tablets with iodide per day to avoid the danger of iodine reserve loss). The other part received placebo tablets. of Se per day was supplemented for the first four months with subsequent increase to of selenium daily. The greatest increase was observed during the first 7 weeks—from initial of serum to of serum and from of urine to of urine—whereas placebo group was without significant differences (Figs. 1, 2). Slight increase appeared during the next 12 weeks of supplementation by Mean values reached serum and urine. These values lie in the range of higher west-european standard, quite near the optimal levels. Prolonged supplementation, even with did not show any substantial increase of Se serum levels. In fact, low decrease in serum Se of the supplemented

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group to was detected in the course of the next 7 weeks. Increase to the mean level Se/l serum was detected after the next six months’ supplementation by Se/day (Fig. 1). Daily urine selenium increased to during 7 weeks of Se/day supplementation with subsequent decrease to Se/day during 6 months of the same doses of Se (Fig. 2). More explanations of the unexpected time-course of urine and serum Se levels during supplementation with Se/day could be suggested. One of them might be decrease of Se intake due to the unregular intake of tablets, the other speculation might be decreased Se absorption in elderly persons in the case of abrupt increase of Se intake. Available data are not sufficient for more detailed conclusions.

3. SUMMARY In the course of 1 year supplementation trial, most of supplemented elderly persons reached the level of Se/l of serum, which is considered as nearly optimal for safeguard of full functions of selenium in the organism. The level of urine selenium increased to Se/l, which is also adequate. Optimum levels were reached within 4 months of supplementation with dose Se/day.

ACKNOWLEDGMENT This work was partly supported by grants IGA MZ. R No. NG/19-3 and NB/4845-3.

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REFERENCES Berry, M.J., Banu, L., and Larsen, P.R., 1991, Type I iodothyronine deiodinase is a selenocysteine-containing enzyme. Nature, 349:438–440. Bortoli, A., Fazzin, G., Marchiori, M., Mello, F., Brugiolo, R., and Martelli, F., 1991, Selenium status and effect of selenium supplementation in a group of elderly women. J. Trace Elem. Electrolytes Health Dis., 5:19–21. Ferry, M. and Roussel, A.M., 1996, Trace elements and aging, in: Therapeutic uses of trace elements (J. Neve, P. Chappuis, and M. Lamand, eds.), pp. 99–105, Plenum Press, New York and London. Flohe, L., Günzler, W., and Schock, H.H., 1973, Glutathion peroxidase: A selenoenzyme. FEBS Letters, 32:132–134. Gratzlova, J., Adameckova, D., Hegyi, L., and Delejova L., 1992, Selenium values in the serum of elderly. Biochem. Clin. Bohemoslov., 21:176–182 (in Slovak). Kvícala, J. and Havelka, J., 1988, Frequency of concentrations of some trace elements in serum by INAA. J. Radioanal. Nucl. Chem., 121:261–270. Kvícala, J., Havelka, J., Zamrazil, V., Cerovská, J., and Cermák, S., 1993, Serum selenium levels and selenium intake estimation from urine excretion in inhabitants of Prague urban area, in: Trace Elements in Man and Animal—TEMA 8 (M. Anke, D. Meissner, and C.F. Mills, eds) pp. 233–234, Verlag Media Touristik, Gersdorf, Germany, Kvícala, J., Zamrazil, V., Soutorová, M., and Tomiška, F., 1995, Correlations Between Parameters of Body Selenium Status and Peripheral Thyroid Parameters in the Low Selenium Region. Analyst, 120:959–965. Kvícala, J., Zamrazil, V., and Tluchor B., 1996a, Deficiency of Selenium in Inhabitants of Highly Polluted Area of North-West Bohemia, in: Therapeutic Uses of Trace Elements (J. Neve, P. Chappuis, and M. Lamand, eds.) pp. 345–350, Plenum Press, New York. Kvícala, J., Zamrazil, V., and Jiránek, V., 1996b, Selenium Deficient Status of Inhabitants of South Moravia, in: Natural Antioxidants and Food Quality in Atherosclerosis and Cancer Prevention (J.K. Kumpulainen and J.T. Salonen, eds.) pp. 177–187, The Royal Society of Chemistry, Cambridge, UK. Kvícala, J., Zamrazil, V., Bílek, R., Soutorová, M., Dvoráková, M., Šimecková, A., Kantorová, I., Pobišová, Z., Cerovská, J., and Jiránek, V., 1997, Low selenium status of inhabitants of South Bohemia and its relation to iodine and thyroid hormone metabolism. Biomarkers and Environment, 1:12–20. Neve, J., 1991, Methods in Determination of Selenium States. J. Trace Elem. Electrolytes Health Dis., 5:1–17. Robberecht, H.J. and Deelstra, H.A., 1984, Selenium in human urine: concentration levels and medical implication. Clin. Chim. Acta., 136:107–120.

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BLOOD AND SKIN ANTIOXIDANT STATUS IN WERNER SYNDROME (4 PATIENTS) M. T. Leccia1,2, M. J. Richard2, G. Borla Darve1, J. C. Béani1,2, A. Favier1, and P. Amblard1,2 1

Service de Dermatologie CHU A. Michallon 38043 Grenoble Cedex 09 France 2 LBSO, CHU A. Michallon 38043 Grenoble Cedex 09 France

1. INTRODUCTION The Werner syndrome (WS) is an autosomal recessive disease characterized by premature aging, genetic instability and a high incidence of cancer. The gene defective in WS is located on chromosome 8 and encodes a protein that shows significant sequence homology to the family of DNA helicases. However, the molecular defects and biochemical events responsible for the clinical phenotype remain to be determined. The free radical theory of aging postulates that unrepairable oxidative cellular damages could participate in cellular senescence. The antioxidant enzyme pathways include superoxide dismutases (SOD) that convert superoxide radicals (O2°) to hydrogen peroxide (H2O2). Thereafter catalase and glutathione peroxidase (GSH-Px) catalyse the conversion of H2O2 to H2O. Previous studies showed that an altered balance in the activity of the SOD to GSH-Px plus catalase ratio induces cellular features of senescence and could be an important determinant of cellular aging. We report in this study four cases of WS patients with characteristic clinical aging features. For these 4 patients, we have evaluated blood (GSH-Px, SOD, catalase, glutathione, Zn, Se, Cu, Mn, Vitamins A, E, C, TBARs) and tissular (GSH-Px, SOD, glutathione, TBARs) antioxidant status using skin biopsies. Our data show that in all cutaneous fibroblasts there is an altered SOD to

Address all correspondence to: Dr M. T. Leccia, Service de Dermatologie, CHU A. Michallon, 38043 Grenoble cedex 9, France; Telephone: (33) 4 76 76 55 08; Fax: (33) 4 76 76 55 58; email: Marie- Therese. [email protected] Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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GSH-Px and catalase activity ratio, suggesting an accumulation of H2O2 in these cells. There is no significant variation for trace element and vitamines in blood. All 4 patients show an increase of erythocyte GSH-Px. The 3 older patients with the more severe symptoms have a decrease of plasmatic glutathione level and an increase of lipid peroxidation markers.

2. PATIENTS AND METHODS Michèle (41 years), Amédée (45 years) and Martine (35 years) are from the same fratry with consanguinity. Suzanne (46 years) is a de novo WS case without consanguinity. All patients express symptoms of premature aging, started around the age of puberty, as there are short stature, wrinkling, thin and slerodermoid skin, leg ulcers in 2 patients, graying and sparse hair, cataract, high-pitched voice and hypogonadism. Michèle and Amédée present diabetes, osteoporosis and vascular calcifications. All patients show an increase of urinary hyaluronic acid excretion confirming the diagnosis of WS. The 4 patients have normal intellectual functions and understanding. Cutaneous fibroblasts from each patient were obtained from a skin biopsy in sun-protected forearm and cultured in classical conditions. Antioxidant status including copper-zinc, manganese SOD, GSH-Px, catalase, glutathione (GSH) and lipid peroxidation markers as TBARs was evaluated. Blood antioxidant status was determined for each patient including antioxidant enzymes SOD, GSH-Px, GSH, trace elements (Se, Zn, Mn, Cu, Fe) vitamines (vitamins A, E, C, and lipid peroxidation products (TBARs).

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3. RESULTS AND DISCUSSION Few studies concerned oxidative damage, antioxidants and WS and results did not show significant variation in antioxidant status. Only one work demonstrated the positive effect of antioxidant enzyme addition on genomic instability in WS lymphocytes in vitro. As previously described in the litterature, the maximal lifespan of cultured cutaneous fibroblasts from the 4 WS patients was significantly reduced and the cells had altered morphology and size. Results presented in Table 1 show in all 4 patients variations of SOD, GSH-Px and catalase activities that lead to an increase in the ratio of SOD to GSH-PX and catalase in cutaneous cells. Catalase activities are decreased in 3 patients. These imbalance in antioxidant enzyme activities constitutes a prooxidant state in these cells leading to an accumulation of H2O2. These cells do not present any modification of lipid peroxidation as evaluated by TBARs. As shown in Table 2, the 4 patients present an increase of GSH-Px activity determined in erythrocytes, whereas plasmatic GSH-Px and SOD present no change. GSH levels are decreased in 3 patients and lipid peroxidation markers concomitantly increased in 2 patients. There is no variation in trace elements and vitamines (results not shown). These results show antioxidant variations corresponding to prooxidant states in skin fibroblasts and in the plasmatic compartment tative mechanism to protect these cells. Interestingly, the patient presenting normal tissular catalase activity and plasmatic GSH level is the youngest and less seriously ill patient. In addition to the genetic evidence of WS, biochemical events such as prooxidant states could represent pathogenic and/or additonal factors for genomic instability and cellular accelerated aging phenotype. It would be of interest to complete these preliminary results by the evaluation of antioxidant status in other WS and since the beginning of the disease. Confirmation of similar modifications in antioxidant systems may be useful in the evolution of therapeutic strategies for aging.

REFERENCES de Haan, J.B., Cristiano, F., Iannello, R., Bladier, C., Kelner, M.J., and Kola, I., 1996, Elevation in the ratio of Cu/Zn-superoxide dismutase to glutathione peroxidase activity induces features of cellular senescence and this effect is mediated by hydrogen peroxide, Hum. Molec. Genet. 5:283–292. Goto, M., Weber, J., Woods, K., and Drayna, D., 1992, Genetic linkage of Werner’s syndrome to five markers on chromosome 8, Nature 355:735–738. Harman, D., 1997, Role of free radical reactions in aging and disease, J. Geriatr. Dermatol. 5:114–127.

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Nordenson, I., 1977, Chromosome breaks in Werner’s syndrome and their prevention in vitro by radicalscavenging enzymes, Hereditas 87:151–154. Yu, C.E., Oshima, J., Wijsman, E.M. et al., 1997, Mutations in the consensus helicase domains of the Werner syndrome gene, Am. J. Hum. Genet. 60:330–341.

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EFFECTS OF ALUMINUM ON GLUTAMATE METABOLISM A Possible Explanation for Its Toxicity in the Aged Rats and in an Experimental Model of Aluminum Overload

Cécile Struys-Ponsar, Olivier Guillard*, and Philippe van den Bosch de Aguilar Université Catholique de Louvain Laboratoire de Biologie Cellulaire Unité Bani, 5, Place Croix du Sud B-1348, Louvain-la-Neuve *Institut des Xénobiotiques et Laboratoire de Toxicologie CHU de Poitiers, 86000 Poitiers France

1. INTRODUCTION To clarify the role that aluminum (Al) may play in neurodegenerative diseases such as Alzheimer’s disease, a huge amount of experimental model have been carried out on many aspect of Al biology. While alterations of key processes in central nervous system neurones have been extensively investigated following Al exposure (Meiri et al., 1993), little is known about the consequences of Al exposure on the astrocyte population. The aim of this work was to investigate the consequence of Al exposure on the metabolism of glutamate in the brain of rats during aging (2, 8, 24 and 34 months) and in an experimental model of Al load by intraperitoneal injection of Al gluconate (667 µg for 3 months.

2. RESULTS During aging, brain Al (measured by GFAAS according to the method of D’Haese et al., 1985) rose significantly in rats from 0.29µg/g at 8 months to 1.43 µg/g at 34 months. This brain Al elevation was not uniform. Analysis of Al concentration in the different brain areas of the rat showed that the level of Al accumulation depends on the area, Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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some areas appearing more vulnerable to Al accumulation such as the hippocampus and the neocortex while others seem to be spared such as the cerebellum and the spinal cord. The level of the Al load reached after intraperitoneal injection of Al gluconate was similar to the concentration observed in the very old rats with values reaching The brain areas which significantly contributed to this Al load were the hippocampus and the neocortex. Changes in glutamate, aspartate and glutamine concentrations were analyzed by HPLC according to the method of Delbarre et al. (1991) in brain regions of Al treated rats and compared to the levels observed in brain regions of 24 month old rats known to accumulate Al during aging. The levels of aspartate, glutamate and its metabolite, glutamine are presented in Tables 2 & 3. Of the three amino acids, only glutamine showed a significant increase in concentration in the hippocampus, parietal cortex, and temporal cortex of 24 month old rats. In these areas, a decrease in the level of glutamate was observed but it was not significant. After Al treatment, glutamine concentrations increased in the neocortex and the hippocampus at a level similar to the level observed in the brain regions of 24 month old rats. Since astrocytes are central to glutamate metabolism, possessing both a high affinity uptake system for glutamate and glutamine synthetase which converts glutamate in the presence of ammonia to glutamine which in turn is taken up after diffusion in the

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extracellular space by neurones and hydrolised to glutamate (Olney, 1994), glutamate uptake, glutamine synthetase (GS) activity and glutamine accumulation were measured in primary rat astrocyte culture exposed to Al chloride (200, 400 and GS activity was significantly increased in astrocytes exposed to 200, 400 and The relative specificity of the effect of Al on GS was examined by examining the sensitivity of two other enzymes, lactate dehydrogenase (LDH) and MTT dehydrogenase. Al exposure induced a significant decrease in MTT dehydrogenase activity at Al concentrations of 400 and whereas it induced a significant increase in LDH activity at concentrations of 400 and Secondly, the effect of two other cations was examined (Table 5). The inhibitory effect of lead acetate on GS activity was observed at 200 and of Al. On the other hand, zinc chloride or cobalt chloride had no effect on GS activity.

3. CONCLUSION Definition of Al toxicity is traditionally presented in terms of neurone injury and glial cells are generally relagated to a secondary role involving reactive or suportive responses. However, the observations of the present work point out the astrocyte population as a potential target for Al toxic action. In vivo, significant increase in glutamine concentration was observed during aging and after Al exposure in brain regions characterized by Al accumulation, the hippocampus and the neocortex. In vitro, exposure of astrocytes to Al significantly increased GS activity. Some steps of glutamate metabolism appeared therefore to increase in the brain of rats after Al exposure.

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REFERENCES Delbarre B., Delbarre G., and Calinon F., 1991, Accumulation of amino acids and hydroxyl free radicals in brain and retina of Gerbil brain after transient Ischemia. J. of ocular Pharmacology, 7(2):147–155. D’Haese P.C., Van de Vyver F.L., de Wolff F.A., and de Broe M.E., 1985, Measurement of aluminium in serum, blood, urine and tissues of chronic hemodialysed patients by use of electrothermal atomic absorption spectrometry. Clin. Chem., 31:24–29. Meiri H., Banin E., Roll M. and Rousseau A., 1993, Toxic effects of aluminum on nerve cells and synaptic transmission, Progress in Neurobiol. 40:89–121. Olney J.W., 1994, New mechanisms of excitatory transmitter neurotoxicity, J. Neural. Transm. 43:47–51.

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ZINC-INDUCED EXCESSIVE GLUTAMATE RELEASE MAY CAUSE ACCELERATED SENESCENCE WITH DEFECT IN LEARNING AND MEMORY IN SENESCENCE ACCELERATED MOUSE

Takeshi Saito, Noriko Nakagawa, Kyoko Takahashi, Dan Li, Yuwako Yamamoto, Hiroko Sasaki, Nozomu Nakamura, 1 Kazuhiro Ogawa, Hiroyoshi Fujita, Masaaki Kurasaki , 1 2 Masashi Okabe , and Toshiyuki Hosokawa Laboratory of Environmental Biology Department of Preventive Medicine Hokkaido University School of Medicine 1 Department of Environmental Medicine and Informatics Graduate School of Environmental Earth Science 2 Center for Research and Development in Higher Education Hokkaido University Sapporo Japan

1. INTRODUCTION It is well known that Zn plays important roles in maintaining brain functions. It is highly concentrated in the giant boutons of hippocampal mossy fibers of the brain. Many histochemical and electron microscopic investigations have indicated that Zn is found in the synaptic vesicles of mossy fiber terminals. The findings of active uptake and release of zinc from hippocampal mossy fibers by neuronal activity have suggested that it may act as a neuromodulator (Assaf and Chung, 1984, Itoh, Saito et al., 1993). Glutamate is considered to be a main neurotransmitter in hippocampal mossy fiber. It is thought that bouton Zn is closely associated with this excitatory amino acid neurotransmission. It is known that exogenously applied inhibits mossy fiber synaptic transmission and has an antagonistic effect on NMDA receptors. Moreover, Zn inhibits glutamate release from hippocampal slices. Recent studies showed changes of it’s level in the brain with advancing aging and suggest that trace elements are closely related to aging process of the brain (Saito, Watanabe, Itoh and Saito, 1994). Thus, changes in Zn Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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metabolism with advancing aging affects the hippocampal crucial functions including learning and memory. Senescence-accelerated mouse (SAM) has been established as a murine model of aging (Takeda, Hosokawa, Takeshita, Irio, Higuchi, Matsushita, Tomita et al., 1981). Shortened life span and early manifestation of various signs of senescence are obtained in prone strains of SAM. The P10 strain (SAM P10) shows deficits in learning and memory and brain atrophy. However, there is little information about the mechanism of brain aging of SAMP 10 with deficits in learning and memory. In the present study, we determined age-dependent differences in Zn concentrations between the control mouse (SAMR1) and SAMP10. Then, we determined the level of releases of glutamate and glycine from CA3 region of the hippocampus and the immunohistochemical localization of NMDA receptors and glial fibrillary acidic protein (GFAP) in the hippocampal CA3 region of both strains at old age to evaluate the critical role of Zn in aging of the brain and to clarify the mechanism of deficits in learning and memory of SAMP10.

2. MATERIALS AND METHODS SAMP10 and SAMR1 strains at young and old age were used for this experiment. Zn concentration was determined by ICP-MS spectrometry. The release of glutamate and glycine was determined by microdialysis-HPLC-ECD method. To visualize the expressions of NMDA receptors and GFAP, immunohistochemical procedures were carried out.

3. RESULTS AND DISCUSSION Age-dependent differences in Zn concentration in the hippocampus were observed in both SAMP 10 and SAMR1 (Table 1). Especially, a significant decrease in Zn concentration was observed in the hippocampus of SAMP 10 at old age. Furthermore, significant increases in the release of endogenous glutamate and glycine were detected in the hippocampal CA3 region of SAMP 10 at old age as compared to that of control (Fig: 1). A significant increase in GFAP in the hippocampal CA3 region of SAMP 10 was detected as compared to the control at old age (data not shown). However, no significant difference in expressions of NMDA receptors was observed in both strains at old age. There are some evidences which link to Zn to brain aging with deficits in learning and memory. Zn modulates long term potentiation (LTP) in the hippocampus, which is

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thought to be one of the criteria of the learning and memory. Furthermore, Guidolin et al. (1992) showed correlation between Zn level in hippocampal mossy fibers and spatial memory in aged rats. However, there is no report to clarify the mechanism of deficits in learning and memory caused by Zn. It was reported that Zn is released with glutamate from presynapse and binds to the NMDA receptors to modulates the neurotransmission of glutamate and prevents neuronal cells injury from neurotoxicity of glutamate in the hippocampus. Moreover, Zn inhibits glutamate release from hippocampal slices. The present results demonstrated that an excessive amount of glutamate release occurs in the hippocampal CA3 region of SAMP10. As Zn inhibits glutamate release from hippocampal slices, it is reasonable to think decrease in Zn at old age of SAMP 10 causes an excessive release of glutamate. Furthermore, NMDA receptors have binding sites for Zn and glycine, and low level of Zn and an excess amount of glycine in extracellular synaptic cleft strengthen glutamatergic neurotransmission via NMDA receptors. Thus, over stimuli by glutamate should induce neuronal cell injury in the hippocampal region of SAMP 10, where central region of learning and memory exists. Thus, we investigated

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the expression of GFAP which is used the evidence for neuronal cell injury. Our result showed that neuronal cell injury was significantly increased in the hippocampal CA3 region of P10 at old age as compared to the control.

4. CONCLUSION Our results demonstrated that an abnormal glutamate neurotransmission caused by Zn deficiency induces neuronal cell injury in the hippocampus of SAMP 10. These neuronal disturbances may cause accelerated aging of brain of SAMP 10 with deficits in learning and memory (Fig. 2).

REFERENCES Assaf, S.Y. and Chung, S.-H., 1984, Release of endogenous from rat brain tissue during activity, Nature 308:734–736. Guidolin, D., Polato, P., Venturin, G., Zanotti, A., Mocchegiani, E., Fabris, N., and Nunzi, M.G., 1992, Correlation between zinc level in hippocampal mossy fibers and Spatial memory in aged rats, Ann. N Y Acad. Sci. 673:187–193. Itoh, T., Saito, T., Fujimura, M., Watanabe, S., and Saito, K., 1993, Restraint stress-induced changes in endogenous zinc release from the rat hippocampus, Brain Res. 618:318–322. Saito, K., Watanabe, S., Itoh, T., and Saito, T., 1994, Dynamics of trace and ultra-trace elements in the brain, Hokkaido Univ. Med. Lib. 31:11–17. Takeda, T., Hosokawa, M., Takeshita, S., Irio, K., Higuchi, T., Matsushita, T., Tomita, Y., Yasuhira, K., Hashimoto, H., Shimizu, K., Ishii, M., and Yamamura, T., 1981, A new murine model of accelerated senescence, Mech. Aging Dev. 17:183–194.

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SERUM SELENIUM AND GLUTATHIONE PEROXIDASE ACTIVITY IN OCTOGENARIAN AND NONAGENARIAN SUBJECTS IN BELFAST

Rea I. M., McMaster D., Murphy A., and Mercer C. Cardiovascular Research Group Queen’s University of Belfast Department of Geriatric Medicine Whitla Medical Building Belfast BT9 7BL, Northern Ireland UK

1. INTRODUCTION Glutathione peroxidase (GSHPx) is a seleno-dependent enzyme which catalyses the removal of hydrogen peroxide thus protecting cells, including endothelial cells against oxidative damage. In this study, selenium and glutathione peroxidase were measured in a cohort of elderly and very elderly subjects since it was considered that subjects who age “successfully” might provide evidence of good anti-oxidant status.

2. SUBJECTS, MATERIALS AND METHODS 2.1. Subjects Elderly subjects were randomly selected from the middle cohort of the Belfast Elderly Longitudinal Free-living Aging STudy (BELFAST) which has been described previously (Rea, 1999). Two hundred and sixty subjects aged 65–100+ years of which 140 were >90 years old, who were “good” for their age, apparently well, free-living, cognitively intact and mobile and met the clinical criteria for Senieur status (Lighart, 1984), were enlisted into the study.

Address all correspondence to: Dr. I., Maeve Rea; Department of Geriatric Medicine, Whitla Medical Building, The Queen’s University of Belfast, 97, Lisburn Road, Belfast BT9 7BL, Northern Ireland, UK; Telephone: 0044-1232-272156; Fax 0044-1232-325839; email: [email protected] Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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2.2. Methods Selenium (Se) and GSHPx were measured in serum and whole blood as described by McMaster et al. (1990). A subsample of 16 subjects completed a 24 hour dietary recall (Rea, 1998) and the dietary data analysis was carried out using the COMPEAT computer programme.

3. RESULTS Se and GSHPx activity in both serum and whole blood fell significantly with increasing age (p < 0.0001) but GSHPx activity in whole blood corrrected for haemoglobin concentration showed less change with age (p = 0.02). Males had significantly higher Se and GSHPx activity compared to females (p = 0.001). Serum Se was significantly associated with GSHPx (p < 0.0001). A small subgroup analysis of dietary intakes (16) showed low serum selenium to be associated with reduced protein and B vitamin intake (p = 0.02).

4. DISCUSSION In this study in Northern Ireland, elderly subjects, selected for good health, showed evidence of reduced selenium and GSHPx status with increasing age. In comparative studies in older subjects there have been variable results. The EVA study of 70 year old free-living subjects in France showed no change in selenium compared to younger adults (Coudray et al., 1997), the PAQUID study (Berr et al., 1993) showed a decline in serum and erythrocyte selenium but no change for GSHPx with age, whereas in institutionalised 80 year old subjects, Ducros et al. (1997) showed a decline in both selenium and GSHPx activity. In this study there was some limited evidence from the small subgroup analysis which suggested that low serum selenium may be associated with reduced protein and vitamin B intake but this finding requires further confirmation. This cross-sectional study suggests that elderly and very elderly community-living subjects have lower selenium and decreased GSHPx status as they age which could compromise their free-radical defence mechanisms but which may be correctable by diet.

REFERENCES Berr, C., Nicole, A., Godin, J., Ceballos-Picot, L., Thevenin, M., Dartigues, J.-R, and Alperovitch, A., 1993, Selenium and oxygen-metabolising enzymes in elderly community residents: A pilot epidemiological study JAGS. 41:143–148. Coudray, C., Roussel, A.M., Arnaud, J., and Favier, A., 1997, Selenium and antioxidant vitamin and lipoperoxidation levels in preaging French population. EVA Study Group. Etude de vielissement arteriel, Biol. Trace. Elem. Res. 57:183–190. Ducros, V., Faure, P., Ferry, M., Couzy, F., Biajoux, L., and Favier, A., 1997, The sizes of the exchangeable pools of selenium in elderly women and their relation to institutionalisation, Br. J. Nutr. 78: 379–396. Lightart, G.L., Corberand, I.X., Fournier, C., Galanaud, P., Hifmans, W., Kennes, B., Muller, P., Hermelink, H.K., and Steinmann, C.G., 1984, Admission criteria for immunogerontological studies in man. The Senieur protocol, Meck Aging. Dev. 28:47–52.

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McMaster, D., Bell, D., Anderson, P., and Love, A.H., 1990, Automated measurements of 2 indicators of human selenium status and applicability to population studies, lin. Chem. 36:211–216. Rea, I.M., McNerlan, S.E., and Alexander, H.D., 1999, CD69. CD25 and HLA-DR activation antigen expression on CD3+ lymphocytes and relationship to serum TNFa, IFNg and sIL-2R levels in aging, Exp. Gerontol, 34:79–93. Rea, I.M., Nelson, S.J., Murphy, A., Ward, M., and McNulty, H., 1998, The use of 24 dietary assessment in 70–96 year old subjects living in the community of Belfast urban area, Eur. J. Clin. Nutr. 52:S41–S42

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LONG TERM TOPICAL ANTIOXIDANT TREATMENT PROVIDES PROTECTION AGAINST CLINICAL SIGNS OF PHOTOAGING L. Vaillant1, L. Declercq2, D. Malvy3, J. C. Béani4, J. Bazex5, D. Maes6, and S. Hercberg3 Department of Dermatology in Tours1 Grenoble4 and Toulouse5 (France) Estée Lauder Companies (Belgium2, USA6) National Coordination SU.VI.MAX3 (France)

1. INTRODUCTION Most photobiological effects in the skin (from sunburn to immunosuppression, photoaging and photocarcinogenesis) are attributed to ultraviolet radiation and are believed to be essentially mediated by reactive oxygen species (Scharffetter-Kochanek et al., 1997). Since sunlight exposure is potentially damaging to the skin, leading to free radical generation and depletion of the cutaneous antioxidant defense system (Podda et al., 1998), it can be assumed that administration of additional antioxidants may represent a promising strategy to reduce photodamage. Topical or systemic antioxidant treatment with antioxidants such as green tea polyphenols (Katiyar et al., 1999), (Lopez-Torres et al., 1998) or a combination of ascorbic acid and (Eberlein-König et al., 1998) has indeed been shown to provide protection against cutaneous damage caused by acute UV exposure. The current study was set up to assess the clinical effect in conditions of normal daily life of a long term topical treatment with an antioxidant blend, containing tocopherol acetate, magnesium ascorbyl phosphate, rosemary extract and other antioxidants.

2. TEST SUBJECTS AND METHODS A test panel of 111 female volunteers was followed in this clinical trial over a period of 18 months. The study was designed to allow a three way double-blind comparison. Twice a day the panelists applied a product, either with or without antioxidants, to their face and left forearm. The right arm served as untreated internal control. Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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The condition of the skin was evaluated at baseline, then every 6 months. Final evaluation was performed after 18 months on the following criteria: skin thickness (high resolution ultrasound, Dermcup 2020®), skin elasticity (Cutometer®), clinical evaluation of lines and wrinkles guided by a photographic scale and image analysis of silicone replicas, epidermal moisturization (clinical Kligman score) and capacitance (Corneometer®).

3. RESULTS 3.1. Skin Lipid Peroxidation Topical application to the forearm of an antioxidant containing formulation immediately leads to a decrease in the lipid peroxide level compared to the vehicle treated control (Fig. 1). The peroxide level remains about 50% lower (P < 0.01) on the treated arm throughout the day and returns to baseline by the next morning.

3.2. Skin Thickness The average full skin thickness was similar in both arms at baseline in both treatment groups. After 18 months of treatment a highly significant 6% difference (P < 0.01) in thickness was observed between the antioxidant treated arm and the untreated control in the verum group. These results suggest that the normal trend of skin thinning with age can be slowed down by a long term treatment with an antioxidant containing formulation. The protective effect was more pronounced on the chronically sun exposed, dorsal side of the forearm. The effect of the vehicle treatment in the control group was minimal.

3.3. Skin Elasticity Elasticity of the skin was evaluated in terms of extensibility (cutometer). In line with the effect on the skin thickness the loss of skin elasticity with age can be partially prevented by a long term treatment with an antioxidant containing formulation. The

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protective effect was again more pronounced on the chronically sun exposed, dorsal side of the forearm (P < 0.05 at Ml8). The effect of the vehicle treatment in the control group was less important.

3.4. Clinical Evaluation of Lines and Wrinkles All 111 panelists participating in the trial were regularly examined by a dermatologist to assess the overall evolution of the signs of photodamage to the face. The clinical score of wrinkle density on the face didn’t change significantly over 18 months. The two treatment groups did, however, show a different trend: there was a tendency towards a slight reduction of wrinkles in the antioxidant treated group over 18 months time, whereas the vehicle control group generally showed more severe wrinkles. The overall assessment of the skin surface topography on silicone replicas taken around the eye area completely confirmed these clinical findings and even showed a marked reduction in wrinkle severity in some panelists of the antioxidant treated group.

4. CONCLUSION The present clinical study was unique in its objective to evaluate the effect of antioxidants on the skin of human volunteers, in conditions of normal daily exposure to the environment. The results strongly suggest that regular topical application of antioxidants leads to long term protection benefits for the skin. The antioxidant blend offers an immediate protection by significantly reducing lipid peroxidation at the skin surface. Treatment over 18 months slows down some of the landmarks of the skin aging process: thinning of the skin, loss of elasticity and formation of lines and wrinkles.

REFERENCES Eberlein-König, B., Placzek, M., and Przybilla, B., 1998, Protective effect against sunburn of combined systemic ascorbic acid (vitamin C) and (vitamin E), J. Am. Acad. Dermatol. 38:45–48. Katiyar, S.K., Matsui, M.S., Elmets, C.A., and Mukhtar, H., 1999, Polyphenolic antioxidant (–)epigallocatechin-3-gallate from green tea reduces UVB-induced inflammatory responses and infiltration of leukocytes in human skin, Photochem. Photobiol. 69(2):148–153. Lopez-Torres, M., Thiele, J.J., Shindo, Y., Han, D., and Packer, L., 1998, Topical application of alphatocopherol modulates the antioxidant network and diminishes ultraviolet-induced oxidative damage in murine skin, Br. J, Dermatol. 138(2):207–215. Podda, M., Traber, M.G., Weber, C., Yan, L.J., and Packer, L., 1998, UV-irradiation depletes antioxidants and causes oxidative damage in a model of human skin, Free Radic. Biol. Med. 24(l):55–65. Scharffetter-Kochanek, K., Wlaschek, M., Brenneisen, P., Schauen, M., Blaudschun, R., and Wenk, J., 1997, UV-induced reactive oxygen species in photocarcinogenesis and photoaging, Biol. Chem. 378(11): 1247–1257.

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LACTULOSE STIMULATES CALCIUM ABSORPTION IN POSTMENOPAUSAL WOMEN DOSE-DEPENDENTLY

E. G. H. M. van den Heuvel, Th. Muijs, W. van Dokkum, and G. Schaafsma TNO Nutrition and Food Research Institute P.O. Box 360, 3700 AJ Zeist Netherlands

Animal studies have indicated that calcium absorption is increased by lactulose, a synthetic disaccharide. As no results of human studies on the influence of lactulose on calcium absorption are available, a study was conducted in a population group (postmenopausal women) who may benefit from the envisaged enhancing effect of lactulose on calcium absorption. Twelve postmenopausal women drank 100ml water with 5 or 10 g of lactulose or a reference substance at breakfast for 9 days. The three treatments were given according to a randomized, double-blind, cross-over design, separated by two 19-d wash-out periods. On the 8th day of each treatment period 44Ca dissolved in orange juice was drank immediately after the solution with the study substance and just before a standard breakfast with 162mg of carrier calcium. Within half an hour 48CA was given intravenously. Before and for 36 hours after isotope administration urine was collected in which the isotope ratios were measured by inductively coupled plasma mass spectrometrmy. From the isotope enrichments, true fractional calcium absorption was calculated. Fractional calcium absorption during the treatments with the reference substance, 5g and 10g of lactulose was (mean ± SD) 27.7 ± 7.7, 30.0 ± 7.6 and 32.2 ± 7.0, respectively. A significant increase in calcium absorption was found between the highest dose of lactulose and the reference treatment (p < 0.01). A non-significant increase in calcium absorption was found when 5 g of lactulose was ingested. Between the dose of lactulose and its positive effect on calcium absorption a significant linear trend existed. In conclusion lactulose increases calcium absorption in postmenopausal women. More research is warranted to find out how lactulose stimulates calcium absorption and whether its able to improve calcium balance in humans and/or decrease the rate of aging bone loss.

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SELENIUM AND COGNITIVE DECLINE IN THE ELDERLY—THE EVA STUDY

C. Berr, B. Balansard, J. Arnaud, A. M. Roussel, and A. Alpérovitch INSERM U360, Hôpital de la Salpêtrière 75651—Paris Cedex 13 and Laboratoire de biologic du stress oxydant Université de Grenoble I 38700, La Tronche France

The aim of this study was to determine whether systemic oxidative stress status is associated with cognitive decline in elderly community subjects. We studied vascular and cerebral aging in 1166 high cognitive functioning subjects aged 60–70 years volunteers in the EVA (“Etude du Vieillissement Artériel”) cohort with a 4-year follow-up. Subjects completed a global cognitive test (MMSE) at baseline and during the 4-year follow-up examination. Blood samples were obtained at baseline to determine plasma levels of selenium. Associations between selenium levels and demographic (age, sex, socio-economic level), lifestyle (alcohol, tobacco), clinical and metabolic (lipids, glycemia) factors were examined. Risk of cognitive decline, defined as a loss of 3 points in MMSE score between baseline and the 4-year follow-up, was assessed by selenium level. Mean level of selenium was equal to 1.10 µmol/l (sd = 0.2). Education and income levels were positively associated to selenium. There was also a positive association between selenium and cholesterol. Age, sex, alcohol and tobacco consumption were not significantly associated with selenium in this sample. Subjects with low levels of selenium (defined as lower than 25th percentile, have an increased risk of cognitive decline (Odds ratio = 1.58 confidence interval 95% (1.08–2.31)) after adjustment for various confounding (sex, age, depressive symptomatology, educational level, alcohol and tobacco consumption, BMI, cholesterol, triglycerides, and initial MMSE score). Analysis did not show a dose-effect relationship between selenium levels and risk of cognitive decline. This result suggest that antioxidant deficiencies may accelerate cognitive decline or be a risk factor for cognitive decline.

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DOES HORMONAL REPLACEMENT THERAPY INFLUENCE ANTIOXIDANT STATUS AND LDL OXIDIZABILITY IN POST-MENOPAUSAL WOMEN? Bureau I.1, Laporte F.1, Ducros V.1, Faure H.1, Rayssiguier Y.2, Favier A.1, and Roussel A. M.1 1

LBSO, Université Joseph Fourier UFR Pharmacie, Grenoble, France; and 2 INRA Theix Unité Maladies Métaboliques et Micronutriments France

It’s now well known that oxidized LDL is widely involved in the process of cardiovascular diseases. In postmenopause, an increased incidence of cardiovascular diseases has been reported, possiby linked with oxidative stress. In contrast, hormonal replacement therapy (HRT) seems to be protecting against cardiovascular diseases. This effect could be partly related to an antioxydant effect, such as a restored antioxidant micronutriment status and/or a decreased LDL oxidation. The aim of this study was to compare the antioxidant micronutrient status in postmenopausal women who receive HRT with those who do not receive therapy and to establish a correlation among antioxydant micronutrient status, LDL oxidizability and estrogen therapy. Healthy postmenopausal women, 55 ± 3 years, were divided into two groups of 30 women, one group reveived HRT and the other group remained untreated. We measured Zn, Se, Cu, vitamin E and C, carotenoids plasma levels as antioxidant, and Fe plasma level as a potential pro-oxydant. LDL oxidation was estimated in vitro (copper ) by the rate of conjugated dienes formation and by lag time, after isolation by ultracentrifugation. The statistical ananlusis of our data showed that HRT did not seem to improve the antioxidant micronutrient status and therefore LDL oxidizability remained unchanged. These preliminary results shoud be confirmed by others parameters indicating the level of oxidative stress such as glutathion, thiols, total antioxidant plasma capacity, superoxide dismutase, glutathion peroxidase.

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METALLOTHIONEIN EXPRESSION AND ITS SIGNIFICANCE IN THE BRAIN AGING OF DOG Akinori Shimada, Masahiko Satoh1, and Chiharu Tohyama1 Department of Veterinary Pathology Tottori University Tottori-shi, Tottori 680-0945, Japan and 1 Environmental Health Sciences Division National Institute for Environmental Studies Tsukuba, Ibaraki 305-0053 Japan

Dogs are known to share a variety of morphological age-related brain changes with humans; the changes include neuronal loss, dystrophic axonal changes, astrogliosis and amyloidosis characterized by the formation of senile plaques in the cerebral cortex (Shimada et al., 1991). To study the roles of metallothioneins (MTs) in the brain, the expression pattern of metallothioneins were compared between the brains with severe age-related changes (aged dogs) and those with mild changes (young dogs). Intense MT-I & -II immunoreactivity as well as signals for MT-I & -II mRNA (Kobayashi et al., 1997) were shown in the hypertrophic astrocytes in the brains with severe age-related changes. These MT-I & -II positive astrocytes dominated in the thalamus, cerebral cortex and medulla, where remarkable age-related changes were observed. In contrast, MT-I & -II expression was considerably weak in the brains of dogs with mild age-related changes. On the other hand, both MT-III immunoreactivity and signals for MT-III mRNA (Kojima et al., 1998) were demonstrated in neurons in the brain regardless of the intensity of the age-related changes. Metal analysis demonstrated higher level of Fe and Cu, which are known to be responsible for the generation of free radicals, in the brain tissue of aged dogs. These results suggest, first, MT-I & -II may be induced in relation to the progress of the age-related changes in the brain, and second, the MT-isotype may play an important role in the protection of the brain tissue from the toxic insults responsible for the brain aging.

Address all correspondence to: Dr. Akinori Shimada, Dept. of Veterinary Pathology, Tottori University, Tottori-shi, Tottori 680-0945, Japan; telephone & fax 857-31-5422; e-mail: [email protected]

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REFERENCES Shimada, A., Kuwamura, M., Umemura, T., Takada, K., Ohama, E., and Itakura, G., 1991, Modified Bielschowsky and immunohistochemical studies on senile plaques in aged dogs. Neurosci. Lett. 129:25–28. Kobayashi, K., Shimada, A., Yamano, Y., and Umemura, T., 1997, Molecular cloning of a canine metallothionein cDNA. J. Vet. Med. Sci. 59:819–823. Kojima, S., Shimada, A., Kodan, A., Kobayashi, K., Morita, T., Yamano, Y., and Umemura, T., 1998, Molecular cloning and expression of the canine metallothionein-III. Canad. J. Vet. Res. 62:148–151.

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VITAMIN AND MINERAL INTAKES AND BIOCHEMICAL STATUS IN A SPANISH AGED WOMEN GROUP. EFFECTS OF BODY MASS INDEX

P. Valera P. Garcia-Garcia, R. M. Ortega, A. Lopez-Sobaler, and A. M. Requejo Instituto de Nutricion y Bromatología (csic-ucm) Facultad de Farmacia Ciudad Universitaria 28040 Madrid

It is well known that aging is linked to nutritional status impairment. Moreover, the elderly usually have a sendentary life style that may contribute to deteriorate even more the situation. Many studies have shown that habitual, moderate physical activity and dietary improvement (i.e. avoiding micronutrients deficiencies) can be useful in achieving bodyweight control in the elderly. Therefore, the aim of the present study is to investigate the relation between the intake and the vitamin and mineral status in an aged non-institutionalized group belonging to Madrid (Spain) and to know the effect of body mass index. A total of 83 elderly women aged 70.8 ± 6.4y were studied and divided in two groups depending on the Body Mass Index (BMI): (1) BMI 25–30kg/m2 (n 53) and (2) BMI> = 30kg/m2(n 30). Anthropometric measurements were taken and nutrient intake was assessed using the 7-day dietary history method. The energy and nutrient content of all the food ingested was determined using Spanish Food Composition Tables. The intakes obtained were compared with dietary recommendations for the Spanish population made by the Department of Nutrition. Serum iron and folic acid, plasma zinc, ascorbic acid and red blood cell folate were analyzed.

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Body mass index of all elderly women was higher than 25kg/m2. Energy, Zn and vitamin B6, D, E, folate intakes were lower than the recommended. However, an adequate nutritional status was observed for iron, folate and ascorbic acid. When BMI effect was studied no modifications were observed in the nutritional status. It might be concluded that in spite of the high basic anthropometric parameters the elderly women showed nutritional impairment. Special care must be provided to the diet of the elderly population.

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BIOMARKERS OF MOUSE AGING Modifications of Minerals and Antioxidant Enzymes

Heng-Kuan Wong, Jacqueline Riondel, and Alain Favier Laboratoire de Biologie du Stress Oxydant (LBSO) UFR de Pharmacie Domaine de La Merci, 38700 La Tronche France

Free radical damage has been reported to contribute to the etiology of many diseases and disorders including various types of cancer, autoimmune diseases, inflammatory dieseases, coronary heart diseases, photodermatoses, cataract, Alzheimer’s disease, alcoholism. There is evidence that free radical damage can contribute substantially to the etiology of the aging process. To investigate the correlation between the oxidative stress and aging, we have determined the levels of lipid peroxidation in the brain, liver, kidneys, spleen and plasma from Swiss albino mice at ages of 4 and 14 months. The results showed that the level of lipid peroxidation (measured as malondialdehyde) was significantly higher in older animals than in young animals. The activities of erythrocyte antioxidant enzymes: glutathion peroxidase (GSH-Px Se) and superoxide dismutase (SOD Cu-Zn) displayed an age-dependent elevation. During aging process a significant increase of plasma Se level was observed but no significant alteration of plasma Zn level was noted. Concerning the reduced glutathione (GSH) levels, no significant alteration was observed in most tissues except an age-dependent decline in the liver correlated with an oxidized glutathione (GSSG) level increase. After extraction from liver the redox sensitive nuclear protein Sp1 showed an agedependent decline of its expression. NF. B considered as a stress oxidative inductible transcription factor displayed a significant increase of its expression and binding capacity during the aging process.

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DOES HORMONAL REPLACEMENT THERAPY INFLUENCE TRACE ELEMENT STATUS IN POST-MENOPAUSAL WOMEN? Bureau I.1, Arnaud J.1, Anderson R.2, Richard M. J.1, Laporte F.1, Favier A.1, and Roussel A. M.1 1

LBSO, Université Joseph Fourier UFR de Pharmacie, Grenoble, France and 2 Beltsville Human Nutrition Research Center Belstville, Maryland USA

Hormonal replacement therapy (HRT) counteracts the increases in the incidence of cardiovascular diseases observed following menopause. These improvements could be linked to increased micronutrient status. Sub-optimal micronutrient status leads to increased pro-oxidant properties which are associated with cardiovascular diseases. The purposes of this study were to compare the trace element status (Cu, Mn, Cr, Se, Zn) in prostmenopausal women who receive HRT with those who do not receive therapy and to establish a correlation among trace element status, estrogen therapy and risk factors of cardiovascular diseases. Healthy postmenopausal women, 55 ± 3 years, were divided into two groups of 30 women, one group received HRT and the other group remained untreated. The Cu, Mn, Cr, Se and Zn plasma and urine concentration and erythrocyte Cu were determined to assess micronutrient status and compared with the risk factors associated with cardiovascular diseases. Untreated postmenopausal women did not show any modification in micronutrient status versus women who received HRT. Parallely, estimation of dietary habits by a 7 days diet record interestingly showed that antioxidant intakes in postmenopausal women without HRT were higher than in postmenopausal women with HRT. This observation could explain the lak of micronutrient status modification in relation with HRT.

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RELATIONSHIP BETWEEN SERUM SELENIUM AND RED CELL AND PLASMA GLUTATHION PEROXIDASE LEVELS Effect of Supplementation with Nutritional Doses of Antioxidants In Elderly and Adult Subjects

Paul Preziosi1, Josiane Arnaud2, Marie-Jeanne Richard2, Pilar Galan1, Denis Malvy3, Serge Briancon4, Anne-Marie Roussel2, Alain Favier2, and Serge Hercberg1 1

Institut Scientifique et Technique de la Nutrition et l‘Alimentation (Conservatoire National des Arts et Métiers) 2 rue Conté, F-75003 Paris, France 2 Laboratoire de Biochimie CHU de Grenoble, France 3 INSERM U330, Université Victor Ségalen Bordeaux2, France 4 Ecole de Santé Publique Faculté de Médecine Nancy, France

Two randomized double-blind trials were performed in order to assess the efficacity of differing combinations of antioxidant nutrients on biochemical parameters of vitamin and trace elememnt status and free radical metabolism in elderly long term hospitalized subjects and in free-living adults. The effects on red cell and plasma glutathion peroxidase levels was assessed. The first study was performed in a total of 756 institutionalized elderly subjects recruited in 26 nursing homes in different areas of France. Four groups were constituted, receiving daily, for 1 year, either vitamins (beta-carotene, 6mg; vitamin C, 120mg; and vitamin E, 15mg), trace elements (zinc, 20mg and selenium, 100.g), trace elements asociated with vitamins, or a placebo. Biochemical indicators of trace elements and vitamin status and free radical parameters were measured before and after 6 months and 1 year supplemention. Mean plasma levels of alpha-tocopherol, gamma-tocopherol, vitamin C, alpha-carotene, bera-carotene and copper increased significantly after 6 months of 450

Relationship between Serum Selenium and Red Cell and Plasma Glutathion Peroxidase Levels

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supplementation in groups receiving vitamins alone or associated with trace elements. Serum selenium concentrations were significantly increased at 6 months of supplementation, and serum zinc only after one year in the trace element groups. Serum lycopene levels were significantly decreased by trace element supplementation. Two-way analysis of variance indicated a vitamin and a mineral effect on GPx activity after 6 and 12 months of supplementation. The second randomized double-blind trial was performed in 401 healthy subjects (166 males aged 45–60 years and 235 females aged 35–60 years). Two groups were compared receiving daily either a combination of vitamins (beta-carotene, 6 mg; vitamin C, 120mg; and vitamin E, 30mg) and trace elements (zinc, 20mg; and selenium, 100mg); or a placebo. Biological markers of vitamin and trace element status and free radical parameters were measured initially, and after 3 months and 6 months of supplemention. Mean serum levels of alpha-tocopherol, vitamin C, beta-carotene, zinc and selenium increased significantly after 3 months of supplementation in the group receiving multivitamins associated with minerals. The percentage of subjects with low serum concentrations decreased in the group receiving supplementation. Compared to baseline plasma GPx concentrations were significantly higher in the supplemented group at 3 and 6 months, as were red cell GPx concentrations at 6 months.

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INTERVENTION STUDIES ON ANTIOXIDANT TRACE ELEMENTS Special Focus on Selenium

Serge Hercberg Institut Scientifique et Technique de la Nutrition et de l‘Alimentation/CNAM 2 rue Conté, F-75003 Paris

During the last twenty years, interventional studies have been conducted to try to demonstrate the causal relationship between antioxidant nutrients and diseases (Hercberg, 1998). In fact, three types of arguments have contributed over the last 20 years to an increase in the interest in antioxidant intake, and their consequences on health in industrialized countries, and consequently have providing the scientific background to develop intervention studies. 1. First, experimental works have supported the possible role of oxidative stress in the determinism of pathologic processes and protection against oxidative damage by antioxidant nutrients (Halliwell et al., 1989; Sies et al., 1992). A large body of evidence suggests that free radical production directly and indirectly plays a major role in cellular processes such as carcinogenesis and atherosclerosis (Niki, 1987; Steinberg, 1991). By their antioxidant functions of protecting organisms against free radical damage, essential micronutrients such as some trace elements (zinc, copper, manganese, and selenium) and some vitamins (vitamin E, vitamin C and beta-carotene) have been considered as helpful in the prevention of diseases initiated or promoted by oxygen radicals, such as cancer and cardiovascular diseases. 2. Second, a large number of epidemiological studies have suggested that low dietary intake or the borderline biochemical status of some antioxidant trace elements and vitamins may be important risk factors for various diseases (Diplock, 1991; Stampfer et al., 1993, 1995; Kohlmeier et al., 1995; Byers and Perry, 1992; Block et al., 1992). 3. And third, a significant percentage of the affluent world population was shown to have a relatively low intake, or borderline vitamin and/or mineral status, which was considered to be a high risk factor for developing diseases of “affluence”, such as cancers and cardiovascular diseases (Hercberg et al., 1994). During the last thirty years, numerous observational studies (ecological, casecontrol and even large prospective studies) have been developed to elucidate the relation Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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between dietary intake or biological status in antioxidants, and risk of chronic diseases. However, while the results of these epidemiological data generally suggest a relationship between antioxidant micronutrient status and risk of cancer and cardiovascular diseases, the proper design of observational studies does not enable conclusions as to a causal effect. 1. Ecological studies compare dietary intakes or biomarkers assessing antioxidant status between populations and morbidity/or mortality in these populations. For example, in the field of trace elements, epidemiologic studies conducted in the late 1960s an 1970s began to find an inverse relationship between selenium intake and cancer mortality at a population level. Schrauzer et al. (1977) reported a 27 country comparison, indicating that total cancer mortality rate and age-corrected mortality due to leukemia and cancers of the colon, rectum, breast, ovaries, and lung varied inversely with estimated per capita selenium intake. Similar results were reported in China (Yu et al., 1985). Pooled blood samples from healthy donors in 19 US states and 22 countries also showed an inverse correlation between blood selenium levels and cancer mortality rates, and there were significant inverse relationships for most of these within the same site as well across the collective 19 states and 22 countries (Shamberger et al., 1976). But, the relationship observed at a population level do not permit to conclude that it exist at an individual level, neither that this relation supply a causality link. It is clear that population differs by a lot of other particularities than antioxidant status. But the convergency of the results of this kind of studies reinforce the hypothesis of an inverse relationship between antioxidant intake or status (particularly for selenium) and some specific causes of mortality. 2. The case-control study compares dieta habits or biological indicators of “cases”, patients with diseases (cancers, cardiovascular diseases), with those of “controls”, subjects diagnosed as not having pathologies. For exemple, Mikac-Devic et al. (Mikac-Devic, 1992), determined serum selenium in patients suffering from colorectal cancer and in a control group of healthy persons from the same environment as the group of patients. Serum selenium concentration was significantly lower in cases compared to control. Similarly, Calautti et al. (1980) described that lowered mean serum concentrations were observed in a group of patients with as compared to normal individuals. The main difficulty when interpretating these studies lies in the impossibility of determining whether the blood concentrations of selenium existed prior to the onset of disease, or were the consequence of the disease process. 3. In prospective studies, dietary information and biological indicators are collected from subjects who are free of disease, and they are subsequently followed for development of disease. Since prospective studies are less prone to bias, they are considered to provide stronger scientific evidence. Excitement about selenium’s anticarcinogenic role rose when Willet et al., in 1983, presented the results of prospective study comparing initial serum selenium concentrations from 111 subjects in whom cancer developed during the subsequent 5 years with those in serum samples from 210 cancer-free subjects matched for age, race and smoking history (Table 1). Patients who developed cancer had a serum selenium concentration of Se/ML (±0.002 SEM) versus noncancer individuals with a concentration of Se/mL (±0.002 SEM). Those in the lowest quintile of serum selenium concentration had a risk twice as high as those in the highest quintile. The association between low selenium level and cancer was strongest for gastrointestinal and prostatic cancers.

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A number of prospective studies have shown that cancer patients are generally of slightly lower selenium status, on average than healthy controls (Combs and Gray, 1998). For example, Clark et al. (1993) found in a decade-long prospective study of selenium status and cancer incidence in a cohort of 1,738 americans that initial plasma selenium concentration was inversely related to subsequent risks of both non-melanoma skin cancer (basal and/or squamous cell carcinoma) and colonic adenomatous polyps. A few years ago, Comstock et al. (Comstock et al., 1992) reviewed case-control differences in prediagnostic serum levels of selenium compared for 10 cancer sites in 10 study population. Serum levels of selenium are generally lower in cases than in controls. The majority of studies reported lower prte-diagnostic serum levels among cases than among controls, but with only two of 24 showing a case-control difference of greater than 10 percent. As concerns the relationship between selenium status and risk of cardiovascular disease, several prospective studies have been conducted, especially in Finland, where serum selenium levels are exceptionally low. Some of them found an increase in cardiovascular morbidity and mortality in subjects with low serum selenium levels (Comstock et al., 1992; Virtamo et al., 1985). A large prospective study observed a relationship between selenium deficiency and an excess risk ot MI as well as death from CHD in Eastern Finland (Salonen, 1982). The finding was subsequently confirmed in another prospective population study (Suadicani et al., 1992) and in a case-control study (Kok et al., 1989). An association was observed between low serum selenium levels and accelerated progression of carotid atherosclerosis in eastern finnish men (Salonen et al., 1992). But other studies did not find such a relationship (Kok et al., 1987; Virtamo et al., 1985; Ellis et al., 1983). These equivocal results may be due to the poor significance of concentrations in serum selenium as a reliable indicator of selenium status. However, in a cross-sectional study conducted in hospitalized subjects with intermediate selenium levels, a significant inverse correlation was observed between plasma selenium and the degree of angiographically defined coronary atherosclerosis (Moore et al., 1984). Finally, many observational studies show that a high intake or high blood concentrations of antioxidant micronutrients (particularly selenium) are associated with reduced risk of diseases. However, they cannot be conclusive for a causal link. Only intervention studies, by specifically changing antioxidant intake, can provide conclusive answers. Most of randomized controlled double-blind trials recently published tested the effect of antioxidant vitamins alone or in a limited association with other nutrients. Few tested the effect of antioxidant trace elements alone or associated with other antioxidant nutrients (Table 2). This may be due to the fact that epidemiologic data issued from

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observational studies are more complete and consistent for antioxidant vitamins than for antioxidant trace elements. However, some consistent data suggest that antioxidant trace elements, particularly selenium, may be implicated in the determinism of chronic diseases. Finally, many case-control studies and prospective studies show that a high intake or high blood concentrations of selenium are associated with reduced risk of cancer at several common sites or cardiovascular diseases. Several prospective studies point to a negative relationship between plasma selenium levels and risk of cancer (Willet et al., 1983; Akesson, 1997; Coates et al., 1988; Fex et al., 1987; Glattre et al., 1989; Menkes et al., 1986; Peleg et al., 1985; Ringstad et al., 1988; Salonen et al., 1984; Van T Veer et al., 1990) or cardiovascular diseases (although this relation was not significant for 4 of them (Akesson, 1997; Coates et al., 1988; Ringstad et al., 1988; Salonen et al., 1984). Some case-control and prospective studies provided no evidence of protective effect of selenium. In case of cancer, it is possible that the level of the selenium status of studied population and may be the specific localisation of studied cancers may explain a part of this discrepancy between studies. For example, with regard to breast cancer, a Finnish study was consistent with the hypothesis that there is a threshold below which low selenium intake increases breast cancer risk, but selenium intake is not likely to be associated with breast cancer in countries with moderate or high levels of selenium intake. Moreover, even when they are consistent, while all these observational studies suggest a relationship at the population and individual level, they do not affirm a causality link. Only randomized trials in which selenium or a placebo is assigned at random may reveal this causal relationship. Different clinical trials conducted in China have first yielded results supportive of anticancer efficacy of selenium. One study involved the use of a daily supplement of Se as Se-enriched yeast (An, 1995). The results indicated that Se supplementation eliminated liver cancer incidence among hepatitis surface antigen carriers (from the control rate of 1.58%) and reduced over a 2-year period the annual incidence of that cancer among first-degree relatives from 414.7–553.2 cases per 100,000 in the control group to 175.4–210.4 per 100,000 in the Se-treated group, in the first and second years, respectively. In another study (44), liver cancer incidence among villages provided salt fortified with 15ppm Se as dropped from 54.8/100,000 to 34.5/100,000 during 1984–1990 while rates in control villages remainded high (54–65/100,000).

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The Nutritional Intervention Trials in Linxian (China), in a general population of 29,584 subjects (mostly non-smokers) with a follow-up of 8 years (Blot et al., 1993) showed, a beneficial effect of nutritional doses of selenium associated with betacarotene (15mg/d) and vitamin E (30mg/d) on total mortality and cancer incidence, particularly that of stomach cancer. A reduction in the incidence of cerebrovascular disease was observed but did not reach statistical significance. As early studies found reduced plasma selenium concentration in patients with skin cancer that were not melanomas, Clark et al. (1996) initiated a randomized trial with 1312 patients with histories of basal cell or squamous cell carcinomas of the skin, and provided them with either an oral supplement of Se/day or a placebo during a mean (SD) of 4.5 (2.8) years. Selenium treatment did not significantly affect the primary end points of incidence of new basal or squamous cell carcinoma of the skin. Selenium treatment was, however, associated with a statistically significant reduction in several secondary end points that were not the focus of the study: total and lung cancer mortality (Table 3); total cancer incidence; colon-rectal cancer; and prostate cancer incidence. Total cancer incidence was 42% lower in the selenium group (p < 0.001). Recently, a total of 974 men with a history of either a basal cell of squamous cell carcinoma were randomized to either a daily supplement of of selenium or a placebo (46). Patients were treated for a mean of 4.5 years and followed for a mean of 6.5 years. Selenium treatment did not protect against development of carcinomas of skin but was associated with a significant reduction in the secondary endpoint of prostate cancer incidence. There were significant health benefits also for the other secondary endpoints of total cancer mortality, end the incidence of total, lung and colorectal cancer. In France an other intervention study implicating antioxidant trace elements is on going (Hercberg et al., 1997; Hercberg et al, 1998). The “SUpplementation en VItamines et Minéraux AntioXydants” (SU.VI.MAX) Study is a randomized double-blind, placebocontrolled, primary-prevention trial designed to test, in France, the efficacy of a daily supplementation with antioxidant minerals (selenium, and zinc, 20 mg) and vitamins (vitamin C, 120mg; vitamin E, 30 mg; and beta-carotene, 6mg) at nutritional doses (one to three times the daily recommended dietary allowances), in reducing several major health problems in industrialized countries, and especially the main causes of premature death (cancers and cardiovascular diseases); 12,735 eligible subjects (women aged 35 to 60, and men aged 45 to 60) were included in 1994 and and will be followed up for eight years. The methodology and design of the SU.VI.MAX Study is bilt on the accumulation of mechanistic and epidemiologic data suggesting that antioxidants act not only

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individually but also cooperatively and in some cases synergistically. The intrinsic chemical reactivity of each antioxidant, their differing locations in the membrane and lipoproteins, their additive or synergistic effects, their multiple interactions and cooperative action led us to hypothesize that, rather than a single antioxidant nutrient, it is a balanced combination of antioxidants that may provide maximal efficacy associated with maximal safety. The objectives and the specific design of this intervention study are linked to its public health aim. The targeted population is the general population (not simply highrisk subjects) and the antioxidant agents tested are being administered at a level which is not pharmacologic and which may be attained by dietary intake of natural sources of these micronutrients and/or eventual enriched foods. The amounts we are testing in the SU.VI.MAX study are those which, in observational studies, have been associated with the lowest risk of diseases. The interest of testing a large combination (associating trace elements, vitamins and beta-carotene), at nutritional dosis, has increased at the light of the results of recently reported randomized controlled trials on populations at high risk, in which single nutrients are given in high doses over short periods, have not yet shown any substantial benefits. Indeed, the Alpha Tocopherol and Beta Carotene (ATBC) Study, which studied male heavy smokers in Finland, observed a significantly higher lung cancer incidence rate in a group receiving relatively high levels of beta-carotene alone or associated with vitamin E. In the same study, subjects receiving vitamin E had significantly more hemorrhagic strokes than controls. These conclusions were supported by the recently published results of the Carotene and Retinol Efficacy Trial (CARET) (Omenn et al., 1996), a study in the United States on asbestos-exposed workers and heavy smokers who are at high risk for lung cancer. Cases of lung cancer were significantly more frequent in the group receiving relatively high levels of beta-carotene associated with vitamin A than in the placebo group. Finally, the Physician Health Study in the USA (Hennekens et al., 1996) did not demonstrate any positive or negative effects of long-term supplementation with quite similar doses of beta-carotene. The only intervention study demonstrating a positive effect was the Nutritional Intervention Trial in Linxian (China) (Blot et al., 1993). This study showed, in a general population, a beneficial effect of nutritional doses of an association of beta-carotene, vitamin E and selenium on total mortality and the incidence of cancer, particularly esophagal cancer. These apparent discrepancies may be explained by the choice of the study population (general or high-risk subjects), the different doses of supplementation (nutritional or higher), the number of antioxidants tested (one, two, or more) and the type of administration (alone or in a balanced association, with or without trace elements). Moreover, some of these intervention studies have reported, in high-risk populations receiving high doses of antoxidant vitamins, a greater relative risk of some pathologies. Therefore, it was interesting to develop randomized, controlled trials on average or low-risk individuals given combination multiple nutrients. The advantage lies in testing a combination of nutrients at levels similar to those contained in a healthy diet associated, in observational study, with the lowest risk of disease. In the SU.VI.MAX study, mean values observed for initial serum vitamin and trace element concentrations were similar to thoses previously described for French adult populations (Table 4). Serum concentrations of beta-carotene, vitamin C were significantly higher in women than in men. No difference was found betwen men and women for plasma vitamin E, zinc and selenium concentrations.

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At baseline, mean values for biochemical markers of vitamin and trace element status did not differ between the two groups for any of the parameters tested. The effect of supplementation by antioxidant micronutrients during a 2 years period, on serum concentrations of biochemical markers assessing vitamin and trace element status was significant for all of the studied nutrients, vitamin C, E, beta-carotene, zinc and selenium: biochemical indicators of vitamin and trace element status reach reasonable levels. These preliminary data thus indicate that supplementation (2 years) with moderate doses of antioxidant vitamins and trace element, in presumaly healthy subjects, clearly though moderately vitamin and mineral status, with blood concentrations reaching concentrations consistent with a positive effect (Table 5). After 2 years of supplementation, indicators of antioxidant status reach reasonable level without reaching concentrations as high as those observed in the Finnish and American intervention studies, which tested relatively high doses of antioxidants, and ended up with higher risk of pathology. In CARET (Omenn et al., 1996), as in ATBC (ATBC, 1994), the relatively high levels of supplementation were associated with a substantial increase in the plasma concentrations of beta-carotene. For example, the initial concentrations of plasma carotene were multiplied by 18 in ATBC and by 12 in CARET. The final concentrations were higher than those associated, in observational studies, with the lower risk of diseases. They were also higher than those observed in the SU.VI.MAX study. These results are consistent with other data we observed in a short term suplementation trial (6 months) developped on adults with same doses of antioxidants (Preziosi et al., 1998). It is important to note that these positive biological effects were observed with daily multi-vitamin and mineral supplements containing one to two times the recommended dietary allowances. These doses corresponded to levels of dietary antioxidant micronutrients associated, in observational studies, with the lowest risk of cancer and cardiovascular diseases.

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In conclusion, we dispose of a background of epidemiology that supports the plausibility of selenium as a risk factor in human cancers and (at a least degree cardiovascular diseases) and a substantial body of experimental data from a variety of animal models that establish that Se can be antitumorigenic (and may be antiathreosclerotic). The effect may be particularly effective in a combination with other antioxidants nutrients, trace elements or vitamins.

REFERENCES Akesson, B., 1997, Plasma selenium in patients with abnormal plasma protein patterns, Trace Elem. Med. 4:77–79. An, P., 1995, Selenium and endemic cancer in China. In: Environmental Bioinorganic Chesmistry of Selenium, pp.91–149, Whager, P.M., Combs, G.F., Jr., Yeh, J.Y., ed, Chisese Academy of Science, Beijing. Block, G., Patterson, B., and Subar, A., 1992, Fruit, vegetables, and cancer prevention, a review of the epidemiological evidence, Nutr. Cancer 18:1–29. Blot, W.J., Li, J.Y., Taylor, P.R. et al., 1993, Nutrition intervention trials in Linxian, China: supplementation with specific vitamin/mineral combinations, cancer incidence, and disease-specific mortality in the general population, J. Natl. Cancer Inst. 85:1483–1492. Byers, T. and Perry, G., 1992, Dietary carotenes, vitamin C, and vitamin E as protective antioxidants in human cancers, Annu. Rev. Nutr. 12:139–159. Calautti, P., Moschini, G., Stievano, B.M., Tomio, L., Clazavara, F., and Perona, G., 1980, Serum selenium levels in malignant lymphoproliferative diseases, Scand. J. Haematol. 24:63. Clark, L.C., Combs, G.F., Jr., Turnbull, B.W., Slate, E., Alberts, D., Abele, D., Allison, R. et al., 1996, The nutritional prevention of cancer with selenium 1983–1993: a randomized clinical trial, JAMA, 276:1957–1963. Clark, L.C., Dalkin, B., Krongrad, A., Combs, G.F., Jr., Burnbull, B.W., Slate, E.H., Witherington, R., Herlong, J.H. et al., 1998, Decreased incidence of prostate cancer with selenium supplementation: results of a double-blind cancer prevention trial, Brit. J. Urol. 81:730–734. Clark, L.C., Hixon, L., Combs, G.F., Jr., Reid, M., Turnbull, B.W., and Sampliner, R., 1993, Plasma selenium concentration predicts the prevalence of colorectal adenomatous polyps, Cancer Epidemiol. Biomarkers Prev. 2:41–46. Coates, R.J., Weiss, N.S., and Daling, J.R., 1988, Serum levels of selenium and retinol and the subsequent risk of cancer, Am. J. Epidem. 128:515–523. Combs, G.F. and Gray, W.P., 1998, Chemopreventive agents: selenium, Pharmacol. Ther. 79:179–192. Comstock, G.W., Bush, T.L., and Helzlsouer, K., 1992, Serum retinol, beta-caroten, vitamin E, and selenium As related to subsequent cancer of specific sites, Am. J. Epidemiol. 135:115–121. Diplock, A.T., 1991, Antioxidant nutrients and disease prevention: an overview, Am. J. Clin. Nutr. 53:189S–193S. Ellis, N., Lloyd, B., and Lloyd, R.S., 1983, Selenium and vitamin E in relation to risk factors for coronary heart disease, J. Epemiol. Community Health 37:213–217. Fex, G., Pettersson, B., and Akesson, B., 1987, Low plasma selenium as a risk factor for cancer death in middle-aged men, Nutr. Cancer 10:221–229. Glattre, E., Thomassen, Y., Thoresen, S.O. et al., 1989, Prediagnostic serum selenium in a case-control study of thyroid cancer, Int. J. Epidemiol. 18:45–49. Halliwell, B. and Gutteridge, J.M.C., 1989, Free Radicals in Biology and Medicine. 2nd ed. Oxford, UK, Clarendon Press. Hennekens, C.H., Buring, J.E., Manson, J.E. et al., 1996, Lack of effect of long-term supplementation with beta-carotene on the incidence of malignant neoplasms and cardiovascular disease, N. Eng. J. M. 334:1145–1149. Hercberg, S., Galan, P., Preziosi, P., Alfarez, M.J., and Vazquez, C., 1998, The potential role of antioxidant vitamins in preventing and cancers, Nutrition 15:513–520. Hercberg, S., Galan, P., Preziosi, P., Roussel, A.-M., Arnaud, J., Richard, M.-J., Malvy, D., Paul-Dauphin, A., Briançon, S., and Favier, A., 1997, Background and rationale behind the SU.VI.MAX Study, a prevention trial using nutritional doses of a combination of antioxidant vitamins and minerals to reduce cardiovascular diseases and cancers, Int. J. Vit. Nutr. Res. 68:3–20.

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Hercberg, S., Preziosi, P., Briançon, S., Galan, P., Triol, I., Malvy, D., Roussel, A.-M., and Favier, A., 1998, A primary prevention trial of nutritional doses of antioxidant vitamins and minerals on cardiovascular diseases and cancers in general population: The SU.VI.MAX Study. Design, methods and participant characteristics. Control Clin. Trials 19:336–351. Hercberg, S., Preziosi, P., Galan, P., Devanlay, M., Keller, H., Bourgeois, C., Potier de Courcy, G., and Cherouvrier, F., 1994, Vitamin status of a healthy French population: dietary intakes and biochemical markers, Int. J. Vit. Nutr. Res. 64:220–232. Kohlmeier, L. and Hastings, S.B., 1995, Epidemiologic evidence of a role of carotenoids in cardiovascular disease prevention, Am. J. Clin. Nutr. 62:13708–13768. Kok, F.J., De Bruidjn, A.M., Vermeeren, R. et al., 1987, Serum selenium, vitamin antioxidants and cardiovascular mortality: A 9-year follow-up study in the Netherlands, Am. J. Clin. Nutr. 45:462–468. Kok, F.J., Hofman, A., Witterman, J.C.M., De Bruijn, A.M., Kruyssen, D.H.C.M., De Bruin, M., and Valkenburg, H.A., 1989, Decrease blood selenium and risk of myocardial infarction, JAMA 261: 1161–1164. Menkes, M.S., Comstock, G.W., Vuilleumier, J.P. et al., 1986, Serum beta-carotene, vitamins A and E, selenium, and the risk of lung cancer, N. Engl. J. Med. 315:1250–125S. Mikac-Devic, M., Vukelic, N., and Kljaic, K., 1992, Serum selenium level in patients with colorectal cancer, Biol. Trace Elem. Res. 33:87–94. Moore, J.A., Nolva, R., and Wells, I.C., 1984, Selenium concentration in plasma of patients with arteriographically defined coronary atherosclerosis, Clin. Chemist. 30:1171–1173. Niki, E., 1987, Antioxidant in relation to lipid peroxidation, Chem. Phys. LIP. 44:227–253. Omenn, G.S., Goodman, G.E., Thorquist, M.D. et al., 1996, Effects of a combination of beta-carotene and vitamin A on lung cancer and cardiovascular disease, N. Eng. J. Med. 334:1150–1155. Peleg, I., Morris, S., and Hames, C.G., 1985, Is serum selenium a risk factor for cancer?, Med. Oncol. Tumor Pharmacother. 2:157–163. Preziosi, P., Galan, P., Herbeth, B., Valeix, P., Roussel, A.M., Malvy, D., Paul-Dauphin, A., Arnaud, J., Richard, M.J., Briançon, S., Favier, A., and Hercberg, S., 1998, Effects of supplementation with a combination of antioxidant vitamins and trace elements, at nutritional doses, on biochemical indicators and makers of the antioxidant system in adult subjects, J. Am. Coll. Nutr. 3:244–249. Ringstad, J., Jacobsen, B.K., Tretli, S., and Thomassen, Y., 1988, Serum selenium concentration associated with risk of cancer, J. Clin. Pathol. 41:454–457. Salonen, J.T., 1982, Association between cardiovascular death and myocardial infarction and serum selenium in a matched-pair longitudinal study. Lancet ii:175–179. Salonen, J.T., Alfthan, G., Huttunen, J.K. et al., 1984, Association brtween serum selenium and the risk of cancer, Am. J. Epidemiol. 120:130–134. Salonen, J.T., Salonen, R., Seppänen, K., Kantola, M., Suntioinen, S., and Korpela, H., 1992, Interactions of serum copper, selenium, and low density lipoprotein cholesterol in atherogenesis, Brit. Med. J. 302:756–760. Schrauzer, G.N., White, D.A., and Schneider, C.J., 1977, Cancer mortality correlation studies. IV. Associations with dietary intakes and blood levels of certain trace elements, notably Se-antagonists, Bioinorg. Chem. 7:35–56. Shamberger, R.J., Tytko, S.A., and Willis, C.E., 1976, Antioxidants and cancer. Part VI. Selenium and age-adjusted human cancer mortality, Arch. Environ. Hlth. 26:231. Sies, H., Staahl, W., and Sundqquist, A.R., 1992, Antioxidant functions of vitamins: vitamin E and C, beta-carotene, and other carotenoids, Ann. N Y Acad. Sci. 669:7–20. Stampfer, M.J. and Rimm, B., 1993, A review of the epidemiology of dietary antioxidants and risk of coronary heart disease, Can. J. Cardiol. 19:14B–18B. Stampfer, M.J. and Rimm, B., 1995, Epidemiologic evidence for vitamin E in prevention of cardiovascular disease, Am. J. Clin. Nutr. 62:1365S–1369S. Steinberg, D., 1991, Antioxidant and atherosclerosis: a current assessment. Circulation, 84:1420–1425. Suadicani, P., Hein, O.O., and Gyntelberg, T., 1992, Serum selenium concentration and risk of ischaemic heart disease in a prospective cohort study of 3,000 males, Atherosclerosis, 96:33–42. The Alpha-Tocopherol, Beta Carotene Cancer Prevention Study Group, 1994, The effect of vitamin E and beta carotene on the incidence of lung cancer and other cancers in male smokers, NEJM 330, 15:1029–1035. Van, T., Veer, P., Van der Wielen, R.P.J., Kok, F.J., Hermus, R.J.J., and Sturmans, F., 1990, Selenium in diet, blood and toenails in relation to breast cancer: a case-control study, Am. J. Epidem. 131:987–994. Virtamo, J., Valkeila, E., Alfthan, G., Punsar, S., Huttunen, J.K., and Karvonen, M., 1985, Serum selenium and the risk of coronary heart disease, Am. J. Epidem. 122:276–282.

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Virtamo, J., Valkeila, E., Alfthan, G., Punsar, S., Huttunen, J.K., and Karvonen, M., 1985, Serum selenium and the risk of coronary heart disease, Am. J. Epidem. 122:276–282. Willet, W.C., Polk, B.F., Morris, J.S. et al., 1983, Prediagnostic serum selenium and risk of cancer, Lancet 2:130–134. Yu, S.Y., Chu, Y.J., Gong, X.L., Hou, C., Li, W.G., Gong, H.M., and Xie, J.R., 1985, Regional variation of cancer mortality incidence and its relation to selenium levels in China, Biol. Trace Elem. Res. 7:21–29.

144

SELENIUM STATUS IN NORTHERN IRELAND AND FRANCE The Prime Study

Dorothy McMaster1, Pedro Marques-Vidal2, Pierre Ducimetière3, Phillipe Amouyel4, Dominique Arveiler5, and Alun Evans1 1

The Queen’s University of Belfast Belfast, N Ireland 2 INSERM CJF 94-06, Toulouse France 3 Coordinating Centre U258 INSERM, Paris, France 4 INSERM CJF 95-05, Institut Pasteur-Lille France 5 Laboratoire d’Epidémiologie et de Santé Publique Strasbourg, France

The findings in investigations into the contribution of low selenium status to the development of coronary heart disease (CHD) have been inconsistent (Salonen et al., 1982; Robinson et al., 1983; Salonen et al., 1985; Virtamo et al., 1985; Miettinen et al., 1987; Ringstad et al., 1987; Kok et al., 1987, Suadicani et al., 1992; Salvini et al., 1995). From earlier collaborative work carried out within the World Health Organization’s MONICA Project (MONitoring of trends and determinants in CArdiovascular diseases) it had been established that the incidence of CHD in Northern Ireland was 3 times higher than in France (Tunstall-Pedoe, 1994). A comparison of Se status in these two countries using matching cohorts could provide additional valuable information. The PRIME Study, set up in 1991, is a Prospective Epidemiological Study of Myocardial Infarction which aims to test the prognostic efficiency of candidate risk factors and to identify factors that could explain this large difference between the two countries. (The PRIME Study Group, 1998.) To date the study has shown that levels of the classical risk factors in each of the cohorts cannot explain the higher incidence of CHD in Belfast than in Address all correspondence to: Dr. Dorothy McMaster; Department of Medicine, Institute of Clinical Science, Grosvenor Road, Belfast, N. Ireland, BT12 6BJ; email [email protected] Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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France. Therefore other possibilities require investigation. Glutathione peroxidase (GPX, EC 1.11.1.9) is a Se dependent, antioxidant, enzyme which exists in several forms and which catalyses the breakdown of hydrogen peroxide and fatty acyl lipid peroxides thus protecting cells, including endothelial cells, against oxidative damage. When Se intake is low (below 50–80ug/d) GPX is correlated to plasma Se and thus a strong correlation indicates that Se status is unsaturated and is below the achievable level (van’t Veer et al., 1991). For epidemiological studies the level of GPX in serum can be used as an adequate marker of selenium status. We have measured GPX in all subjects to compare Se status in Northern Ireland with France and have included serum Se in a limited number of subjects. The PRIME Study is based in Northern Ireland and in the South, East and North of France. Men aged 50–59 years have been recruited from both industrial and community settings and have been examined for evidence of CHD at a baseline screening examination. Recruitment began in May 1991 and closed in January 1994 and involved four MONICA centres: Belfast n = 2,748, Lille n = 2,627, Strasbourg n = 2,611 and Toulouse n = 2,610. All subjects gave informed consent and the relevant local ethical committees granted permission. Se status was estimated as described previously (McMaster et al., 1990). The table shows clearly that Se status in Northern Ireland was significantly higher than in France (p < 0.001) and in both countries current smokers had lower levels of Se and GPX than ex- or non-smokers (p < 0.001). Spearman rank correlation analysis gave significant positive relationships (p < 0.001) between Se and GPX in both countries: r = 0.37 (N Ireland) and 0.39 (France). A strong inverse relationship was found between the amount of tobacco smoked and Se status: r = –0.24 and –0.16 for Se and r = –0.12 and –0.08 for GPX in N Ireland and France respectively, (p < 0.001). However, Se status was not different in subjects with a history of heart disease at baseline to those without. A prospective study based on a representative sample of the population is the preferred investigation to determine the predictive ability of classical or new risk factors for CHD. Whereas most previous studies have used only measurements of serum Se to indicate Se status we have used the biological activity of a Se dependent enzyme. In both countries the strong positive correlation between Se and GPX clearly indicates that Se status is unsaturated. The finding that Se status in Belfast men at entry to this study was

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significantly higher than in the French men indicates that this particular candidate risk factor is unlikely to contribute to the development of CHD in Northern Ireland. The mechanism by which cigarette smoking increases the risk of CHD is not clear. We explored the possibility of an association between smoking and lowered Se status. While smoking in Belfast was more frequent, smokers in both countries had significantly lower Se status than non-smokers or ex-smokers. In addition there was an inverse relationship between the amount of tobacco smoked and GPX status suggesting that in heavy smokers the antioxidant capacity of GPX may be compromised. The finding that Se status was the same in men with a history of CHD as in those without throws no further light on the subject. The higher Se status found in the N Ireland population, historically at high risk for CHD, argues against a major impact of this candidate risk factor on the CHD geographical gradient. The PRIME Study is organised under an agreement between INSERM and the Merck, Sharpe and Dohme-Chibret Laboratory. We wish to thank Dominique Courbon for facilitating the data analysis.

REFERENCES Kok, F.J., de Bruijn, A.M., Vermeeren, R., Hofman, A., van Laar, A., de Bruin, M., Hermus, R.J.J., and Valkenburg, H.A., 1987, Serum selenium, vitamin antioxidants, and cardiovascular mortality: a 9-year follow-up study in the Netherlands, Am. J. Clin. Nutr. 45:462–468. McMaster, D., Bell, N., Anderson, P., and Love, A.H.G., 1990, Automated measurement of two indicators of human selenium status, and applicability to population studies, Clin Chem. 36:211–216. Miettinen, T.A., Alfthan, G., Huttunen, J.K., Pikkarainen, J., Naukkarinen, V., Mattila, S., and Kumlin, T., 1987, Serum selenium concentration related to myocardial infarction and fatty acid content of serum lipids, BMJ. 287:517–519. Ringstad, J., Jacobsen, B.K., and Thomassen, Y., 1987, The Tromsø Heart Study: Relationships between the concentration of selenium in serum and risk factors for coronary heart disease, J. Trace Elem. Electrolytes Health Dis. 1:27–31. Robinson, M.F., Campbell, D.R., Sutherland, W.H.F., Herbison, G.P., Paulin, J.M., and Simpson, F.O., 1983, Selenium and risk factors for CHD in New Zealand, NZ. Med. J. 96:755–757. Salonen, J.T., Alfthan, G., Huttunen, J.K., Pikkarainen, J., and Puska, P., 1982, Association between cardiovascular death and myocardial infarction and serum selenium in a matched-pair longitudinal study, Lancet 1982:175. Salonen, J.T., Salonen, R., Penttilä, I., Herranen, J., Jauhaiainen, M., Kantola, M., Lappeteläinen, M.S., Mäenpää, P.H., Alfthan, G., and Puska, P., 1985, Serum fatty acids, apolipoproteins, selenium and vitamin antioxidants and the risk of death from coronary artery disease, Am. J. Cardiol. 56:226–231. Salvini, S., Hennekens, C.H., Morris, J.S., Willett, W.C., and Stampfer, M.J., 1995, Plasma levels of the antioxidant selenium and risk of myocardial infarction among U.S. Physicians, Am. J. Cardiol. 76:1218–1221. Suadicani, P., Hein, H.O., and Gyntelberg, F., 1992, Serum selenium concentration and risk of ischaemic heart disease in a prospective cohort study of 3,000 males, Atherosclerosis, 96:33–42. The PRIME Study Group (prepared by JWG Yarnell), 1998, The PRIME Study: classical risk factors do not explain the severalfold differences in risk of coronary heart disease between France and Northern Ireland, Q. J. Med. 91:667–676. Tunstall-Pedoe, H. et al. On behalf of WHO MONICA Project., 1994, Myocardial infarction and coronary deaths in the World Health Organization MONICA Project. Circulation; 90:583–612. van’t Veer, P. and Alfthan, G., 1991, Biomarkers of Se, workshop report, in: Biomarkers of Dietary Exposure, (F.J. Kok, P. van’t Veer, eds.). Proceedings of the 3rd meeting on Nutritional Epidemiology, pp. 106–118, Smith Gordon and Company Ltd., London. Virtamo, J., Valkeila, E., Alfthan, G., Punsar, S., Huttunen, J.K., and Karvonen, M.J., 1985, Serum selenium and the risk of coronary heart disease and stroke, Am. J. Epidemiol. 122:276–281.

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DETERMINANTS OF BLOOD SELENIUM CONCENTRATIONS IN FRANCE (SU VI MAX STUDY) J. Arnaud1, A. M. Roussel1, P. Preziosi2, P. Galan2, S. Hercberg2, A. Favier1, and the SU VI MAX group 1

Laboratoire de Biochimie C CHUG, BP 217, 38043 Grenoble Cedex 9 France 2 ISTA, CNAM, 2 rue Conté 75003 Paris, France

The presented results are part of a randomized double-blind placebo-controlled prospective study: SU VI MAX. The aim of this study was to determine the potential beneficial effect of a daily antioxidant supplementation at low doses (Zinc: 20mg, Selenium: Vitamin E: 30mg, Vitamin C: 120mg and 6mg) fro 8 years on the major causes of premature death (i.e. cancers and cardiovascular diseases). The present paper is focused on selenium status of the volunteers at the enrollment, in 1994. Three thousand six hundred and seventy nine middle-aged men (45–60 years) and 5,503 women (35–60 years) were selected. Lifestyle and occupation were assessed by questionnaire. Selenium was determined in serum by electrothermal atomic absorption spectrometry (Perkin Elmer 4,100 ZL, Norwalk, CT) using Seronorm Trace Element (Nycomed, Oslo, Norway) as internal quality control. Serum selenium values lower than were observed in only 0.63% of males and in 1.18% of females. Women had lower serum selenium walues than men vs p = 0.0001). In women serum selenium concentrations increased with age (p = 0.0001), whereas it remained similar in men. Smoking decreased the serum selenium concentration in men but not in women. There were regional differences in serum selenium concentration (Table 1), with lower values in the East than in the West and lower values in the south than in the North. Occupation had an effect on serum selenium concentrations. In men, serum selenium concentrations were linked to income. In women, the differences observed according to occupation were more complexe Contraceptive method had an effect on serum selenium concentrations: women who used oral contraceptive agents had a higher selenium than women who had an intrauterin device and women who did not use contraceptive agents (p = 0.0001). 467

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SELENIUM STATUS AND ANTIOXIDANT ENZYME ACTIVITIES IN HIGH SCHOOL CHILDREN FROM AN ENDEMIC GOITER AREA B. Giray1, T. Teziç2, Y. Gedik3, A. Öktem3, and F. Hincal1 1

University of Hacettepe Faculty of Pharmacy Department of Toxicology Ankara, Turkey 2 Dr. Sami Ulus Children’s Hospital Ankara, Turkey 3 Karadeniz Technical University Faculty of Medicine Department of Pediatrics Trabzon, Turkey

It is now well recognized that besides iodine a second essential trace element, selenium, is involved in the regulation of thyroid hormone system. Both of these elements are inadequately available for man and life stock in great parts of the world. Existing data suggest that none of the regions of Turkey is free of endemic goiter and the prevalence rate is very high in the East Black Sea Region. In this study, we have aimed to determine selenium status and antioxidant enzyme activities in a group of goitrous high school children living in two towns, away from the sea coast in the East Black Sea Region, in order to investigate the relation of the antioxidant status with iodine deficiency. Subjects were selected by a simple random sampling technique after screening of the whole population of the high schools of the two towns by neck palpation. The overall prevalance rate of goiter was found to be 39.6%. The results of goitrous group (n = 48, aged 15–18) were compared with those of non- goitrous healthy children (n = 49) from the same populations, and an outside control group (n = 24) from another region of low rate of prevalence was also used. Selenium levels were found to be significantly (p < 0.01) low in children with goiter compare to both in- region and out- region healthy controls (67.1 ± 10.9; 75.0 ± 14.6; respectively) of the same gender and age. Antioxidant enzyme activities of erythrocytes were also significantly (p < 0.01) lower in goitrous children than non-goitrous children of the endemic goiter area and out-region healthy controls (GSHPx: 14.69 ± 5.27, 18.50 ± 5.45, 19.01 ± 469

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5.12U/g Hb; SOD: 24.5 ± 3.81, 27.02 ± 4.29, 27.43 ± 2.56U/g Hb; CAT: 186.29 ± 38.60, 208.30 ± 33.08, 217.51 ± 24.51 K/g Hb, respectively). Overall results have, thus, indicated the presence of an oxidative activity in goitrous children, and suggested that not only selenium deficiency but also a selenium intake of suboptimal range might be a critical factor in areas where iodine deficiency prevails. However, whether the high level of oxidant stress encountered in goitrous populations is a cause or a consequence of the thyroid destruction needs to be carefully investigated. This study has been supported by the Eczacibasi Research and Award Fund.

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IODINE CONTENT OF WATER AND SELENIUM LEVEL IN CORN AND WHEAT CONSUMED IN AN ENDEMIC GOITER AREA

B. Giray and F. Hincal University of Hacettepe Faculty of Pharmacy Department of Toxicology Ankara, Turkey

Representative drinking water samples were collected from the different sources that available in two towns in the East Black Sea Region of Turkey where prevalence rate of endemic goitre is very high. The two towns are ~50km apart from each other, ~40–50km away from the sea coast and at an altitude of ~300m. In agreement with the urinary iodine data that we have collected from a group of 97 high school children, iodine content of water was very low (average: range: reflecting a moderate to severe iodine deficiency in the region. Selenium content of corn and wheat grains produced and/or consumed in the same region was also measured as an indicator of the main dietary selenium source of the population. Corn crops collected from different villages had average selenium content of with a range of The average concentration of selenium in wheat samples received from Trabzon Office of Turkish Grain Board, representing the main staple food distributed and consumed in the region, was with a range of Wheat is by far the most important staple food in Turkey. The per capita average daily consumption of wheat and wheat products in Turkey is about two times as high as most Western countries. It has been estimated that ~480g wheat or wheat product are consumed daily by average socioeconomic class Turks and this amount corresponds to almost 42% of daily diet. Most of wheat is consumed as bread, however, in Black Sea Region, corn bread is replaced largely (in rural areas) or at least partially with wheat bread, due to the fact that corn is produced in the area, but not the wheat. The recent demonstrations that three isozymes of iodothyronine 5'- deiodinase, are selenoenzymes has led to the recognition of a key role for selenium in thyroid hormone metabolism. When one considers the selenium content of the staple foods and their proportion in the average diet, in addition to the relatively poor bioavaliability of 471

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selenoproteins available in corn (selenocystine, selenocysteine), it can be concluded that the selenium daily intake in such a suboptimal range may not be considered as safe for an area that moderate to severe iodine deficiency exists. This study has been supported by the Eczacibasi Research and Award Fund.

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SELENIUM DEFICIENCY IN SOUTH-WEST BOHEMIA J. Kvícala1, V. Zamrazil1, and V. Jiránek2 1

Institute of Endocrinology Národní 8, 116 94 Praha 1 Czech Republic 2 Immunotech, a.s., Radiová 1 102 27 Praha 10 Czech Republic

Essential role of selenium for animal and man has been well established during past three decades by epidemiological studies, in vivo and in vitro experiments, as well as by elucidation of several regulatory and protective functions of selenium compounds. Selenium as Se-cysteine is the main component of active centre of Se-dependent GSHPeroxidases (GSH-Px), one of the main parts of antioxidative defence system of the organism. Low concentrations of GSH-Px on the basis of low selenium status of the population may contribute to the increased occurrence of so called “oxidative diseases” like cardiovascular diseases, malignity, inflammations, or neurological diseases. One type of GSH-Px plays the role of Peroxinitrite Reductase and protects cells against peroxinitrite-mediated reactions. Selenoprotein P localised in serum has probably also antioxidative influence, together with the function of Se reserve. Severe deficiency of selenium (especially with concurrent iodine deficit) may cause serious difficulties in the thyroid hormone regulation of the body. Recent results proved substantial role of selenium as Se-cysteine in key hormones of thyroid hormone metabolism and regulation— Deiodinases. Selenium compounds eliminate toxic and carcinogenic effects some heavy metals and organic compounds. Selenium plays probably substantial role in brain function. Recent results on subcellular distribution of selenium in the cell may demonstrate very important functions of selenium in the cell nuclei. Function of reproductive organs is dependent upon sufficient selenium status as well as immune functions. Because of all these for the life necessary functions of selenium and, on the other hand by the pilot studies at the end of eighties indicated deficit of selenium in our population, series of epidemiologic studies started to establish and localise selenium deficiency in the Czech Republic. Presented paper contributes to the estimation of selenium status of the subpopulation in South-West Bohemia by the analyses of the most often used selenium indexes. Serum and urine selenium for both sexes in the age between 6 and 473

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65 years has been analysed. Hair Se analysis was also performed for the group of men between 36 and 49 years (which group proved to have the highest selenium indices in the majority of searched regions). Results were for serum selenium 53 ± 10 ug/l (n = 30), urine selenium 12 ± 4 ug/l (n = 27), and hair selenium 0.257 ± 0.054 ug/g (n = 32). All three indices measured in this group were very low and proved serious selenium deficiency. Lower serum selenium concentrations were detected both for younger and older categories. Age-related differences have been found for both sexes also in the case of urine selenium. This work was partly supported by grants IGA MZ. R No. 3417-3 and GA. R No. 311/96/1609.

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EFFECT OF COPPER SUPPLEMENTATION IN MIDDLE AGED PEOPLE ON PLASMA ANTI-OXIDANTS AND RED BLOOD CELL OXIDIZABILITY Foodcue Study

E. Rock, A. Mazur, Y. Rayssiguier, C. Kehoe*, J. M. O’Connor*, M. P. Bonham*, and J. J. Strain* Unité Maladies Métaboliques et Micronutriments I.N.R.A-C.R.N.H. 63122 Saint Genès Champanelle France *NICHE, University of Ulster Cromore Road, Coleraine, BT52 1SA Northern Ireland

Copper (Cu) is an essential trace element and its deficiency may be involved in a number of degenerative and inflammatory diseases. Although overt Cu deficiency is not a significant nutritional problem for populations, the estimated intake of around 1 to 2mg/d may not be optimal. Cu is considered as an antioxidant in vivo but it also has prooxidant activity in vitro and accumulation of tissue Cu may lead to oxidative stress. The objective of the study was to provide data on the significance of increased dietary Cu as a pro- or anti-oxidant in vivo in free living healthy middle aged men and women. Free radical-induced hemolysis of red blood cells (RBC) in vitro was used to delineate such pro- or antioxidant activities. Twenty six subjects were supplemented daily for 6 week periods with capsules containing either placebo or CuSO4 (3mg) or Cu aminoacid chelate (3mg or 6mg Cu-AA). Analyses were performed on blood collected at the end of each 6 week period. Oxidizability of RBC was assessed following hemolysis induced by an azo initiator (AAPH). Plasma antioxidants were determined by HPLC methods. The mean time necessary for 50% hemolysis induced by AAPH was significantly higher after supplementation with 3mg CuSO4/d and 6mg Cu-AA as compared with placebo periods. No difference was found after supplementation with 3mg Cu-AA. 475

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Decreased susceptibility of RBC to peroxidation occurred without changes in erythrocyte (Cu-Zn)SOD activity. Among the other indices of Cu status measured, diamine oxidase was the only putative index which was significantly increased following Cu supplementation. Analysis of plasma antioxidants showed that decreased hemolysis was significantly and positively correlated with increased α- and β- carotene in the plasma. Together, these data suggest that intake of copper as high as 7mg/d has no pro-oxidant activity and may result in protection of red blood cells against oxidation. The decreased oxidizability of RBC did not result from increased (Cu, Zn)SOD activity and may occur through other mechanisms such as changes of membrane antioxidant content. Thompson and Joseph Ltd., Norwich, England are acknowledged for the supply of Cu supplements. Grants from EU: Fair CT95-0813.

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PLASMA AND URINE SELENIUM OF COWS FROM VARIOUS REGIONS OF THE CZECH REPUBLIC AND ITS COMPARISON WITH CORRESPONDING HUMAN POPULATION SELENIUM INDEXES J. Kvicala1 and V. Kroupová2 1

Institute of Endocrinology Národní 8, 116 94 Praha 1 Czech Republic 2 South-Bohemia University Faculty of Agriculture Studentská 13, 370 05 Ceské Budejovice Czech Republic

Selenium is one of the most beneficial trace elements for animal and men. Regulatory functions of selenoproteins (regulation of thyroid hormone metabolism, prostacycline, thromboxane, and prostaglandine synthesis regulation), their protective functions (deactivation of toxic and carcinogenic heavy metals and organic compounds, antioxidative and antiradical effect of Se-dependent GSH-Peroxidases and other selenoproteins), and till now observed but not elucidated effects upon brain functions, reproductive functions and immune functions, may be fulfilled only in the conditions of satisfactory selenium status and intake. Intake of selenium both for animal and men is limited by its presence in the food chain. The first and basic limitation is its level and bioavailability in the soil. Soil level is determined by the constitution of the rocks and bioavailability depends of the type of selenium compounds and of some other influences like acid rains and types of plants. It is especially important for herbivorous, because most plants do not concentrate compounds of selenium like animals. Carnivorous and omnivorous have increased intake of selenium because of its functional concentration in the bodies of herbivorous, even when also carnivorous and omnivorous including human population of the regions with low soil selenium suffer from selenium deficiency. Two belts of the low selenium area pass through the Europe—from North to South (from Scandinavia to Balkan) and from East to West (from Belorussia to France). The 477

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Czech Republic lies just in the crossing of the belts and some preliminary signs showed serious deficiency of the selenium in our country. One of them was occurrence of muscle dystrophy in the livestock in south regions of the Czech Republic, especially during spring months, reason of which might be selenium deficit. We have started to detect selenium status of the cows in various regions not only because of their poor health status. Milk, milk products and beef meat are one of the main sources of selenium for humans, and previous works on selenium status revealed poor selenium indices (content of selenium in serum, urine, and hair) of our population. Bovine plasma and urine were analyzed by the use of neutron activation analysis and fluorimetry. Quality assurance control was carried out by the use of the NIST SRM—bovine plasma and urine. So low values of bovine liquids as 2–5 ugSe/l plasma and 0.5 ugSe/l urine were detected in some places of south regions of the Czech Republic. Even when nutrition of human population is affected to a great deal by the exchange of both raw materials for food industry and provisions, some similarities in the ratio of selenium indexes between regions have been found for cattle and human population. This work was partly supported by grants IGA MZ. R No. 3417-3 and GA. R No. 311/96/1609.

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A COMPARATIVE STUDY OF BLOOD ANTIOXIDANT PARAMETERS IN TWO PORTUGUESE URBAN POPULATIONS A. M. Viegas-Crespo1, M. C. Santos1, M. L. Pavão2, P. A. Lopes1, and J. Nève3 1

Fac. Sciences, University Lisbon, Campo Grande 1700 Lisbon, Portugal University Azores 9500 Ponta Delgada, Portugal 3 Free University Brussells B-1050 Brussells, Belgium

This work forms part of a Praxis XXI project intitled “Blood parameters associated with antioxidant function in human populations from Portuguese regions”. The aim of the study is to evaluate serum concentrations of several trace elements (Se, Cu, Zn, and Mn) and the activities of enzymes (superoxide dismutase and glutathione peroxidase) associated with antioxidant defenses, as well as serum peroxidation indices and the serum lipid profile. These parameters will be analysed taking into account the age, sex and life habits in healthy groups from each population. Groups with cronic diseases, such as hiperlipidemic and diabetic ones, will be studied as well. The target populations will be from Lisbon (Mainland) and from Ponta Delgada (Azores Archipelago) and aged 20 to 60 years. These urban populations will be compared with those from several rural and fishing regions, which have been studied before. Furthermore an intra and interpopulational comparative study including all the groups will be carried out. Preliminary results have been obtained before, for both populations, concerning serum selenium concentrations (mean ± SD) using atomic absortion spectrometric procedures: (Lisbon—Mainland); (Ponta Delgada—Azores Archipelago). 479

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It can be observed that men have higher levels of the element than women for both populations (P < 0.01), however these levels are in the same range as values obtained in other European countries (1). REFERENCE 1. Robberecht, H. and Deelstra, H., 1994, J. Trace Elements Electrol. Health Dis., 8, 129.

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COMPARISON OF TRACE ELEMENTS AND MACRONUTRIENTS IN BREAST MILK OF WOMEN FROM EIGHT DIFFERENT GEOGRAPHICAL LOCATIONS

G. M. Radzanowski, J. Jackson, K. Pramuk, and S. M. Kaup Wyeth Nutritionals International Radnor, Pennsylvania 19087 USA

An adequate supply of trace elements and macronutrients is essential for the normal growth and development of the newborn infant. Human milk is believed to provide all of the nutrients necessary for optimal infant growth. Historically, the trace element and macromineral composition of human milk has been invaluable for defining the mineral requirements of newborn infants. Breast milk composition, however, is variable. Human milk differs among women and across stages of lactation. Moreover, the composition of human milk is influenced by such factors as maternal nutrition and maternal age. The purpose of this study was to determine the normal range of trace elements and macrominerals found in breast milk among women of different geographical locations. Samples were collected from over 375 nonsmoking women (age 18–40 years, exclusively breastfeeding healthy full term infants 1–12 months in age) in eight countries around the world: Australia, Canada, Chile, China, Japan, Mexico, Philippines, and United Kingdom. The concentration of calcium (Ca), phosphorus (P), magnesium (Mg), sodium (Na), potassium (K), copper (Cu), iron (Fe), and zinc (Zn) were determined by inductively coupled plasma atomic emission spectrometry. Selenium (Se) analysis of the milk was conducted by graphite furnace atomic absorption spectroscopy. Regional differences in the trace element and macromineral content of human milk were observed. Human milk from Philippine women contained twice as much Se as human milk from women in China. Interestingly, Ca and P levels were higher in milk from Mexico and China than milk from Canada and Chile. Also, human milk from Chile contained twice as much Cu as did milk from Canada. Stage of lactation influenced human milk mineral composition. In general, milk levels of Cu, Fe and Zn decreased as the stage of lactation increased. Understanding these variations in the mineral composition of human milk is important in addressing the nutrient needs of the human neonate.

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SELENIUM CONCENTRATION IN HUMAN MILK AND THE DAILY SE INTAKE BY BREAST-FED INFANTS IN THE WESTERN PART OF POLAND

B. A. Zachara and A. Pilecki Department of Biochemistry The Ludwik Rydygier Medical University

24 Kar.owicza Str., 85-092 Bydgoszcz Poland

Interest in the importance of selenium (Se) in human milk during lactation has increased in recent years. It has been established that Se is specially essential for infants who require this micronutrient for normal growth and development. The aim of our study was to determine the Se concentration in human milk in lactating women in the Western part of Poland and to calculate the daily Se intake of breast-fed infants. The study group comprised 144 lactating women. Most of them were living in cities with over 50,000 residents in the Western part of Poland. The mothers were healthy and had uncomplicated pregnancies and deliveries. All belonged to the low or middle income class. Milk samples were collected in the period between 14 and 28 days after delivery. The daily Se intake by breast-fed infants was calculated on the basis of Se concentration in milk and milk volume consumed in the first month of life. Se concentration was measured by the fluorometric method of Watkinson with 2,3-diaminonaphthalene as a complexing reagent. The mean Se concentration in the milk of lactating women is 10.71 ± 2.87 ng/ml. No significant differences were observed between the provinces; however, the highest values were obtained in the seaside resorts (11.09 ± 2.58 ng/ml) and in some towns of Lower Silesia (11.38 ± 3.13 ng/ml). In the cities of the middle-west part of Poland, the Se level in milk was not significantly lower (10.04 ± 3.03 ng/ml) than in the above-mentioned regions. The calculated mean daily Se intake by breast-fed infants is 6.43 and ranged from 5.64 to 7.39 mg/day. The mean daily Se intake in the region studied by us is lower than the recommended value (10mg/day) established by the US National Research Council. Similar, or even lower values, were calculated in New Zealand (5 mg Se/day), Belgium (7.2), Spain (4.5) and some other countries, where the Se intake was below 6 mg/day. On 482

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the other hand, in Japan, Germany and Scotland, the daily Se intake by breast-fed infants exceeded 15 mg/day. Our results show that the Se concentration in milk is uniform and the daily Se intake of breast-fed infants living in the west part of Poland is lower than the recommended dose of this element. The reason for this fact seems to be the low Se content in the soil and, consequently, in the foodstuff in this region. Breast milk is the only source of Se during the first few weeks of life. The insufficient level of Se in mother’s milk may be the cause of some Se-deficient diseases in infants. This study was supported by the State Committee for Scientific Research (KBN), No. 4 P05E-070 13.

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EFFECT ON BIOCHEMICAL MARKERS OF TWO-YEARS SUPPLEMENTATION WITH ANTIOXIDANTS IN THE SU.VI.MAX STUDY Interest of Nutritional Doses

Serge Hercberg1, Henri Faure2, Paul Preziosi1, Josiane Arnaud2, Marie-Jeanne Richard2, Pilar Galan1, Denis Malvy3, Serge Briancon4, Anne-Marie Roussel2, and Alain Favier2 1

Institut Scientifique et Technique de la Nutrition et l’Alimentation (Conservatoire National des Arts et Métiers) 2 rue Conté, F-75003 Paris France 2 Laboratoire de Biochimie CHU de Grenoble, France 3 INSERM U330 Université Victor Ségalen Bordeaux2, France 4 Ecole de Santé Publique Faculté de Médecine, Nancy France

The “Supplementation en Vitamines et Minéraux AntioXydants” (SU.VI.MAX) Study is a randomized double-blind, placebo-controlled, primary-prevention trial which started in 1994 in France. This epidemiologic study is designed to test the efficacy of a daily supplementation with antioxidant vitamins (vitamin C, 120 mg; vitamin E, 30 mg; and beta-carotene, 6mg) and minerals (selenium, 100 mg; and zinc, 20 mg) at nutritional doses, in reducing the main causes of premature death (cancers and cardiovascular diseases). The antioxidant agents tested are at a non-pharmacologic level that may be reached by dietary intake of natural sources of these micronutrients and/or eventual enriched foods. 12,735 eligible subjects (women aged 35 to 60, and men aged 45 to 60) were included in 1994 and will be followed up for eight years. The effect of supplementation by antioxidant micronutrients during a 2 years period, on serum concentrations of biochemical markers assessing vitamin and trace 484

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element status was significant for all of the studied nutrients, vitamin C, E, beta-carotene, zinc and selenium. After 2 years of supplementation, biochemical indicators of vitamin and trace element status reach reasonable level without reaching concentrations as high as those observed in intervention studies, which tested relatively high doses of antioxidants, and ended up with higher risk of pathology. Our preliminary data thus indicate that supplementation (2 years) with moderate doses of antioxidant vitamins and trace element, in presumaly healthy subjects, clearly though moderately vitamin and mineral status, with blood concentrations reaching concentrations consistent with a positive effect.

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IODINE NUTRITION OF FRENCH ADULTS ISSUED FROM THE SU.VI.MAX COHORT Pierre Valeix1, Marjorie Zarebska1, Paul Preziosi1, Bruno Pelletier2, and Pilar Galan1 1

Institut Scientifique et Technique de la Nutrition et l’Alimentation (Conservatoire National des Arts et Métiers) 2 rue Conté, F-75003 Paris France 2 Lipha-Santé, 37 rue Saint Romain F-69379 Lyon, France

The status of iodine nutrition was evaluted in France on a sample of 12,014 persons (4,860 men and 7,154 women) issued from the SU.VI.MAX cohort. The media urinary iodine (UI) concentration for the entire male population was 8.5. g/100 ml, 8.2.g/100 ml for the female sample. The percentage of subjects with UI > 10. g/100 ml was lower among the 55- to 60-yr-old age group; 34.8% of men and 34.4% of women, respectively, were above the critical threshold 10.g/100 ml used as an indicatio of an iodine-replete population). Conversely, with increased age, the proportion of the population with UI concentrations below 5 .g/100 ml increased from 14.6 to 16.8% among males, and from 15.5 to 22.8% among females (P = 0.001). Higher proportions of women aged 45 to 60 fell into the iodine deficiency range (UI < 5. g/100 ml) when compared to men of those ages (21.9% vs 15.9%, P = 0.001); 17.8% of women of child-bearing age (35–45) has UI below 5. g/100 ml. Median UI displayed wide significant regional variations. Median UI concentrations were higher among residents living in the northwest and west of France than among those living in the center and east, independently of age and sex. Data clearly identified a west to east gradient in the population risk for iodine deficiency for both sexes when focusing attentin on the groups excreting UI concentrations of less than 5.0. g/100 ml. In all regions, the percentage of subjects with UI concentrations < 5.0.g/100 ml was always greater among women aged 45 to 60 than among men of those ages. The central region of France (Auvergne, Limousin) and the northeastern areas (Alsace, Lorraine, Champagne-Ardennes, Franche-Comté) had the highest percentages with 32.9% and 24.4% of women, and 22.2% and 22.5% for men, respectively, excreting less than 5.0. g/100 ml. Theage-related decrease in iodine intake, although modest in degree, might be the result of food-patterns changes paralleling the observed physiological reduction in energy 486

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intake, but could also be due to the present public awareness of the need for a volontary reductio on added salt to control hypertension. Regional variartions in median UI concentrations are in part a reflection of fluctuations in the total iodine content of foodstuffs and pasture grasses, which depends on the quantities supplied to soil by the weathering of primary bedrocks, by the atmosphere in the form of rain or aerosols, and more recently, by agricultural practices and food processing. The results definitively underlined the risk for the French adult population of being exposed to mild iodine deficiency, although iodized household salt was introduced on a voluntary basis from 1952 onwards (10–15 mg NaI/kg salt). Surveillance of iodine nutrition in pregnant women to prevent the occasional risk of intellectual impairment should be emphasized, and a nationwide effort to promote iodized salt and to provide women of child-bearing age with advice on dietary sources of iodine should be implemented. In addition, efforts to improve iodine nutrition throught generalized iodine prophylaxis would reduce radiation-induced damage to the thyroid in case of nuclear accident. This study was supported in part by Lipha-Santé, a subsidiary of the Merck Group, F-69379, Lyon, Cedex 08, France.

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DIETARY IRON INTAKE AND IRON STATUS OF FRENCH ADULTS PARTICIPATING IN THE SU.VI.MAX COHORT Pilar Galan1, Paul Preziosi1, Bernadette Fieux1, Marjorie Zarebska1, Serge Briancon2, Denis Malvy3, Anne-Marie Roussel4, Alain Favier4, and Serge Hercberg1 1

Institut Scientifique et Technique de la Nutrition et l’Alimentation (Conservatoire National des Arts et Métiers) 2 rue Conté, F-75003 Paris France 2 Ecole de Santé Publique Faculté de Médecine Nancy, France 3 INSERM U330, Université Victor Ségalen Bordeaux 2, France 4 Laboratoire de Biochimie CHU de Grenoble, France

Dietary iron intakes and iron status were assessed in a national sample of adults living in France and participation in the SU.VI.Max cohort. Biochemical data (serum ferritin and hemoglobin concentrations) were obtained for 6.648 women 35–60 years old and for 3.283 men 45–60 years old. Assessment of iron dietary intakes was realized on a sample of 3.111 women and 2.337 men who reported six 24-h dietary recors during a one year period; 22.7% of menstruating women and 5.3% of post-menopausal women presented a total depletion of iron stores (serum ferritin <15 . g/l). Iron-deficient anemias were found in, respectively, 4.4% and less than 1% of these women. Three-quarters of the anemias were related to iron deficiency in menstruating women. In men, iron depletion and iron deficiency anemia were very rare. Post-menopausal women had much higher serum ferritin levels than menstruating women. In menstruating women, those using intra uterine devices had significantly lower srum ferritin levels than those without contraception, and much lower than those using oral contraception. The frequency of iron depletion reached 28.1% in women using intra uterine devices, but only 13.6% in those using oral contraceptives. 488

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Mean iron intake was 16.7 ± 5.7 mg/d in men and 12.3 ± 3.4 mg/d in women. Heme iron represented respectively 11.0 and 10.4% of iron intake. Ninety-three percent of menstruating women had dietary iron intakes lower than RDA; 52.6% consumed less than 2/3 of these RDA. In post-menopausal women and in mn, respectively, 27.7% and 3.6% had dietary intakes lower than RDA. Energy consumption was positively correlated with iron intake There was a positive correlation (adjusted for menopausal status, oral contraception and intrauterine practices) between serum ferritin and meat (r = 0.18; p < 0.001) and fish intake (r = 0.05; p < 0.02) and a negative correlation between serum ferritin and dairy products (r = –0.10, p < 0.001). Hemoglobin was also positively correlated with meat intake (r = 0.06, p < 0.002). After adjustment for menauposal status and contraceptive habits, and using the multiple linear regression models, the relationship between meat and fish consumption and serum ferritin remained positively significant. Serum ferritin was negatively correlated with dairy products. There was also a relationship between hemoglobin and meat intake. Serum ferritin was positively correlated with total dietary intake of iron (r = 0.09; percentage of heme iron and total intake of animal proteins It was negatively correlated with calcium and fiber intales Hemoglobin was positively correlated with total iron intake (r = 0.05; p < 0.05) and with animal proteins (r = 0.04; p < 0.05). Multiple linear regression, after adjustments for reproductive status, confirmed these correlations. Coffee, tea and vitamin C intake, known to influence non-heme iron absorption, were not significantly correlated with serum ferritin or hemoglobin after appropriate adjustments. This work is supported by the EPIFER Group composed by several firms: Robapharm, Kellogg’s, Candia, Besnier, Innothera, Behring. This work has also received a grant from VOLVIC.

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INSULIN ENHANCING EFFECTS OF VANADIUM

John H. McNeill Faculty of Pharmaceutical Sciences The University of British Columbia] Vancouver, B. C. Canada V6T 1Z3

1. INTRODUCTION Vanadium is a group V transitional element which is widely distributed in nature, in soil, water, air, plants, and animals. It is the twenty-first most abundant element in the earthís crust; the average content (135ppm) is 0.014% and is close to that of zinc. In sea water, it is much less abundant (in the range of 2–30 ppb) but it is found in marine organisms, especially in ascidians (sea squirts) which concentrate vanadium to levels >1 g/kg dry weight. Drinking water contains vanadium levels of 0–6 ppb, whereas plants and animals have levels of 0–5 ppm (Waters, 1977). Due to its wide use in industry as a catalyst, atmospheric levels of vanadium are increasing (Hudson, 1964). In humans, the total body pool of vanadium is estimated to be around 100–200 g (Byrne and Kosta, 1978). Since it comprises less than 0.01% body weight, vanadium is classified as a trace mineral. The approximate tissue vanadium concentrations in humans are (ng/g): liver, 5–19; kidney, 3–7; bone and teeth,
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in vanadium as a cholesterol lowering agent in the 1950ís (Curran et al., 1959) but the major finding (Cantley et al., 1977) that vanadium could inhibit Na+K+ ATPase triggered a great deal of research (see Nechay et al., 1986). It became apparent that vanadium could inhibit a number of biologically important enzymes, particularly phosphatases. Many of these enzymes were involved in carbohydrate metabolism and a pattern of in vitro insulin-like activity of vanadium compounds began to emerge in the late 1970ís and early 1980ís (seeTsiani and Fantus, 1997; Verma et al., 1998).

2. IN VITRO EFFECTS OF VANADIUM A listing of the in vitro effects of vanadium on enzymes and processes involved in carbohydrate and lipid metabolism has been published (Poucheret et al., 1998). Note that the list is representative and is not intended to be complete (see Tsiani and Fantus, 1997). Note also that the effects of vanadium mimic those of insulin. Vanadium, unlike insulin, apparently does not stimulate protein synthesis in skeletal muscle or adipocytes (Clark et al., 1985; Fantus et al., 1989).

3. IN VIVO EFFECTS OF VANADIUM Since vanadium has insulin mimetic effects in vivo we reasoned that administering it to diabetic rats should result in insulin-like effects provided that a suitable dosage schedule was used. This proved to be correct and in 1985 (Heyliger et al.) we were able to show that sodium vanadate, added to the drinking water, reduced the elevated blood glucose of streptozotocin (STZ) diabetic rats to control levels. Blood glucose was not reduced in control rats but insulin values were decreased indicating that vanadate could mimic or enhance the effects of endogenous insulin. Diabetes-induced cardiomyopathy was prevented by the vanadium treatment. These results were confirmed by Meyerovitch et al., (1987) who also demonstrated that vanadium increased glucose uptake into muscle and liver in vivo. Brichard et al., (1988) first described a dose-response relationship between vanadium and its glucose lowering effects. Because vanadium in its vanadyl form was reported to be less toxic than the vanadate form a large number of studies have been carried out in vivo using vanadyl sulfate (Poucheret et al., 1998; Tsiani and Fantus, 1997). Vanadyl treatment normalized plasma glucose, triglycerides, cholesterol, creatinine and thyroid hormone levels in STZ-rats and also prevented cataracts and cardiomyopathy from occurring (Ramanadham et al., 1989a; 1989b). Other in vivo effects of vanadium in STZ diabeteshave been discussed (Poucheret et al., 1998). Vanadium has also been shown to have insulin-like effects in other models of diabetes (or pre-diabetes) including the Zucker fatty rat, Zucker diabetic fatty rat, ob/ob mouse and the Biobreeding (BB) rat (see Poucheret et al., 1998; Verma et al., 1998). In obese models of diabetes which resemble type 2 diabetes, vanadium treatments decrease elevated glucose, lipids and insulin levels and have positive effects on the oral glucose tolerance test. Those effects can occur without affecting body weight but body weight is decreased at higher doses (Yuen et al., 1996). The BB rat is a genetic model of type 1 diabetes. Vanadium will not produce an effect unless exogenous insulin is also provided indicating that in vivo the effects of vanadium are insulin-enhancing rather than insulin mimetic. Vanadium treatment markedly reduces the insulin required for euglycemia in the BB rat (Battell et al., 1992).

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The inorganic forms of vanadium are not well absorbed and attempts have been made to improve the efficacy of vanadium by synthesizing organic vanadium compounds which are better absorbed and thus more potent. There is now an extensive literature on this topic which is beyond the scope of this article (Poucheret et al., 1998; Tsiani and Fantus, 1997; Orvig et al., 1995; Thompson et al., 1998; Sakurai et al., 1998). We have extensive experience with one of these compounds, bis(maltolato)oxovanadium(iv) (BMOV) (McNeill et al., 1992). BMOV is better absorbed and 2–3 times more potent than vanadyl sulfate and is less toxic. Other organic vanadium compounds (see reviews) also exhibit these qualities.

4. MECHANISM OF ACTION It is generally believed that the principle mechanism of action of vanadium is to inhibit tyrosine phosphatases in the insulin signalling pathways thus preventing the inactivation by dephosphorylation of insulin stimulated protein kinases (see Tsiani and Fantus, 1997).

5. TOXICITY There are several reports in the literature regarding the oral toxicity of vanadium. Domingo (Domingo et al., 1995) has taken a very negative stance with regard to mammalian toxicity. They cite animal studies in which vanadium has produced death, tissue damage, liver and kidney enzyme changes reproductive and developmental toxicity and hematological and biochemical changes. He regards tissue retention as toxicity. Others have been less negative in drawing conclusions from their own work and from reviewing the literature (Thompson et al., 1998; Tsiani and Fantus, 1997; Leonard and Gerber, 1998). All chemicals can be toxic but in doses that lowered elevated blood glucose vanadium did not produce toxicity in animals, control or diabetic, that had received vanadyl sulfate for one year. No tissue damage, hematological, histological or biochemical changes were noted. It is now recognized that vanadium can decrease fluid intake and when this occurs in STZ animals the rats will die of dehydration. If vanadium is withdrawn, the animals rehydrated and vanadium started again at a lower dose, which can be gradually increased, no toxicity occurs (Thompson et al., 1998). Tissue retention of vanadium cannot be regarded as toxicity unless some adverse cellular or biochemical event occurs. The evidence does not support the thesis that this in fact happens. Leonard and Gerber (1998) conclude that vanadium is a weak mutagen and is not carcinogenic. Vanadium at high doses can harm the fetus mainly by damaging the mother. They further state that because of poor transfer of vanadium into the fetus malformations occur only at high doses. The use of any drug in pregnancy must be done only after weighing the risk/benefit ratio. Diarrhea and intestinal cramping is a side effect of vanadium in both animals and man (Thompson et al., 1998).

6. EFFECTS IN HUMANS There are now several reports of the use of both vanadate and vanadyl in diabetic and non-diabetic humans. The studies have involved only small numbers of patients, mostly type 2 diabetics, and for short periods of time up to 6 weeks. The area has been

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recently reviewed by Goldfine et al. (1998). The results are promising but not spectacular and show that vanadium treatment, up to 100mg V (given as vanadyl sulfate) per day, can improve insulin sensitivity, decrease fasting HbAlc, plasma glucose and hepatic glucose output. Glycogen synthesis was increased but glucose oxidation was not. The results were positive but not totally consistent among the various studies. Diarrhea and gastrointestinal disturbances were noted in all studies but patients in general tolerated the vanadium well. Type 1 diabetics showed a 14% decrease in insulin requirement in one study (Goldfine et al., 1998) but no effect was seen in another (Aharon et al, 1998). Vanadyl sulfate had no effect in obese non-diabetic subjects (Halberstam et al., 1996).

7. THERAPEUTIC POTENTIAL Vanadium compounds do appear to have therapeutic potential in diabetes, both type 1 and type 2. The latter has shown the best response to vanadium but, in type 1, it may at least be possible to decrease the amount of insulin required. Since vanadium appears to affect carbohydrate and lipid metabolism by enhancing the effects of insulin by a unique mechanism, vanadium compounds may provide an additional means to treat the disease either alone or in combination with other drugs. Potency is a problem with the naturally occurring vanadium compounds. However the synthetic organic vanadium complexes may provide a solution to this problem (Srivastiva, 1995).

ACKNOWLEDGMENTS Work in our laboratory was supported by NSERC and MRC (Canada), Canadian Diabetes Association, Heart and Stroke Foundation of B.C. and Yukon. My thanks to my colleagues, graduate students, postdoctoral fellows and technicians who have contributed to these studies.

REFERENCES Aguis, L. and Vaartjes, W.J., 1982, The effects of orthovanadate on fatty acid synthesis in isolated rat hepatocytes, Biochem. J. 202:791–794. Aharon, Y., Mevorach, M., and Shamoon, H., 1998, Vanadyl sulfate does not enhance insulin action in type 1 diabetes, Diabetes Care 21:2194. Battell, M.L., Yuen, V.G., and McNeill, J.H., 1992, Treatment of BB rats with vanadyl sulfate, Pharmacol. Commun. 1:291–301. Brichard, S.M., Okitolonda, W., and Henquin, J.C., 1988, Long-term improvement of glucose homeostasis by vanadate treatment in diabetic rats, Endocrinology 123:2048–2053. Bruck, R., Prigozin, H., Krepel, Z., Rotenberg, P., Shechter, Y., and Bar-Meir, S., 1991, Vanadate inhibits glucose output from isolated perfused rat liver, Hepatology 14:540–544. Byrne, A.R. and Kosta, L., 1978, Vanadium in foods and in human body fluids and tissues, Sci. Total Environ. 10:17–30. Byrne, A.R. and Kosta, L., 1979, On the vanadium and tin contents of diet and human blood, Sci. Total Environ. 13:87–90. Cantley, L.C.Jr., Josephson, L., Warner, R., Yanagisawa, M., Lechend, C., and Guidotti, G., 1977, Vanadate is a potent (Na,K)-ATPase inhibitor found in ATP derived from muscle, J. Biol. Chem. 252:7421– 7423.

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Chasteen, N. Dennis (Ed), 1990, Vanadium in Biological Systems: physiology and biochemistry, Kluwer Academic Publishers, Boston, 225pp. Clark, A.S., Fagan, J.M., and Mitch, W.E., 1985, Selectivity of the insulin-like actions of vanadate on glucose and protein metabolism in skeletal muscle, Biochem. J. 232:273–276. Clausen, T., Andersen, T.L., Sturup-Johansen, M., and Perkova, O., 1981, The relationship between the transport of glucose and cations across cell membranes in isolated tissues. XI. The effect of vanadate on 45Ca-efflux and sugar transport in adipose tissue and skeletal muscle, Biochim. Biophys. Acta 646:261–267. Cornelis, R., Versieck, J., Mees, L., Hoste, J., and Barbier, F., 1981, The ultra trace element vanadium in human serum, Biol. Trace Elem. Res. 3:257–263. Curran, G.L., Azarnoff, D.L., and Bolinger, R.E., 1959, Effect of cholesterol synthesis inhibition in normocholesterolemic young men, J. Clin. Invest. 38:1251–1261. Degani, H., Gochin, M., Karlish, S.J.D., and Shechter, Y., 1981, Electron paramagnetic resonance studies and insulin-like effects of vanadium in rat adipocytes, Biochemistry 20:5795–5799. Domingo, J.L., Gomez, M., Sanchez, D.J., Llobet, J.M., and Keen, C.L., 1995, Toxicology of vanadium compounds in diabetic rats: the action of chelating agents on vanadium accumulation, Mol. Cell. Biochem. 153:233–240. Duckworth, W.C., Solomon, S.S., Liepnieks, J., Hamel, F.G., Hand, S., and Peavy, D.E., 1988, Insulin-like effects of vanadate in isolated rat adipocytes, Endocrinology 122:2285–2289. Fantus, I.G., Kadota, S., Deragon, G., Foster, B., and Posner, B.I., 1989, Pervanadate [Peroxide(s) of vandate] mimics insulin action in rat adipocytes via activation of the insulin receptor tyrosine kinase, Biochemistry 28:8864–8871. Goldfine, A.B., Willsky, G., and Kahn, C.R., 1998, Vanadium salts in the treatment of human diabetes In: Vanadium Compounds: chemistry, biochemistry and therapeutic applications, Amer. Chem. Soc. p. 353–368. Gomez-Foix, a.M., Rodriguez-Gil, J.E., Fillat, C., Guinovart, J.J., and Bosch, F., 1988, Vanadate raises fructose 2,6-bisphosphate concentrations and activates glycolysis in rat hepatocytes, Biochem. J. 255:507–512. Guzman, M. and Castro, J., 1990, Simultaneous stimulation of fatty acid synthesis and oxidation in rat hepatocytes by vanadate, Arch. Biochem. Biophys. 283:90–95. Halberstram, M., Cohen, N., Schlimovich, P., Rossetti, L., and Shamoon, H., 1996, Oral vanadyl sulfate improves insulin sensitivity in NIDDM but not in obese nondiabetic subjects, Diabetes 45:659–666. Heyliger, C.E., Tahiliani, A.G., and McNeill, J.H., 1985, Effect of vanadate on elevated blood glucose and depressed cardiac performance of diabetic rats, Science 227:1474–1477. Hudson, T.O.F., 1964, Vanadium: Toxicology and biological significance, Elsevier Publishing Co., Amsterdam. Jackson, T.K., Salhanick, A.I., Sparks, J.D., Sparks, C.E., Bolognino, M., and Amatruda, J.M., 1988, Insulinmimetic effects of vanadate in primary cultures of rat hepatocytes, Diabetes 37:1234–40. Leonard, A. and Gerber, G.B., 1998, Mutangenicity, carcinogenicity and teratogenicity of vanadium, In: Vanadium in the Environment, Part Two: Health effects, John Wiley & Sons, New York, p. 39–53. Lyonnet, Martz, and Martin, 1899, Líemploi therapeutique des derives du vanadium, La Presse Medicale, April 22, p. 191–192. McNeill, J.H., Yuen, V.G., Hoveyda, H.R., and Orvig, C., 1992, Bis(maltolato)oxovanadium(iv) is a potent insulin mimic, J. Med. Chem. 325:1489–1491. Meyerovitch, J., Farfel, Z., Sack, J., and Shechter, Y, 1987, Oral administration of vanadate normalizes blood glucose levels in streptozotocin-treated rats, J. Biol. Chem. 262:6658–6662. Mountjoy, K.G. and Flier, J.S., 1990, Vanadate regulates glucose transporter (Glut-1) expression in NIH 3T3 mouse fibroblasts, Endocrinology 127:2025–2034. Nechay, B.R., Nanninga, L.B., Nechay, P.S., Post, R.L., Grantham, J.J., Macara, I.G., Kubena, L.F., Phillips, T.D., and Nielsen, F.H., 1986, Role of vanadium in biology, Feb. Proc. 45:123–132. Nriagu, J.O. (Ed), 1998, Vanadium in the Environment, Part Two: Health effects, John Wiley & Sons, New York, 401pp. Orvig, C., Thompson, K.H., Battell, M., and McNeill, J.H., 1995, Vanadium compounds as insulin mimics, In: Metal Ions in Biological Systems (H Sigel and A Sigel, Eds), Marcel Dekker, Inc, Basel, V. 31, Chapter 17:575–594. Paquet, M.R., Romanek, R.J., and Sargeant, R.J., 1992, Vanadate induces the recruitment of GLUT-4 glucose transporter to the plasma membrane of rat adipocytes, Mol. Cell. Biochem. 109:149–155. Poucheret, P., Verma, S., Grynpas, M.C., and McNeill, J.H., 1998, Vanadium and diabetes, Mol. Cell. Biochem. 188:73–80.

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Ramanadham, S., Brownsey, R.W., Cros, G.H., Mongold, J.J., and McNeill, J.H., 1989b, Sustained preventions of myocardial and metabolic abnormalities in diabetic rats following withdrawal from oral vanadyl treatment, Metabolism 38:1022-1028. Ramanadham, S., Mongold, J.J., Brownsey, R.W., Cros, G.H., and McNeill, J.H., 1989a, Oral vanadyl sulfate in treatment of diabetes mellitus in rats, Am. J. Physiol. 257:H904–H911. Sakurai, H., Fujii, K., Fujimoto, S., Fujisawa, Y., Takechi, K., and Yasui, H., 1998, Structure-activity relationship of insulin-mimetic vanadyl complexes with VO(N202) Coordination Mode, In: Vanadium Compounds: chemistry, biochemistry and therapeutic applications (Tracey, A.S. and Crans, D.C., Ed), Amer. Chem. Soc. p. 344–352. Shechter, Y., 1990, Insulin-mimetic effects of vanadate. Possible implications for future treatment of diabetes, Diabetes 39:1–5. Shechter, Y and Ron, A., 1986, Effect of depletion of phosphate and bicarbonate ions on insulin action in rat adipocytes, J. Biol. Chem. 261:14945–14950. Sigel, H. and Sigel, A. (Ed), 1995, Metal Ions in Biological Systems 31: Vanadium and its role in life, Marcel Dekker Inc, New York, 779pp. Simonoff, M., Llabador, Y., Peers, A.M., and Simonoff, G.N., 1984, Vanadium in human serum, as determined by neutron activation analysis, Clin. Chem. 30:1700–1703. Srivastiva, A.K., 1995, Potential use of vanadium compounds in the treatment of diabetes mellitus, Exp. Opin. Invest. Drugs 4(6):525–536. Srivastiva, A.K. and Chiasson, J.L. (Ed), 1995, Vanadium Compounds: Biochemical and therapeutic applications, Mol. Cell, Biochem. 153:1–244. Tamura, S., Brown, T.A., Dubler, R.E., and Larner, J., 1983, Insulin-like effect of vanadate on adipocyte glycogen synthase and on phosphorylation of 95,000 dalton subunit of insulin receptor, Biochem. Biophys. Res. Commun. 113:80–86. Thompson, K.H., Battell, M., and McNeill, J.H., 1998, Toxicology of vanadium in mammals, In: Vanadium in the Environment, John Wiley and Sons, New York Chap. 31, p. 21–38. Thompson, K.H., Yuen, V.G., McNeill, J.H., and Orvig, C., 1998, Chemical and pharmacological studies of a new class of antidiabetic vanadium complexes, In: Vanadium Compounds: chemistry, biochemistry and therapeutic applications (Tracey, A.S. and Crans, D.C., Ed), Amer. Chem. Soc. p. 329–343. Tolman, E.L., Barris, E., Burns, M., Pansisni, A., and Partridge, R., 1979, Effects of vanadium on glucose metabolism inn vitro, Life Sci. 25:1159–1164. Tracey, A.S. and Crans, D.C. (Ed), 1998, Vanadium Compounds: chemistry, biochemistry and therapeutic applications, Amer. Chem. Soc. 381pp. Tsiani, E. and Fantus, G., 1997, Vanadium Compounds: Biological actions and potential as pharmacological agents, Trends Endocrinol. Metab. 8:51–58. Ueki, H., Sera, M., and Tanaka, K., 1989, Stimulatory release of lipoprotein lipase activity from rat fat pads by vanadate, Arch. Biochem. Biophys. 272:18–24. Ueki, H., Sera, M., Tominaga, N., Morita, T., Sugino, E., and Hibino, S., 1992, Inhibition of increasing effect of vanadate on glycogen content and lipoprotein lipase activity in fat pads by 5-N,N-hexamethylene amiloride, Chem. Pharm. Bull. 40:542–543. Verma, S., Cam, M.C., and McNeill, J.H., 1998, Nutritional factors that can favorably influence the glucose/insulin system: vanadiumî. J. Amer. Col. Nutrition 17(1):11–18. Waters, M.D., 1977, Toxicology of vanadium, In: Advances in Modern Toxicology, Vol. 2, Toxicology of Trace Elements, (Goyer RA, Mehlman MA, Ed), Hemisphere, Washington, D.C. pp. 147–189. Yuen, V.G., Pederson, R.A., Dai, S., Orvig, C., and McNeill, J.H., 1996, Effects of low and high dose administration of bis(maltolato)oxovanadium(iv) on fa/fa Zucker rats, Can. J. Physiol. Pharmacol. 74: 1001–1009.

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IS ZINC ESSENTIAL TO MODULATE INSULIN SENSITIVITY?

Patrice Faure, Serge Halimi, Sophie Bouvard, Olivier Ramon, Karipne Lalane, Anne Marie Roussel, and Alain Emile Favier

Non insulin dependant diabetes mellitus (NIDDM) is a public health problem because of its growing prevalence in most of the countries and its subsequent complications. Prevalence of diabetes in developed countries is estimated to be 2–3%. Diabetic complications are a heterogeneous group of clinical disorders affecting microvascular (retina, kidney and peripheral nerves) and macrovascular system. Diabetic retinopathy is a leading cause of blindness in active population of developed countries. Epidemiological data suggest a strong relationship between the level of glycemia and the incidence and progression of the vascular diseases in individuals with insulin-dependent diabetes mellitus (IDDM) and NIDDM. The purpose of this review is to consider whether an essential trace element as zinc plays a role in the modulation of insulin activity. Actually, through the description of syndrome X, it is known that insulin resistance per se is correlated to vascular complications (Reaven et al., 1988). Therefore the importance of zinc in prediabetic states must be taken into account regarding its potential capacity to protect vascular tissues.

1. ZINC AND GLUCOSE HOMEOSTASIS Zinc has numerous potential targets to modulate insulin activity (Brandao-Neto J et al., 1990). For instance, zinc is a cofactor of enzymes implicated in glucose metabolism. One can advance the hypothesis that zinc may be associated with the energy homeostasis via its interaction to neoglucogenic and glycolysis enzymes(Cooper et al., 1996, Hedges et al., 1995). Therefore this trace element could have opposite effects on enzyme activity and play a role of activator or inhibitor depending on its level. A zinc dependant binding of phosphofructoskinase 1 to an inactivating protein has been previously demonstrated (Brand et al., 1991). In the muscle zinc cation plays a role of activator at a low concentration as well as inhibitor at a high concentration for phosphofructokinase

Address all correspondence: Dr P. Faure; LBSO Laboratoire de Biologie du Stress Oxydant, Hôpital A. Michallon, BP 217 38043 Grenoble; France Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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activity (Tamaki et al., 1983). The inhibitory effect of zinc on muscle phosphofructokinase activity, and therefore on glycolysis can be reversed by hystidine (Ikeda et al., 1980) which underlines that the effect of zinc is linked to the nutritional environment. Moreover zinc has been found in high concentrations in the endocrine pancreas as well as in the insulin molecule. Selective zinc deprivation in rats has demonstrated that this trace element also plays a role in insulin synthesis and activity. We previously shown that zinc depletion from insulin decreases its activity in rats (Faure et al., 1994). Zinc can also modulate insulin receptor transcription through zinc finger proteins like Spl, containing three zinc fingers necessary for its binding on GC boxes. Actually the gene coding for insulin receptor contains four Spl clusters. Deletion and analysis of the insulin receptor promoter shown that Spl binding sites are necessary to activate insulin receptor expression (Araki et al., 1991). On HeLa cells, we have recently shown that zinc modulates insulin receptor expression in the presence of glucose. On the other hand we shown that Spl is inactivated by glucose and protected by zinc preincubation.

2. ZINC AND CLINICAL OR EXPERIMENTAL NIDDM OR INSULIN RESISTANCE 2.1. NIDDM and Zinc Status: Is this the Appropriate Question? Numerous studies focused on the zinc status during NIDDM and could not give definitive contribution, except concerning the existence of a hyperzincuria (Chausmer, 1998). Plasma zinc level seems to depend on the duration of the disease and the associated complications. In these conditions it is not clear which comes first: the effects of insulin resistance on zinc status or the effects that follow the alterations in zinc homeostasis on insulin activity. Confounding many of the human and animal studies is the effect of the metabolic state on zinc distribution in the insulin sensitive tissues. In streptozotocine diabetes model, hyperglycemia/hyperinsulinemia is associated with reduced zinc in liver, kidney and muscle (Raz et al., 1988). In a model of prediabetic state (high fructose fed rats, HFD) we recently observed a decreased zinc status in muscle and liver. The improvement of insulin sensitivity was accompanied by a raise in liver zinc (Faure et al., 1999a). In human, acute metabolic disorders of diabetes are associated with an increased hypercincuria and a transitory hypozincemia. These modifications are correlated to the degree of hyperglycemia, but not glycosuria (Mc Nair et al., 1981). On the other hand Golik et al. (1993) noticed that the association of diabetes with congestive heart failure aggravates urinary zinc loss. In the case of insulin dependant diabetes mellitus, hypozincemia is observed during a ketotic state and corrected by insulin treatment, in parallel to an improvement of insulin sensitivity (Faure et al.,). Then we advance the hypothesis that zinc exchanges from liver, muscles and insulin sensitive tissues occur during diabetes leading to a relative tissue zinc reduction. These exchanges are important during hyperglycemia and all acute metabolic disorders.

2.2. The Effects of Zinc Supplementation or Zinc Deprivation on Insulin and Free Radical Activities In man few studies focused on the effects of long term zinc supplementation. In 1995 we supplemented Insulin dependent diabetic patients (Faure et al., 1995a), and observed a decrease of lipid peroxidation and an improved Se GsH-Px activity. This result

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concerning Se GsH-Px results from the protective effect of zinc against the glycation of this enzyme. More recently, Roussel et al. (in publication) observed a beneficial effect of zinc (30mg/day, 6 months) on lipid peroxidation in NIDD patients. In patients suffering from cirrhosis, Marchesini et al. (1998) noticed that a long-term zinc supplementation at a high level (200 mg, 3 times per day, 60 days) improved by 30% glucose disposal. The same zinc level in patients without liver disease would result in an opposite effect on glucose disposal. In the different studies it is interesting to observe that the effect of zinc was raised when complications were associated. In animals, Zinc deprivation decreases insulin activity (Faure et al., 1991) and nutritional zinc supplementation improves fasting insulinemia and glycemia in genetically obese (ob/ob) mice (Chen et al., 1998). On the other hand, zinc supplementation worsens body fat accumulation in both genetically obese mice and dietary-obese mice (Chen et al., 1997). The mechanism involved remains unclear and could be linked to the effect of zinc on thermogenesis and on food consumption. In high fructose fed rats, we recently observed that zinc 100ppm does not influence insulin activity, contrary to zinc 50ppm leading to an increased insulin activity. On the other hand, It is interesting to observe that zinc 50ppm improves antioxidant defense system more especially blood reduced glutathione. This result can be compared to the one obtained with a reference antidiabetic drug, metformin, as we observed using the same model (Faure et al., 1999b). Regarding zinc depletion, very low zinc (<3ppm) leads to a prediabetic state in rats but also to a dramatic metabolic disorder (Faure et al., 1991). Marginal zinc deprivation has various effects depending on the experimental model and the measured parameters. In any case, zinc depletion does not lead to a patent diabetic state but to an isolated insulin resistance, close to syndrome X. Therefore at the light of previous data, it seems important to evaluate the protective effect of zinc on glucose induced cytotoxicyty, including vascular cells.

3. ZINC AND CELL PROTECTION AGAINST GLUCOSE INDUCED CYTOTOXICITY 3.1. The Involvement of Free Radicals in Glucose Induced Cytotoxicity Free radicals in tissue are generated by both enzymatic and non enzymatic pathways leading to the formation of reactive compounds by reduction or oxidation. Reactive oxygen species in most cells can be involved in physiological functions, but they may also cause cellular injury by hydroperoxidation, ischemia/reperfusion, inflammation, ionizing radiation exposure, diabetes and aging (Halliwell and Gutteridge, 1986). On the other hand, a number of enzymatic and nonenzymatic cellular antioxidative defenses protect cells from the attack of oxygen free radicals. The most important and versatile protector is the glutathione (GSH) more especially in diabetes (Thormaley et al., 1996). GSH is present in most mammalian cells and plays an important role in many biological processes such as sulfur-containing amino acid metabolism, and participates to the cellular defense system against oxidative stress by reducing disulfide linked proteins and other cellular molecules, or by scavenging free radicals and reactive oxygen intermediates (Forman et al., 1996). There are strong evidences that glucose, under physiological conditions, is prone to oxidation and consequently generates hydrogen peroxide and reactive intermediates such hydroxyl-free radicals (Hunt et al., 1988). It has been suggested that oxidative stress plays an important role in tissue damage associated with diabetes and that peroxide formation

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is increased parallely to elevated glucose oxidation. In different studies, it has been suggested that variations in glucose concentrations are sufficient to induce cell death through a free radical-mediated mechanism, delay in various phase of the cell cycle of human endothelial cells, inhibition of endothelial cell replication, and to induce an oxidative stress in smooth muscle cells (Bouvard et al., 1999) 3.2. The Role of Zinc in Cell Protection

3.2.1. Zinc and Free Radical Activity. Zinc may be involved in regulation of cell numbers by its role in both proliferation and death by apoptosis. There have been several reports dealing with inhibitory and/or toxic effects of zinc in vitro in various kinds of cells, for example, HeLa cells (Borovansky and Riley, 1989) and fibroblasts (Richard et al., 1993). The majority of these reports suggest that in vitro becomes cytotoxic above (Borovansky and Riley, 1989). Zinc does not undergo single electron redox reactions and therefore does not participate in radical reactions directly; and since it binds to thiols it is regarded as a stabilizer rather than an inhibitor. Moreover, it has been suggested that zinc is protective against free radical injury (Bray et al., 1990). Using In vitro systems, zinc has been shown to have an antioxidant role through two mechanisms protections. The first is the protection of sulfhydryl groups against oxidation. This was shown primarily in studies on the enzyme levulinate dehydratase (Seehra et al., 1981). The second mechanism involves the prevention of °OH and production by transition metals. This probably occurs by competition between Zn and prooxidant metals (e.g., copper, iron) for binding sites (Girotti et al., 1985), resulting in a decrease of their ability to transfer electrons. 3.2.2. Experiments Using Zinc as a Cell Protector toward Glucose. We were interested to explore the sensivity of HeLa-Tat versus parental cell lines to glucose. The first effects of cytotoxicity were observed at 20 mM for HeLa-Tat and 30 mM for HeLa wild. At these concentrations, glucose induced 50% of cytotoxicity in both cell lines. HeLa-Tat cells were more sensitive to the cytotoxic activity of glucose than the parental cell line, and at weaker concentration. The only difference between the two cell lines being the presence of Tat. Actually, this protein is believed to induce a higher sensitivity towards high glucose concentrations. Zinc has been shown to have antioxidant properties, but has not previously been explored in glucose oxidant effects. In the presence of zinc, when HeLa wild and HeLa-Tat were incubated with high glucose concentrations, zinc reversed the effect of glucose. The viability was significantly increased in the both cell lines. So, zinc protected HeLa wild as well as HeLa-Tat against glucose cytotoxicity. Concerning the intracellular thiol groups in HeLa wild and in HeLa-Tat, zinc was able to protect cellular thiols groups content against a high glucose concentrations by binding to thiols. In parental cells at 30 mM glucose with zinc, the cellular thiols groups content was significantly increase compared to the cellular thiol groups at 30 mM glucose and become superimposable to the initial value (5mM glucose). In HeLa-Tat at 20 mM glucose with zinc, the cellular thiols groups content was significantly increased compared to the cellular thiol groups at 20 mM glucose but not become superimposable to the initial value (5mM glucose). It is to be noted for HeLa-Tat that at 30 mM glucose with zinc, the intracellular concentration of zinc is similar to control but that intracellular thiol groups are diminished. Interestingly, even with the same supplement concentration in zinc, HeLa-Tat presented lower intracellular zinc content compared to HeLa wild.

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Regarding smooth vascular muscle cells (SVMC), in a model of primary culture from rat aorta, we recently shown that zinc at a very low level is efficient to decrease by 40% the glucose induced cell proliferation. This confirms the capacity of zinc to protect vascular tissues from the deleterious effect of glucose. The zinc targets on vascular protection are numerous including fibrinolysis, platelet aggregation and lipid peroxidation (Faure et al., 1995b). Meanwhile the mechanisms involved are complex and certainly include the action of zinc on insulin activity. Actually it is now demonstrated that insulin resistance leads to vascular modifications as endothelial cell apoptosis and SVMC proliferation, resulting in functional vascular changes. Through the understanding of the direct role of insulin resistance in the development of vascular degenerative lesions in NIDDM, a new approach and a new utilization of zinc can be envisaged in the nextfuture. In particular it seems important to use zinc at a early stage of the disease to delay as much as possible the vascular cell modifications. More study are required, in particular during prediabetic state like syndrome X to experiment zinc as an insulin activity modulator. The interest of zinc as an antioxidant is confirmed, more especially to protect cell against the glucose induced oxidation.

REFERENCES Araki, E, Murakimi, T., Shirotani, T., Kanai, F., Shinohara, Y., Shimada, F., Mori, M., Shichiri, M., and Ebina, Y., 1991, A cluster of four Sp1 binding sites required for efficient expression of the human insulin receptor gene. J. Biol. Chem. 6:3944–3948. Borovansky, J. and Riley, P.A.C., 1989, cytotoxicity of zinc in vitro. Chem Biol Interact 69:279–291. Bouvard, S., Faure, P., Roucard, C, Favier, A., Halimi, S. Zinc protects HeLa Wild and HeLa-Tat cultured cell lines from the glucose induced cytotoxicity, Biochem J, In press. Brand, I.A., Heinickel, A., 1991, Key enzymes of carbohydrate metabolism as target of the 11.5 KDA Zn(2+)-binding protein (parathymosin), J Biol Chem, 266:20984–20989. Brandao-Neto, J., Veira, J.G.H., Shuhawa, T., Russo, E.M.K., Piesco, R.V., and Curi, P.R., 1988, Interrelation ships of zinc with glucose and insulin metabolism in humans, Biol Trace Elem Res, 24:73–81. Bray, T.M. and Bettger, W, 1990, The physiological role of zinc as antioxidant. Free Radic. Biol. Med. 8:281–291. Chausmer, A.B., 1998, Zinc, insulin and diabetes, J Am College Nutr, 17:109–115. Chen, M.D., Liou, S.J., Lin, P.Y., Yang, V.C., Alexander, P.S., and Lin, W.H., 1998, Effects of zinc supplementation on the plasma glucose level and insulin activity in genetically obese (ob/ob) mice, Biol Tr Elem Res, 61:303–311. Chen, M.D., Lin, P.Y., Chen, P.S., Cheng, V., and Lin, W.H., 1997, Zinc attenuation of GDP binding to brown adipocytes mitochondria in genetically obese (ob/ob) mice, Biol Tracel Elem Res, 57:139–145. Cooper S.J., Leonard, G.A., McSweeney, S.M., Thompson, A.W., Naismith, J.H., Qamar, S., Plater, A., Berry, A., and Hunter, W.N., 1996, The crystal structure of a class II fructose 1-6-biphosphate aldolase shows a nevel binuclear metal-binding active site embedded in a familiar fild. Structure. 4:1303–1315. Faure, P., Roussel, A.M., Martini, M., Favier, A., and Halimi, S., 1991, Insulin-sensitivity in zinc depleted rats: Assessment with the euglycemic hyperinsulinic clamp technique. Diab. Metab 17:325–331. Faure, P., Corticelli, P., Richard, M.J., Arnaud, J., Coudray, C., Halimi, S., Favier, A., and Roussel, A.M., 1993, Lipid peroxidation and trace element status in diabetic ketotic patients: Influence of Insulin therapy. Clinical Chemistry, 5:789–793. Faure, P., Lafond, J.L., Rossini, E., Halimi, S., Favier, A., and Blache, D., 1994, Evidence for the role of zinc in insulin protection against free radical attack: molecular and functional aspects. Biochem. Biophys Acta 1209:260–264. Faure, P., Benhamou, P.Y., Perard, A., Halimi, S., and Roussel, A., 1995a, Lipid peroxidation in insulin dependant diabetic patients with early retinal degenerative lesions: effects of an oral Zn supplementation, Eur J Clin Nutr, 49:282–288. Faure, P., Durand, P., Blache, D., Favier, A., and Roussel, A.M., 1995b, Effects of a long-term zinc deficient diet on rat fatty acid composition, platelet aggregation and arachidonic acid metabolism. Biometal, 8:80–85.

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Faure, P., Bouvard, S., Rossini, E., Favier, A., and Halimi, S., 1999a, High vitamin E amount leads to a modification of zinc and copper tissue distribution in high fructose-fed rats exhibiting an insulin resistance, Trace Elem Electrol, 16:26–32, Faure, P., Rossini, E., Wiernsperger, N., Richard, M.J., Favier, A., and Halimi, S., 1999b, An insulin Sensitizer Improves Both the Free Radical Defense System Potential and Insulin Sensitivity in High-Fructose Fed Rats. Diabetes. 48:353–357. Forman, H.J., Liu, R.M., and Shi, M.M., 1996, Glutathione synthesis in oxidative stress. In Biothiols in health and disease. Packer L, Cadenas E, Eds. NY, p 189–210. Girotti, A.W., Thomas, J.P., and Jordan, J.E., 1985, Inhibitory effect of zinc(II) on free radical lipid peroxidation in erythrocyte membranes. J Free Radic Biol Med, 1:395–401. Golik, A., Cohen, N., Ramot, Y., Maor, J., Moses, R., Weissgarten, J., Leonov, Y., and Modai, P., 1993, Type II diabetes mellitus, congestive heart failure and zinc metabolism, Biol Trace Elem Res, 39:171–175. Halliwell, B. and Gutteridge, J.M.C, 1986, Oxygen free radical in relation to biology and medicine. Biochem Biophys, 501:501–14. Hedges, D., Proft, M., and Eutian, K.D., 1995, Cat 8, a new zinc cluster-encoding gene necessary for derepression of gluconegenic enzymes in the yeast Sacharomices cerevisiae, Moll Cell Biol. 15:1915–1922. Hunt, J.V., Dean, R.T., and Wolff, P., 1988, Hydroxyl radical production and autoxidative glycation: glucose oxidations as the cause of protein damage in the experimental glycation model of diabetes mellitus and ageing. Biochem J, 256:205–212. Ikeda, T., Kimura, K., Morioka, S., and Tamaki, N., 1980, Inhibitory effects of Zn2+ on muscle glycolysis and their reversal by histidine. J Nutr Sci Vitaminol. 26:357–366. Marchesini, G., Biuganesi, E., Ronchi, M., Flamia, R., Thomaseth, K., and Pacini, G., 1998, Zinc supplementation improves glucose disposal in patients with cirrhosis, Metabolism, 47:792–798. Me Nair, P., Kiilerich, S., Christiansen, C., Christiansen, M., Madsbad, S., and Transbil, I., 1981, Hyperzincuria in insulin treated diabetes mellitus-its relation to glucose homeostasis and insulin administration, Clinica Chimica Acta, 112:343–348. Raz, I., Adler, J.H., and Havivi, E., 1988, Altered tissue content of trace metal in diabetic hyperinsulinemic sand rats, Diabetologia, 31:329–333. Reaven, G.M., 1988, Banting Lecture: Role of insulin resistance in human disease, Diabetes 37:1595–1607. Richard, M.J., Guiraud, P., Leccia, M.T., Beani, J.C., and Favier, A., 1993, Effect of zinc supplementation on resistance of cultured human skin fibroblasts toward oxidant stress. Biol Trace Elem Res, 37:187–199. Seehra, J.S., Gore, M.G., Chaudhry, A.G., and Jordan, 1981, PM5-Aminolevulinic acid dehydratase. The role of sulphydryl groups in 5-aminolevulinic acid dehydratase from bovine liver. Eur J Biochem, 114: 263–269. Tamlaki, N., Ikeda, T., and Funatsuka, A., 1983, Zinc as activating cation for muscle glycolysis. J Nutr Sci Vitaminol 29:655–662. Thornalley, P.J., McLellan, A.C., Lo T.W., Benn, J., and Sonksen, PH., 1996, Negative association between erythrocyte reduced glutathione concentration and diabetic complications. Clin Sci 5:575–582.

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ROLE OF CHROMIUM IN GLUCOSE INTOLERANCE, DIABETES, TOTAL PARENTERAL NUTRITION, AND BODY COMPOSITION Richard A. Anderson1, William Cefalu2, Khursheed N. Jeejeebhoy3, and Gilbert R. Kaats4 1

Nutrient Requirements and Functions Laboratory Beltsville Human Nutrition Research Center Beltsville, Maryland 2 University of Vermont College of Medicine Burlington, VT 3 University of Toronto St. Michael’s Hospital Toronto, Ontario, Canada, and 4 Health and Medical Research Foundation 4900 Broadway, San Antonio, Texas

Chromium has been shown within the past five years to be involved in the control of glucose intolerance, type 2 diabetes mellitus (type 2 DM), gestational diabetes, steroidinduced diabetes, and neuropathy. Carefully controlled studies also support the role of supplemental Cr in the regulation of body composition. This brief review will discuss recent developments in these areas.

CHROMIUM, GLUCOSE INTOLERANCE AND DIABETES Numerous studies have demonstrated that inefficiency in insulin action (e.g. insulin resistance) may precede the development of type 2 DM by many years. Although it is well

Address all correspondence to: Dr. Richard A. Anderson, USDA, ARS, BHNRC, NRFL; Bldg. 307, Rm. 224, BARC-East; Beltsville, MD 20705-2350; telephone 301-504-8091; fax 301-504-9062; E-mail [email protected] Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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established that nutritional intervention and exercise greatly improve insulin resistance, forming the cornerstone of prevention studies, the long term success of maintaining diet and exercise regimens in an effort to control weight is poor. In this regard, a clinical improvement in insulin resistance secondary to pharmacological or nutritional supplements is an attractive approach for human intervention trials. Chromium has been postulated as one such nutritional intervention based on evidence from both animal and human studies. Chromium supplementation has been shown to improve insulin action in nondiabetic obese subjects with a family history of type 2 DM (Cefalu et al., 1999). The study involved 29 subjects (14 men, 15 women) in a double-blind, randomized, placebocontrolled trial using Cr picolinate of Cr per day) or placebo for 8 months. Clinical and metabolic evaluations consisted of insulin sensitivity and glucose effectiveness, measurement of glucose tolerance and insulin response to an oral glucose tolerance test (75 g OGTT) and 24-hour glucose and insulin profiles. Anthropometric measures and magnetic resonance imaging (MRI) assessed abdominal fat distribution. Fasting plasma glucose and insulin levels and measures of glycemia (glycated hemoglobin and fructosamine) were also assessed. The Cr picolinate group showed a significant increase in insulin sensitivity at midpoint (p < 0.05) and at the end of the study (p < 0.005) compared with controls, which had no significant changes. No change in glucose effectiveness was seen in either group. There were no significant effects of Cr picolinate on body weight, abdominal fat distribution or body mass index. Improvements in insulin sensitivity without a change in body fat distribution suggest that Cr may alter insulin sensitivity independent of a change in weight or body fat percentage, thereby implying a direct effect on muscle insulin action. Definitive double-blind, placebo-controlled trials are currently being conducted to confirm this observation in patients with type 2 DM to evaluate the effects of Cr supplementation on insulin action and glycemic control. Supplemental Cr as Cr picolinate improved the blood glucose, insulin, cholesterol and hemoglobin A1C, in a dose dependent manner, in people with type 2 DM (Anderson et al., 1997). One thousand of Cr as Cr picolinate was shown to have larger beneficial effects than per day. Study design was double-blind, four months in duration and involved approximately 180 people. Hemoglobin A1c decreased from 8.5 ± 0.2% in the placebo group to 7.5 ± 0.2% in the group receiving of Cr per day to 6.6 ± 0.1% in the group receiving per day. A one year follow-up study involving more than 800 people with type 2 DM confirmed this study and demonstrated that supplemental Cr per day as Cr picolinate) improved blood glucose after one month or less and improvements continued for the remaining nine months (Cheng et al., 1999). The requirements for Cr are related to the degree of glucose intolerance with per day of supplemental Cr being adequate to improve glucose variables of people who are mildly glucose intolerant (Anderson et al., 1991). However, people with more overt impairments in glucose tolerance or diabetes usually require more than of Cr per day. Daily intake of of Cr per kg body weight was also more effective than in women with gestational diabetes (Jovanovic et al., 1999). People with diabetes usually need more than per day of Cr to display beneficial effects of supplemental Cr. Dietary and physical stresses have been shown to increase Cr losses which may be associated with the onset of chronic diseases such as type 2 DM and cardiovascular

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diseases. The stresses associated with steroid administration given in the treatment of diseases such as allergies, arthritis and other inflammatory conditions were shown to increase Cr losses (Ravina et al., 1999). To test if the increased losses of Cr could be related to the onset of steroid-induced diabetes, three patients with steroid-induced diabetes were supplemented with Cr of Cr as Cr picolinate three times per day). Supplemental Cr led to a reversal of the steroid-induced diabetes despite a 50% decrease in medication (Ravina et al., 1999). To confirm these observations, the effects of supplemental Cr on 50 patients with steroid-induced diabetes were determined. Blood glucose in 47 of 50 subjects with steroid-induced diabetes decreased from greater than 13.9mmol/L (250mg/dL) to less than 8.3mmol/L (150mg/dL) following supplementation of Cr for less than one week. In order to be admitted to the study, blood glucose had to be greater than 13.9mmol/L and be uncontrolled by conventional treatments. When Cr supplementation was initiated, hypoglycemic treatments were reduced by 50%. Once blood glucose was normalized, supplemental Cr was reduced to Elimination of the supplemental Cr resulted in a return to elevated levels of blood glucose.

CHROMIUM AND PARENTERAL NUTRITION In the seventies, a patient on long term total parenteral nutrition (TPN) developed glucose intolerance and neuropathy associated with Cr deficiency (Jeejeebhoy et al., 1977). This patient was the first convincing case of human Cr deficiency and established the nutritional role of Cr in humans. TPN is an interesting model, in which patients receive purified diets with the potential of causing deficiencies if an essential nutrient is either not given or given in insufficient quantities. Often because trace elements are found as contaminants in macronutrients e.g. zinc in protein, deficiencies may not occur even if the trace element is not added to the diet because of its presence as an unknown contaminant. This is not the case in patients receiving TPN because there is little contamination of the infused purified macronutrients. Previously, zinc and copper deficiencies were observed in patients on TPN. Therefore the occurrence of Cr deficiency is not unexpected in patients on TPN. Since the description of the first case (Jeejeebhoy et al., 1977), several other cases have been reported (Freund et al., 1979; Brown et al., 1986). Verhage et al., (1996) reported that a 40 year-old male with Crohn’s disease, short bowel syndrome and high output end-jejunostomy developed peripheral neuropathy, ataxia, postural tremor and muscle weakness following 6 months of parenteral nutrition and metronidazole therapy (total dose, 188 g). Nerve conduction studies revealed a sensory motor peripheral neuropathy of the axonal type. The neurological syndrome was initially attributed solely to metronidazole. Stress associated hyperglycemia and impaired intravenous glucose tolerance test (IVGTT) were demonstrable with a reduced fractional glucose clearance rate of 0.86% per minute (reference value, >1.36 ± 0.15% per minute). Although the serum Cr level was within the laboratory normal range, the unexplained glucose intolerance and the neurological syndrome raised the possibility of Cr deficiency. Dramatic symptom resolution was evident within 4 days of intravenous Cr repletion infused at a rate of of Cr per day as the chloride salt. IVGTT normalized during Cr supplementation. After Cr supplementation the fractional clearance of glucose rose to 2.37% per minute. Repeated nerve conduction studies, performed 3 weeks after the initial study and 8 days after initiation of Cr supplementation, were normal.

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SERUM OR PLASMA LEVELS DURING TPN In children receiving exclusive TPN for 28.4 months, the serum Cr levels were lower than controls. In the control children, the serum Cr levels averaged _ and the mean Cr in patients on TPN was (Jeejeebhoy et al., 1977). In contrast, Moukarzel et al. (1992) noted that children on parenteral nutrition had high Cr levels with a mean of as compared with a control mean concentration of They ascribed these high levels to contaminating Cr in TPN solutions. In a review by Anderson (1995), it was shown that the Cr content of the components of TPN were mainly high in blood products and protein hydrolysates. Therefore, depending on the use of these products, the Cr intake may vary widely. It is clear that patients on parenteral nutrition may develop a clinical syndrome of unexpected impaired glucose utilization and hyperglycemia with insulin resistance. In some patients, neuropathy or encephalopathy was demonstrated, and in all cases, Cr infusion resulted in clinical and metabolic responses. However, if the plasma Cr levels in previous studies are compared with currently accepted normal levels (about ), they were all elevated. In fact, the low levels reported in children by Dahlstrom et al. (1986) were almost three times the current control levels. Therefore, there is no relationship between “high” plasma levels and a syndrome of hyperglycemia and neuropathy responsive to Cr. Blood Cr levels are not in equilibrium with tissue levels or stores. The data suggest that the most reliable way to diagnose Cr deficiency is to determine if hyperglycemia and/or neuropathy respond to Cr. Raised plasma levels during TPN cannot be interpreted to indicate adequate Cr nutrition. Finally, these data indicate that further studies are required in patients on TPN to ascertain if tissue levels of Cr, perhaps in white cells, are indicative of the syndrome of Cr deficiency.

CHROMIUM AND BODY COMPOSITION Deposition of body fat appears to be regulated in part by insulin and it has been suggested that insulin resistance, or insulin deficiency, can affect this process and lead to greater deposition of excess body fat. Furthermore, since insulin also affects entry of glucose and amino acids into muscle cells, insulin resistance could also disrupt the maintenance or building of muscle tissue (Eckel, 1992). It has been suggested that supplemental Cr offers the potential of improving body composition by enhancing insulin utilization and thereby slowing down the deposition of excess body fat and facilitating the maintenance and/or addition of muscle tissue. Although there are contradictory findings in the scientific literature on the effects of Cr on body composition, studies are often plagued with methodological difficulties e.g. the use of sub-standard measures of body composition, failure to distinguish fluid depletion from depletion of muscle mass, small sample sizes, high drop-out or non completion rates, and the failure to control for differences in diet and physical activity. The effects of these methodological flaws become more pronounced and troublesome when attempting to measure changes that are typically small and take some time to detect. To reduce methodological shortcomings, we analyzed only studies where subjects consumed at least of Cr per day as Cr picolinate, in randomized, placebo controlled studies. Body composition was measured using hydrostatic or immersion densitometry or Dual Energy X-ray Absorptiometry. Data were adjusted to reflect changes occurring over 60-days and expressed as net change in fat free mass (FFM) due to Cr

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above that achieved in placebo groups. Data were also analyzed using an index of Body Composition Improvement (BCI) that is based on the assumption that losses of body fat and gains in FFM are positive treatment outcomes, whereas the opposite comprise negative outcomes. The BCI is the net result of combining scores of these outcomes. Two of the 19 groups in Fig. 1 (Groups 5 and 11) are graphs depicting adjustments to Groups 4 (Kaats et al., 1998) and 10 (Lukaski et al.,1996) to correct for differences in caloric intake (see Fig. 2). In group 4, although the treatment group’s fat loss compared with placebo was significant before and after the adjustment (p > 0.001), the adjustment for caloric intake more than quadrupled the magnitude of the fat loss. In Group 10, the adjustment resulted in changing a 0.1 kg change in fat mass in the placebo group to a 1.29kg fat loss in the group consuming Cr. Of the 17 groups, 14 treatment groups lost more fat and had greater body composition improvements than their corresponding placebo groups. In three instances, the placebo group lost more fat than the treatment group. A comparison between a combined treatment and combined placebo/control group revealed that the treatment group had significantly greater (P < 0.001) depletion of body fat and improvements in body composition (P < 0.001) than the combined placebo/control group. It is concluded that, although small, Cr picolinate can lead to significant improvements in lean-to-fat ratios and the greater the experimental control, the greater the likelihood of detecting positive changes in body composition with Cr supplementation.

SUMMARY In summary, supplemental Cr has been shown to have beneficial effects on people with glucose intolerance, diabetes, obesity and neuropathy. The majority of the well

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controlled studies involving Cr and body composition also show beneficial effects of Cr on lean body mass. However, these conditions are due to a number of other causes that may be unrelated to Cr, therefore Cr will only be of benefit to those whose maladies are related to suboptimal Cr status.

REFERENCES Anderson, R.A., 1995, Chromium and parenteral nutrition. Nutrition 11:83–86. Anderson, R.A., Cheng, N., Bryden, N.A., Polansky, M.M., Chi, J., and Feng, J., 1997, Elevated intakes of supplemental chromium improve glucose and insulin variables of people with type II diabetes. Diabetes 46:1786–1791. Anderson, R.A., Polansky, M.M., Bryden, N.A., and Canary, J.J., 1991, Supplemental-chromium effects on glucose, insulin, glucagon, and urinary chromium losses of chromium and zinc. Eur. J. Appl. Physiol. 63:146–150. Brown, R.O., Forloines-Lynn, S., Cross, R.E., and Helzer, W.D., 1986, Chromium deficiency after long-term total parenteral nutrition. Dig. Dis. Sci. 31:661–664. Cefalu, W.T., Bell-Farrow, A.D., Stigner, J., Wang, Z.Q., King, T., Morgan, T., and Terry, J.G., 1999, Effect of chromium picolinate on insulin sensitivity in vivo. J. Trace Elem. Exptl. Med. 12:71–84. Cheng, N.-Z., Xixing, Z., Shi, H., Wu, W., Chi, J., Cheng, J., and Anderson, R.A., 1999, Follow-up survey of people in China with type 2 diabetes mellitus consuming supplemental chromium. J. Trace Elem. Exptl. Med. 12:55–64. Dahlstrom, K.A., Ament, M.E., Medkin, M.G., and Meurling, S., 1986, Serum trace elements in children receiving long-term parenteral nutrition. J. Pediatr. 109:625–630. Eckel, R.H., 1992, Insulin resistance: an adaptation for weight maintenance. Lancet 340:1452–1453. Freund, H., Atamian, S., and Fischer, J.E., 1979, Chromium deficiency during total parenteral nutrition. JAMA 241:496–498. Jeejeebhoy, K.N., Chu, R.C., Marliss, E.B., Greenberg, G.R., and Bruce-Robertson, A., 1977, Chromium deficiency, glucose intolerance, and neuropathy reversed by chromium supplementation in a patient receiving long-term total parenteral nutrition. Am. J. Clin. Nutr. 30:531–538. Jovanovic, L., Gutierrez, M., and Peterson C.M., 1999, Chromium supplementation for women with gestational diabetes mellitus. J. Trace Elem. Exptl. Med. 12:91–98.

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Kaats, G.R., Blum, K., Pullin, D., Keith, S.C., and Wood, R.A., 1998, A randomized double-masked placebo controlled replication and extension of the effects of chromium picolinate supplementation on body composition. Curr. Ther. Res. 59:379–388. Lukaski, H.C., Bolonchuk, W.W., Siders, W.A., and Milne, D.B., 1996, Chromium supplementation and resistance training: Effects on body composition, strength and trace element status of men. Am. J. Clin. Nutr. 63:954–965. Mourkarzel, A.A., Song, M.K., Buckman, A.L., Vargas, J., Gass, W., McDirmid, S., Reyen, L., and Ament, M.E., 1992, Excessive chromium intake in children receiving total parenteral nutrition. Lancet 339:385–388. Ravina, A., Slezak, L., Mirsky, N., Bryden, N.A., and Anderson, R.A., 1999, Reveral of corticosteroid-induced diabetes with supplemental chromium. Diabet. Med. 16:164–167. Verhage, A.H., Cheong, W.K., and Jeejeebhoy, K.N., 1996, Neurologic symptoms due to possible chromium deficiency in long-term parenteral nutrition that closely mimic metronidazole-induced syndrome. J. Parent. Enteral. Nutr. 20:123–127.

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EVALUATION OF ZINC IN CHILDREN WITH TYPE 1 DIABETES MELLITUS L. F. C. Pedrosa1, A. Spínola-Castro2, M. Matsumoto3, J. Len3, F. Schwartzman3, L. P. Camargo 3, and S. M. F. Cozzolino4 Department of Nutrition Federal University of Rio Grande do Norte Natal1 Department of Pediatrics2 Division of Pediatric Endocrinology3 Federal University of São Paulo Department of Food and Experimental Nutrition4 Faculty of Pharmaceutical Sciences University of São Paulo, São Paulo Brazil

Since Scott and Fisher (1938) found that insulin crystals included an average of 0.5% zinc, studies were conducted to investigate the role for such element in insulin secretion and metabolism. Alterations in zinc metabolism were reported in both types of the diabetes (Mooradian and Morley, 1987). Abnormal findings have been correlated to the duration of the disease, metabolic control, and to the presence of chronic complications (Canfield, Hambidge and Johnson, 1984; Pedrosa et al., 1999). Prior studies have attemped to show that hyperzincuria should induce a deficiency or at least a marginal Zn status (Cunningham, Markle and Brown, 1994). Plasma Zn is the most frequently measured index of status Zn, but it is an insensitive diagnostic criterion because fluctuations in plasma zinc have been related with food intake and complex metabolic adjustments (King et al., 1994). Measurement of Zn concentrations in other peripheral blood cells, including erythrocyte and leukocyte also have been evaluated in experimental protocols. The aim of this study was to assess the nutritional status of the zinc in children with Type 1 Diabetes mellitus (T1DM), with regard to clinical parameters, diet and metabolic control.

RESEARCH DESIGN AND METHODS Thirty two children with T1DM (15 boys and 17 girls) aged 8.6 years (range, 3.4 to 12.3) who were diabetic for 4.3 years and 19 nondiabetics (ten boys and nine girls) Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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aged 7.9 years (range, 4.2–11.8) were included in this study. Informed consent was given by the parents of all subjects and experimental design was approved by Ethics Committee of Federal University of São Paulo. No patients suffered from any other major disease apart from diabetes. All subjects of the both groups were prepubertal state (Tanner evaluation) and none took any mineral supplementation or medicine. Venous blood samples were drawn in the morning after an overnight fast. Plasma and erythrocyte were separated from whole blood by centrifugation. A 24-hr urine sample was collected in metalfree glassware. Dietary register for 3 consecutive days was performed. Anthropometric measurements were taken for Score-Z classification. Metabolic control was assessed by fasting glycemia, glycosilated hemoglobin and urinary glucose. Blood and urinary glucose were assayed by colorimetric method. HbA1 C was determined by a microchromatographic technique. The zinc analysis in urine, plasma and erythrocyte were carried out by atomic absorption spectrophotometry. Date are reported as median and ranges. Nonparametric Kruskal Wallis test was used to compare groups. The correlations between variables were tested by the Pearson correlation coefficient.

RESULTS The energy intake of the both groups was less than recommended 2,000 kcal/day, in adition to inadequacy of calories distribution derived from macronutrients. The zinc intake of the groups averaged 9.5 ± 3.6 (T1DM) and 6.5 ± 4.2 (Control) was bellow the recommended allowance. Score-Z classification demonstrated that the major of the children with diabetes were with Height/Age indicator between 3SD, Md, and this behavior was different in control subjects. The data of metabolic control index showed poor control of the disease. The median of the HbA1C in T1DM group was 12% (range, 7–22), fasting glycemia levels was (ranged 50–510) and also was observed high values of the urinary glucose. Plasma zinc was not different between groups but the plasma Zn binding capacity (PZBC) in diabetic children was significantly more than in nondiabetic children (p < 0.017) when it was calculated in relation to plasma total proteins. There were no significantly differences in concentration of erythrocyte zinc between groups, but HbA1C values and erythrocyte zinc were inversely correlated (r = –0.404, p < 0.005). The T1DM children excreted significantly more zinc over a 24-h period than control group (p < 0.001).

DISCUSSION In clinical managment of the diabetes, the nutritional therapy has a strong influence in metabolic control. Adequate caloric intake is necessary to ensure normal growth rate and availability of protein. In this study we found TiDM patients consumed diets with low energy levels, in addition a distribution of calories derived from protein, lipids and carbohydrate was inadequate. High protein intake also was observed. This finding is troubling due to the elevated incidence of renal failure in course of the disease. On the other hand, low protein utilization derived from disproportion of energy/protein rate may affect gluconeogenesis, worsening metabolic control (Malik and Jaspan, 1989). Dietary zinc intake was below recommendation in both groups. Our results agree with previous studies that reported low amounts of zinc in typical Brazilian diets (Pedrosa and Cozzolino, 1990; Dantas and Cozzolino, 1990). Data of anthropometric measurements

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in T1DM children demonstrated tendency to “Stunting” malnutrition. This reflects decreased linear growth and insufficient height for present age. The reported relationship between zinc and growth was proved in practice of the supplementation (Allen, 1994). Study on kinetic of zinc in children with T1DM showed that increased zinc clearance which may be secondary to glomerular hyperfiltration, was inversely correlated to their growth rate (Nakamura et al., 1991). Plasma zinc was above normal range in both groups but not different. Despite this result the PZBC (mgZn/g protein) in T1DM childen was significantly higher. As expected from the literature (Heise, 1988; Cunningham, Markle and Brown, 1994) our patients with T1DM displayed a hyperzincuria that approached twice the level of Zn excretion by nondiabetic children. The underlying mechanisms for hyperzincuria is still obscure, however we observed a positive correlation between plasma zinc and urinary zinc excretion (r = 0.509). If plasma Zn and PBZC were elevated together with urinary zinc losses, indicate at least, a compensatory mechanism against chronic deficiency. In spite of this especulation, an inverse correlation between erythrocyte zinc and glycosilated hemoglobin was detected in T1DM patients. In conclusion these results suggest that alterations status zinc in diabetes can lead to a zinc intracellular deficiency, and this was attributed to the degree of metabolic control.

REFERENCES Allen, L.H., 1994, Nutritional influences on linear growth: a general review. Eur. J. Clin. Nutr. 48(Suppl. 1):S75–S89. Canfield, W.K., Hambidge, K.M., and Johnson, I.K., 1984, Zinc nutriture in type I diabetes mellitus: relationship to growth measures and metabolic control. J. Pediatr. Gastroenterol. Nutr. 3:577—584. Cunningham, J.J., Fu, A., Mearkle, L., and Brown, G., 1994, Hyperzincuria in individuals with InsulinDependent Diabetes Mellitus: concurrent zinc status and effect of high-dose zinc supplementation. Metabolism 43:1558–1562. Dantas, R.P.D. and Cozzolino, S.M.F., 1990, Biodisponibilidade de zinco em dieta regional de São Paulo. Arch. Latinoam. Nutr. 40:221–230. Heise C.C., King J.C., Costa F.M., and Kitzmiller J.L., 1988, Hyperzincuria in IDDM women. Relationship to measures of glycemic control, renal function, and tissue catabolism. Diabetes Care 11:780–786. King, J.C., Hambidge, M., Wescott, J.L., Keran, D.L., and Marshall, G., 1994, Daily variation in plasma zinc concentration in women fed meals at six-hour intervals. J. Nutr. 124:508–516. Malik, R.L. and Jaspan, J.B., 1989, Role of protein in diabetes control. Diabetes Care 12:39–40. Mooradian, A.D. and Morley, J.E., 1987, Micronutrient status in diabetes mellitus. Am. J. Clin. Nutr. 45:877–895. Nakamura T., Higashi A., Nishiyama S., Fujimoto, S., and Matsuda I., 1991, Kinetics of zinc status in children with IDDM. Diabetes Care 14:553–557. Pedrosa, L.F.C. and Cozzolino, S.M.F., 1990, Biodisponibilidade de zinco em dieta regional do Nordeste. Rev. Farm. Bioquim. Univ. de São Paulo 26(2):123–133. Pedrosa, L.F.C., Ferreira, S.R.G., Cesarini, P.C., and Cozzolino, S.M.F., 1999, Influence of glycemic control on zinc urinary excretion in patients with Type 1 Diabetes. Diabetes Care 22(2):362–363. Scott, D.A. and Fisher, A.M., 1938, The insulin and zinc content of normal and diabetic pancreas. J. Clin. Invest. 17:725–728.

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ZINC SULPHATE INDUCED METALLOTHIONEIN IN PANCREATIC ISLETS OF MICE AND PROTECTED FROM STREPTOZOTOCIN-INDUCED DIABETES

Patricia Ohly, Claudia Dohle, *Josef Abel, and Helga Gleichmann Clinical Department, Diabetes Research Institute and *Division of Toxicology Medical Institute of Environmental Hygiene at the Heinrich-Heine-University 40225 Düsseldorf, Germany

Type 1 diabetes in man results from destruction of insulin-producing in the pancreatic islets of Langerhans, most likely mediated through a T cell-dependent chronic autoimmune process (Atkinson and MacLaren, 1994). This process appears to be initiated by environmental agents triggering T cell-dependent inflammatory reactions with mononuclear cell infiltrates of the islets. A model of experimentally induced T cellmediated immune diabetes is that induced by multiple low-doses of streptozotocin (MLD-STZ), a naturally occurring diabetogen. Intraperitoneal injections of STZ, 40 mg/kg body wt, given on 5 consecutive days, will affect target molecules (Wang et al., 1998) and elicit local, T helper (Th 1) cell inflammatory immune reactions (Klinkhammer et al., 1988). It is assumed that reactive oxygen intermediates (ROI) are critically involved as non-specific mediators in the process of T cell-dependent destruction induced with MLD-STZ. The generation of hydrogen peroxide by STZ leading to DNA fragmentation was found in vitro and ex vivo in islets by Takasu et al. (1991). The authors concluded that contributed to the sequence of events for the DNA lesion. Based on these findings we proposed that hydroxyl radicals which are the most toxic of the species of ROI, are the ultimate mediators of damage in MLDSTZ-induced diabetes. Therefore, we assume that lesions can be pre-

Address all correspondence to: Dr. Patricia Ohly; Diabetes-Forschungsinstitut; Auf’m Hennekamp 65; D-40225 Düsseldorf, Germany; telephone: 0049-211-3382-234; fax: 0049-211-3382-603; email: [email protected] Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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vented by upregulation of metallothionein (MT) in islets, since MT has been demonstrated to be a specific intracellular antioxidant for by Sato and Bremner (1993). Previously, we and our colleagues first reported on constitutive levels of MT protein and its upregulation by zincsulphate in pancreatic islets ex vivo (Zimny et al., 1993) and in vitro (Ohly et al., 1998). Furthermore, we found that treatment of C57BL/6 and male mice with drinking water enriched with prevented development of MLD-STZ diabetes (Ohly et al.,1999). drinking water given ad libitum for one week significantly induced MT in islets as determined by the saturation assay ex vivo (Eaton and Cherian, 1991). The indices of MT upregulation calculated as mean value ±SEM were 2.3 ± 0.5 for C57BL/6 and 7.5 ±1.6 for mice. By using the reverse transcription (RT) polymerase chain reaction (PCR), we found that the mRNA of the isoforms MT1 and MT-2 are expressed in pancreatic islets of C57BL/6 and mice. For prevention of MLD-STZ diabetes, administration of drinking water was started one week before the first injection of STZ and was continued to be given for several weeks until the end of the experiments. As shown in Fig. 1, our current working hypothesis is that the beneficial effect of drinking water in preventing MLD-STZ diabetes results from significant upregulation of MT in pancreatic islets which provides protection against injury mediated by MLD-STZ-generated being produced from by the Fenton reaction. It is assumed that this protective effect is due to the potential of MT. Probably, the glucosetransporter 2 (GLUT2) of the which has been reported to be the selective target molecule of MLD-STZ (Wang et al., 1998) is rescued from MLD-STZ toxicity. Noteworthy, GLUT2 is pivotal for transport of extracellular glucose into the to stimulate the chain reactions for adequate insulin incretion to maintain extracellular glucose homeostasis. Consequently, pronounced reduction of GLUT2 expression results in loss of adequate response to its physiologic secretagogue glucose and development of hyperglycemia. In summary, proved to be a potent inducer of MT in pancreatic islets and was able to abrogate loss of function in diabetes induced with MLD-STZ. Current

Zinc Sulphate Induced Metallothionein in Pancreatic Islets of Mice

investigations aim to evaluate whether in our experimental systems MLD-STZ and are scavenged by upregulated MT.

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are generated by

REFERENCES Atkinson, M.A. and MacLaren, N.K., 1994, The pathogenesis of insulin-dependent diabetes mellitus, N. Eng. J. Med. 331:1428–1436. Eaton, D.L. and Cherian, M.G., 1991, Determination of metallothionein in tissues by cadmium-hemoglobin affinity assay, Methods Enzymol. 205:83–88. Klinkhammer, C., Popowa, P., and Gleichmann, H., 1988, Specific immunity to streptozotocin: Cellular requirements for induction of lymphoproliferation, Diabetes 37:74–80. Ohly, P., Wang, Z., Abel, J., and Gleichmann, H., 1998, Zincsulphate induced metallothionein in pancreatic islets and protected against the diabetogenic toxin streptozotocin, Talanta 46:355–359. Ohly, P., Wang, Z., Dohle, C., Abel, J., and Gleichmann, H., 1999, Zincsulphate protects from streptozotocininduced diabetes mellitus in mice: A function of metallothionein?, in: Metallothionein IV, (C.D. Klaassen, ed.), pp. 421–428, Birkhäuser, Basel, Switzerland. Sato, M. and Bremner, I., 1993, Oxygen free radicals and metallothionein, Free Radical Biol. and Med. 14:325–337. Takasu, N., Komiya, I., Asawa, T., Nagasawa, Y., and Yamada, T., 1991, Streptozotocin- and alloxan-induced generation and DNA fragmentation in pancreatic islets, Diabetes 40:1141–1145. Wang, Z. and Gleichmann, H., 1998, Glucose transporter 2 in pancreatic islets: crucial target molecule in diabetes induced with multiple-low-dose streptozotocin in mice. Diabetes 47:50–56. Zimny, S., Gogolin, F., Abel, J., and Gleichmann, H., 1993, Metallothionein in isolated pancreatic islets of mice: induction by zinc and streptozotocin, a naturally occurring diabetogen, Arch. Toxicol 67:61–65.

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WATER CONTENT OF MINERALS ASSOCIATED WITH TYPE 2 DIABETES MELLITUS OF SAMPLES COLLECTED ON THE NAVAJO RESERVATION

Judith Hallfrisch, Claude Veillon, Kristine Patterson, A. David Hill, Irene Benn, Bessie Holiday, Ruby Ross, Sylvia Zhonnie, Frances Price, and Ann Sorenson Beltsville Human Nutrition Research Center Beltsville, Maryland USA Navajo Senior Centers Aneth, Oljato, Navajo Mountain Red Mesa, Navajo Reservation Window Rock, Arizona Utah State University Logan, Utah

1. INTRODUCTION There are over 500 distinct Native American tribes in the United States. They comprise 1.9 million people, many of whom live on reservations in 34 of the 50 states (Gohdes, 1995). Many of these tribes have higher rates of non-insulin-dependent diabetes mellitus than the rest of the US population (Will et al., 1997). The Navajo Nation is the largest single group of Native Americans containing over 250,000 people and the Navajo people reside on the largest reservation with over 25,000 square miles of land in Utah, New Mexico, and Arizona. Previous research has indicated that though food intake records may indicate low levels of certain minerals (Ballew et al., 1997), traditional Navajo food preparation methods such as the addition of juniper ash (Christiensen et al., 1997) and drinking water (Hallfrisch et al., 1998) may provide substantial levels of essential minerals. Juniper ash was found to provide 285 mg of Ca, 33 mg of Mg, and 3mg of Fe per gram of ash. Water consumption of two liters/day was found to provide up to 100mg of Ca, 2.2 mg of Zn, 80 mg of Mg, and 1 mg of Fe. These overlooked sources of intake may partially explain why even at seemingly low intake levels, Navajos have relatively low hip fracture rates Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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when compared to the general population. Although NIDDM was rare in Navajos before World War II, recent results suggest that it affects 40% of those over 45 years. While there is clearly a genetic predisposition for development of the disease, a number of minerals have been associated with the development or severity of NIDDM (Blostein-Fujii et al., 1997; Mooradian and Morley, 1987). These include Cr, Cu, Zn, V, Mo, Mg, Cd, and Mn. This study was conducted as part of a larger examination to determine genetic, dietary and lifestyle contributors to the development and severity of NIDDM in this high risk population.

2. METHODS In order to minimize contamination of samples collected in the field, trace mineralfree graduated plastic tubes to which nitric acid was added were used. The 10ml graduation was marked with indelible ink. Trace mineral-free plastic transfer pipettes were used to add water from each site up to the graduation line. These tubes were distributed to the Senior Center Directors of the four Navajo chapters. Each director received 28 tubes to collect samples from 14 different sites on two occasions at least one week apart. These tubes were returned either to JH directly or by priority mail to the Beltsville Human Nutrition Research Center. Samples were stored at—70 °C until analyzed. Powder-free gloves were used in all procedures. Samples were analyzed in duplicate using atomic absorption spectrophotometry and inductively coupled plasma mass spectrophotometry (Veillon, 1986). Water from the tap at Utah State University was used as a non-reservation control. Nonparametric one way analysis of variance among the four chapters sampled was conducted.

3. RESULTS Of the 112 sample tubes distributed, 105 were returned. One of these samples apparently leaked during transport and the tube was empty when it arrived. A total of 104 samples from 53 different sites were analyzed. Approximately 50% of Navajo households have running water. Of the sites collected, 8 were classified as “running water;” 3 “waterpoint” or spring; 6 from wells, 7 from storage barrels. Most of the rest of the samples were collected at one site, either a well or spring or tap, and transported to households and stored there in barrels. Mn and Cu content of water (assuming intake of 2L/day) were minimal, ranging from 0 to about 0.2 mg. Cr was higher in water from Oljato and Red Mesa than from Aneth. Cd levels of water were within standards, but were higher in Navajo Mountain than the other three chapters (1.9 vs £ ). V content of water in Red Mesa was much higher than the other chapters (217 vs £ ,). Mo content of water at Navajo Mountain was lower than the other chapters (1.27 vs 3 ).

4. DISCUSSION It is clear from the results of these analyses that there is wide variability in the water content of minerals both around the reservation and even between duplicate samples from the same site. Whether these differences or differences in the ratios of the minerals

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contributes to NIDDM is yet to be determined. The largest difference noted was in the content of V in Red Mesa compared to the other chapters. The required amount of V has not been established, but is probably 15–30.g/day. Patients with NIDDM given 100 mg/day for four weeks, 50 times as much as would be ingested in water in Red Mesa, had improved glucose control (Boden et al., 1996). Further examination of the rates of NIDDM will determine whether V affects diabetes in Red Mesa. Cr has been known since the 1950s to control blood glucose and has been used in recent studies to improve glucose tolerance (Anderson, 1992); however, since the amounts detected in water were only about 1–2% of usual intake, it is unlikely that the small differences among chapters would have a clinical effect. Mo content was also low enough to be unlikely to have an effect. Overall diet will be determined to see if intakes are marginal or low. Though Navajo Mountain had significantly lower levels in water, the small contribution to requirements would also be unlikely to have an effect on glucose tolerance. Cadmium interacts with a number of minerals and its toxicity can be enhanced by smoking and marginal intakes of manganese, zinc, copper, and calcium (Kostial, 1986). Cadmium water content of Navajo Mountain which was highest of the four chapters is lower than the 3–4. g estimated to be consumed in drinking water of the average United States adult and unlikely to affect glucose intolerance. These data will be used to assess effects of mineral intakes on NIDDM in Navajos.

REFERENCES Anderson, R.A., 1992, Chromium, glucose tolerance, and diabetes. Biol. Trace Elem. Res. 32:19–24. Ballew, C., White, L.L., Strauss, K.F., Benson, L.J., Mendlein, J.M., and Mokdad, A.H., 1997, Intake of nutrients and food sources of nutrients among the Navajo: Findings from the Navajo Health and Nutrition Survey. J. Nutr. 117:2085S–2093S. Blostein-Fujii, A., DiSilvestro, R.A., Frid, D., Katz, C., and Malarkey, W., 1997, Short-term zinc supplementation in women with non-insulin-dependent diabetes mellitus: effects on plasma 5'-nucleotidase activities, insulin-like growth factor I concentrations, and lipoprotein oxidation rates in vitro, Am. J. Clin. Nutr. 66:639–642. Boden, G., Chen, X., and Ruiz, J., 1996, Effects of vanadyl sulfate on carbohydrate and lipid metabolism in patients with non-insulin-dependent diabetes mellitus. Metab. Clin. Exp. 45:1130–1135. Christensen, N.K., Sorenson, A.W., Hendricks, D.G., and Munger, R., 1998, Juniper ash as a source of calcium in the Navajo diet, J. Am. Diet. Assoc. 98:333–335. Gohdes, D., 1995, Diabetes in North American Indians and Alaska Natives. In: Diabetes in America. U.S. Department of Health and Human Services, NIH Publication no 95–1468:683–701. Kostial, K., 1986, Cadmium, in ed. W. Mertz, Trace elements in animal and human nutrition, vol. 2. Pp. 319–345. Academic Press, Inc., London. Mooradian, A.D. and Morley, J.E., 1987, Micronutrient status in diabetes mellitus, Am. J. Clin. Nutr. 45:877–895. Veillon, C., 1986, Trace element analysis in biological samples, Anal. Chem. 58:851A–866A. Will, J.C., Strauss, K.F., Mendlein, J.M., Ballew, C., White, L.L., and Peter, D.G., 1997, Diabetes mellitus among Navajo Indians: Findings from the Navajo Health and Nutrition Survey. J. Nutr. 117:2106S–2114S.

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DIETARY COPPER DEFICIENCY CAUSES ELEVATION OF EARLY AND ADVANCED GLYCATION END-PRODUCTS

J. T. Saari and G. M. Dahlen US Department of Agriculture Agricultural Research Service Grand Forks Human Nutrition Research Center Grand Forks, North Dakota 58202 USA

1. INTRODUCTION Glycation (nonenzymatic glycosylation) is the harmful interaction between sugars and protein that includes the binding of the acyclic form of a sugar to specific amino acids on a protein to form a Schiff base, rearrangement of the Schiff base to form an Amadori product (known as early products), followed by cross-linking and subsequent degradation of proteins to form advanced glycation end-products (Reiser, 1991). The hypothesis that glycation may contribute to damage associated with dietary copper deficiency has depended largely on indirect evidence such as reduced glucose tolerance (Keil and Nelson, 1934), enhancement of defects by dietary enrichment with fructose (Reiser et al., 1983; Saari et al., 1995), reduction of defects by food restriction (Saari et al., 1993; Saari et al., 1995) and inhibition of defects with the advanced glycation inhibitor aminoguanidine (Saari, 1994). Observation of an elevated percentage of glycated hemoglobin (Hb A1) (Klevay, 1982) in copper-deficient rats has thus far provided the only direct evidence of an increase in glycation. The objective of the present study was to corroborate the enhancement of early glycation in copper deficiency by measurement of another early glycation end-product, serum fructosamine, and to determine whether the glycation hypothesis could be supported further by measurement of an advanced glycation end-product, pentosidine. Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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2. METHODS Male, weanling Sprague-Dawley rats were fed a copper-adequate (CuA, 6.4mg Cu/kg diet) or a copper-deficient diet (CuD, 0.4mgCu/kg diet) for five weeks. Each rat was anesthetized, blood was withdrawn from the inferior vena cava and the heart and liver were excised. Hematocrit was determined by using an automated cell counter. Heart and liver samples were lyophilized, digested with nitric acid and hydrogen peroxide and measured for mineral content by inductively coupled argon plasma emission spectroscopy. Hemoglobin was measured by using a commercial kit (# 441, Sigma, St. Louis, MO USA), which utilizes a cation exchange resin to separate Hb A from Hb and spectrophotometric measurement (415nm) of each fraction. Hb is expressed as a percentage of total Hb A. Serum fructosamine was measured by using a commercial kit (# 465, Sigma), which uses a colorimetric test based on the ability of glycated serum proteins to reduce nitroblue tetrazolium. Determination of serum pentosidine was done by high pressure liquid chromatography (HPLC) that utilized sample preparation techniques of Odetti et al. (Odetti et al., 1992) and column switching to enhance separation (Takahashi et al., 1996).

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3. RESULTS Evidence of copper deficiency in rats fed a CuD diet is provided in Table 1, which shows commonly observed signs of severe copper deficiency: depression of liver and heart copper concentrations, reduced body weight, anemia, elevation of heart weight relative to body weight and an increase in liver iron concentration. The effects of dietary copper on early products of glycation, hemoglobin and fructosamine, and on the advanced glycation end-product, pentosidine, are shown in Fig. 1–3, respectively. All three products of glycation are elevated by severe dietary copper deficiency.

4. DISCUSSION The elevation of fructosamine and pentosidine in the serum of copper-deficient rats provides additional direct evidence for the occurrence of glycation in dietary copper

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deficiency and further supports the view that glycation may contribute to defects associated with copper deficiency.

REFERENCES Keil, H.L. and Nelson, V.E., 1934, The role of copper m carbohydrate metabolism, J. Biol. Chem. 106:343–349. Klevay, L.M., 1982, An increase in glycosylated hemoglobin in rats deficient in copper, Nutr. Rep. Int. 26:329–334. Odetti, P., Fogarty, J., Sell, D.R., and Monnier, V.M., 1992, Chromatographic quantitation of plasma and erythrocyte pentosidine in diabetic and uremic subjects, Diabetes 41:153–159. Reiser, K. M. , 1991, Nonenzymatic glycation of collagen in aging and diabetes, Proc. Soc. Exp. Biol. Med. 196:17–29. Reiser, S. , Ferretti, R. J. , Fields, M., and Smith, J.C., 1983, Role of dietary fructose in the enhancement of mortality and biochemical changes associated with copper deficiency in rats, Am. J. Clin. Nutr. 38:214–222. Saari, J.T., 1994, Implication of nonenzymatic glycosylation as a mode of damage in dietary copper deficiency, Nutr. Res. 14:1689–1699. Saari, J.T., Bode, A. M ., and Dahlen, G.M., 1995, Defects of copper deficiency in rats are modified by dietary treatments that affect glycation, J. Nutr. 125:2925–2934. Saari, J.T., Johnson, W.T., Reeves, P.G., and Johnson, L.K., 1993, Amelioration of effects of severe dietary copper deficiency by food restriction in rats, Am. J. Clin. Nutr. 58:891–896. Takahashi, M., Hoshino, H., Kushida, K., Kawana, K., and Inoue, T., 1996, Direct quantification of pentosidine in urine and serum by HPLC with column switching, Clin. Chem. 42:1439–1444.

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THE USE OF STABLE ISOTOPE TRACERS TO EXPLORATE ZINC AND SELENIUM METABOLISM IN INSULIN DEPENDANT PATIENTS M. Bertouze1, P. Faure2, V. Ducros2, S. Halimi2, and A. Favier2 1

Service de Diabétologie Endocrinologie, Nutrition Hopital A Michallon, Grenoble France 2 LBSO, Grenoble, France

Both severe and mild zinc and selenium deficiency states may occur in diabetic patients. There is however a need for improved techniques to assess these trace element status in this population. Isotopic techniques may provide an answer to this problem but the available radioactive isotopes are unsuitable for use in man. So despite the difficulties linked to their measurement, the utilization of stable isotope could provide interesting data concerning trace element status and bioavailibity in diabetic patients. The question arises to the choice of a tracer technique available in a clinical situation. The measurement of bioavailability is possible using two different isotopes (for instance IV and orally). This method can provide interesting informations about the absorption, the retention and the elimination of trace elements and the interaction between trace elements. In practical, it is difficult to use it in patients especially if they are not hospitalized because of the difficulties to collect urinary and fecal samples for several days. The other method is to study the pools that exchange rapidly with plasma as it is the more important for physiological functions and can provide information about the total pool of a trace element. This method has the advantage to be suitable for study in polypathological patients as it requires a single injection of the isotopes followed by several blood sampling. Therefore we propose to describe this technique and to report the results we obtained in 6 diabetic patients compared to 6 controls, concerning the decay curve, the half life and the clearance of zinc and selenium. This clinical trial could give original informations concerning the suitability of the isotopic pool measurement in clinical situations. Moreover these data are of interest as they provide new informations concerning trace element metabolic specificities in insulin dependant diabetic patients, which is necessary to evaluate their real need in zinc and selenium intake. 527

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ZINC MODULATES THE GLUCOSE INDUCED RAT AORTIC SMOOTH CELL PROLIFERATION S. Bouvard1, P. Faure1, A. Favier1, M. Leconte2, and S. Halimi1 1

LBSO, Université J Fourier Grenoble, France 2, Lipha-Lyon, France

The pathogenesis of diabetic vasculopathy is highly complex, and there may be differences between micro and macrovascular lesions. However, the major primary metabolic abnormality that causes both is hyperglycemia. The chronic hyperglycemia is an important factor in microvasculopathy, but also demonstrated its importance in macrovascular disease (1). Vascular pathology involves both functional and structural changes in the endothelial layer, such as altered endothelium-dependent dilatation and reduced endothelial proliferation. When endothelial cells are damaged, vascular smooth muscle cells hyperplasia may be induced by a large number growth factors, cytokines and vasoregulatory agents, resulting in atherosclerosis (2). Increased vascular smooth muscle cells growth and proliferation in the intima of vascular tissues is one key feature of atherosclerosis, and increased proliferation was also demonstrated in vascular smooth muscle cells cultured in high glucose concentrations (3). Although the pathogenesis of diabetic vasculopathy is probably multifactoriel in origin, studies suggest a role for glucose-induced oxidative stress (4) Zinc, an essential trace element, stimulates the proliferation of cultured vascular endothelial cells in a manner which depends on endogenous basic fibroblast growth factor (bFGF) (5). This suggests that zinc may be a preventive factor for atherosclerosis. However, it is unclear whether zinc affects the proliferation of vascular smooth muscle cells or not. The aim of the present study is to clarify whether or not zinc modulates smooth muscle cell proliferation induced by high glucose concentrations. Rat aortic smooth muscle cells (RASMC) were harvested from male Wistar rats by enzymatic dissociation according to the method of Günther (6). The cells were grown in F10 medium containing 5% fetal calf serum. Supplementation assay were performed by adding Zn as zinc chlorure to the medium (final concentration 8mM). Cells were incubated in Zn-supplemented media for 21 days before being harvested or submitted to glucose concentration. In this study incubation of RASMC in the presence of 25 mM glucose resulted in stimulation of cell growth, generation of oxidative stress (decrease of intracellular thiols, 528

Zinc Modulates the Glucose Induced Rat Aortic Smooth Cell Proliferation

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increase of F2-isoprsotanes). The proliferation of RASMC in the presence of high glucose concentration was inhibited by zinc. The intracellular thiols of Zn-supplemented cells was higher than that of non supplemented and the formation of F2-isoprostanes was decreased. In conclusion zinc can modulate glucose induced RASMC proliferation, and therefore contributes to vascular protection. This result is of interest in the comprehension of the mechanisms of vascular lesions linked to diabetes mellitus.

REFERENCES Kuusisto J., Mykkanen L., Pyorala K., and Laasko M., Diabetes, 43, 960–967 (1994). Ross R., Nature (London), 362, 801–809 (1993). Oikawa S., Hayasaka K., Hashizume E., Kotake H., Midorikawa H., sekikawa A., Kikuchi A., and Toyota T., Diabetes, 45 (suppl.3), S114–S116 (1996). Baynes JW., Diabetes, 40, 405–412 (1991). Kaji T., Fujiwara Y., Yamamoto C., Sakamoto M., and Kozuka H., Life Sci., 55, 1781–1787 (1994).

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METABOLIC EFFECTS OF DIETARY CHROMIUM-L-METHIONINE IN HORSES AND BEEF CATTLE M. T. Socha1, S. L. Ralston2, R. Raub3, E. B. Kegley4, C. K. Swenson1, A. B. Johnson1, and T. M. Fakler1 1

Zinpro Corporation, Eden Prairie, Minnesota Rutgers, The State University of New Jersey New Brunswick 3 Kansas State University Manhattan and 4 University of Arkansas Fayetteville 2

A series of three studies was conducted to determine the metabolic effects of Cr from chromium-L-methionine (CrMet) on glucose metabolism in horses and beef cattle. In Study 1, 28 stock-type horses were used in a completely randomized block design to determine the effect supplemental dietary CrMet on glucose tolerance (Intravenous Glucose Tolerance Test, IVGTT) and insulin sensitivity (Intravenous Insulin Challenge Test, IVICT). Supplemental Cr was fed at a rate to supply 0, 0.005, 0.01 or 0.02mgCr/kg body weight (BW). There were significant linear and quadratic effects (P < 0.05) of treatment on area under the glucose curve from 0 to 240 minutes post glucose infusion. Horses receiving either 0.005 or 0.02mgCr/kg BW had smaller areas under the curve for serum insulin for 0 to 100 and 0 to 240 minutes (P < 0.05) post glucose infusion. In addition, the area under the curve for serum insulin from 0 to 240 min after glucose infusion was smaller (P < 0.05) for horses receiving 0.02mgCr/kg BW than for horses receiving 0.01 mgCr/kg BW. In study 2, mares >20yrs of age were used in two separate trials (n = 12 mares/trial) to determine if CrMet would affect glucose (GLU) and insulin (INS) responses in aged horses, The mares were individually fed alfalfa/whole corn plant cubes and pelleted concentrate (ES: Equine Senior, Purina Mills, Inc) in two daily feedings, with 1.36kg ES and ½ of the cubes offered at 0800h, the rest given at l,600h. The CrMet was incorporated into an alfalfa based pellet and fed daily (l,600h) to provide: 0mg (Control), 0.01 mg (Trt 1) or 0.02mgCr/kg BW (Trt2). Mares were randomly assigned to treatment groups, blocked according to their insulin responses to pre-supplementation intravenous dextrose challenge (IVGTT). Mares with insulin responses >250 IU/mL were 530

Metabolic Effects of Dietary Chromium–L-Methionine in Horses and Beef Cattle

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classified as insulin resistant (IR), those with lower responses considered non-IR. In addition, the glucose and insulin response of each mare to a meal of 1.36kg ES was also determined before supplementation was initiated. Mares were fed their assigned treatments for 4 weeks then subjected to an IVGTT and to a meal of ES. Supplementation of CrMet had no effect on the GLU or INS responses to IVGTT (P > 0.10). After ES, GLU response was lower (P < 0.01) at 30min and peak insulin response was lower (P < 0.03) in horses on Trt2 compared to Trt 1 or Control. Rate of GLU clearance after peak response to ES was faster (P < 0.03) in horses on Trt 1 than those on Trt2 or Control. After ES, Trt2 mares had smaller areas under the insulin (P < 0.02) and glucose (P < 0.05) curves than those in Trt1, with Control mares being intermediate. CrMet supplementation may improve some indices of GLU/INS response in aged horses. Responses to meals of concentrates may be a more sensitive test of glucose/insulin tolerance than the IVGTT in aged mares. In Study 3, 36 crossbreed steers were fed a control diet or the diet supplemented with 400 or 800ppb Cr as CrMet. Supplemental CrMet increased the glucose clearance rate from 0 to 10min after IVICT (linear effect, P < 0.05). Glucose half-life 0 to 15 min post insulin infusion was also decreased by supplemental CrMet (linear effect, P < 0.03). Serum insulin concentrations were increased by supplemental CrMet post glucose infusion. These data clearly indicate that CrMet alters glucose and insulin metabolism in horses and beef cattle.

167

ZINC PROTECTS HELA CELLS AGAINST THE GLUCOSE INDUCED CYTOTOXIC EFFECT

P. Faure, S. Bouvard, A. Favier, and S. Halimi LBSO, Université J Fourier, Grenoble France

There are strong evidences that glucose, under physiological conditions, is prone to oxidation and consequently generates hydrogen peroxide and reactive intermediates such hydroxyl-free radicals. It has been suggested that oxidative stress plays an important role in tissue damage associated with diabetes and that peroxide formation is increased parallely to elevated glucose oxidation. Variations in glucose concentrations are sufficient to induce cell death through a free radical-mediated mechanism delay in various phase of the cell cycle of human endothelial cells and inhibition of endothelial cell replication. Zinc is an essential metal, necessary to the function of many enzymes participating in a wide variety of metabolic processes. Zinc may be involved in regulation of cell numbers by its roles in both proliferation and death by apoptosis. There have been several reports dealing with inhibitory and/or toxic effects of zinc in vitro in various kinds of cells, for example, HeLa cells, fibroblasts. Moreover, it has been suggested that zinc is protective against free radical injury. Therefore in the present study, we experimented the effects of zinc on the glucose induced cytotoxic effect. In order to experiment the role of free radical production in hexose induced cytotoxicity, we compared the effects of glucose on HeLa -wild and HeLa tat cells. Actually HeLa tat cells exhibit a lower antioxidant defense system. Regarding its antioxidant properties, the protective effect of zinc was also experimented in both models. High glucose concentration (20 and 30 mM) led to a significant decrease in cell viability. This effect was more important in HeLa Tat cells than in the wild cell line. These glucose concentrations also reduced cytosolic thiol groups in both cell lines, but the drop was significantly more important in HeLa Tat cells. The addition of zinc in the cell medium led to a significant increase in cell viability in both cell lines, parallely to a protection of cytosolic thiol molecules. The glucose induced apoptosis was also measured by the acridine orange-ethydium broming staining and was significantly higher in both cell lines. Zinc exhibited a protective effect against glucose induced apoptosis. A negative correlation was observed between cytosolic thiol groups and the apoptosis. 532

Zinc Protects Hela Cells against the Glucose Induced Cytotoxic Effect

533

In conclusion, the present study demonstrated that glucose becomes a toxic agent modifiying the redox cycle of cultured HeLa Wild and HeLa-tat cell lines and leads to apoptotic and necrotic cell death. This toxic effect is higher when sulfur compounds are low. Zinc has a protective effect in this system and protects cytosolic thiol groups. Similar studies in vascular cells could give new data concerning the role of zinc in the vascular protection, and its importance in the diabetic patients.

168

OXIDATIVE STRESS AND ANTIOXIDANT TRACE ELEMENT STATUS IN PATIENTS WITH NONINSULIN-DEPENDENT DIABETES

Kerkeni A., Roussel A. M., Othmane A., Majhoub S., Zouari N., Najjar F., Arnaud J., Favier A., and Anderson R. A. Faculté de Médecine, Monastir, Tunisie Hôpital Bourguiba, Sfax, Tunisie LBSO, Université J. Fourier, Grenoble France Human Nutrition Research Center Beltsville, MD

A Number of studies suggest an interrelationship between the antioxidant status and development of complications in diabetes. Indeed, a few of works show that diabetes could modify trace elements metabolism and increase the antioxydant deficiency that may contribute to enhance lipid peroxidation that is a mechanism generally recognized as being the most important in the pathogenesis of several pathological disorders such as atherosclerosis. The aim of the present study was to evaluate the oxidative stress and antioxydant trace element status in patients with non insulin dependent diabetes. 95 patients: 52 men and 43 women (mean age 50.3 ± 9.2 years; mean weight 79.0 ± 14.2kg), with non-insulin-dependent diabetes mellitus (NIDDM) and with disease duration of less than 10 years, were investigated. The patients were taking their usual medications and had not clinical signs of diabetic complications (without diabetic retinopathy and creatine lower than Venous whole blood was collected at 8 A.M. after an overnight fast into polypropylene tubes free of trace elements for measurement of glucose, glycosylated hemoglobin, (HbA1c), lipids, apolipoproteins A1 (apo A) and B (apo B), plasma antioxydant trace elements (Zn, Cu, Se), plasma Ca and Mg, erythrocyte selenium dependent glutathione peroxidase (Se GPX) and copper-zinc superoxide dismutase (SOD) and plasma thiobarbituric acid reactive substances (TBARs). Whole blood was centrifuged, plasma and erythrocytes were stored at –80 °C until analysis. All data are expressed as mean ± standard deviation. Statistical significance was defined as p < 0.05. 534

Oxidative Stress and Antioxidant Trace Element Status

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Patients with type II diabetes mellitus exhibited as anticipated significantly higher blood levels of glycemia, total cholesterol and triglycerides than in control health subjects. The plasma zinc concentration was significantly lower in diabetic subjects than in control subjects (9.04 ± 1.23 versus The plasma zinc was negatively corelated with TBARs (r = –0.26 p < 0.05). We had found normal plasma Cu concentration (14.05 ± 3.10 versus but it was significantly lower in men than in women (12.84 ± 2.38 versus It is probable that plasma Cu levels do not reflect necessarily body Cu status but greater inflammatory conditions in diabetes. The plasma selenium concentration was found It appears that diabetic subjects do not have Se deficiency contributing to the known complications of diabetes mellitus. Moreover there was correlation between Se concentrations and glycosylated hemoglobin (r = 0.37 p < 0.01). A significant decrease in plasma Ca and Mg concentrations was observed in diabetic patients (Ca = 82.04 ± 8.83 versus 98.74 ± 2.6mg/l and Mg = 14.95 ± 1.92 versus Hypomagnesemia and hypocalcemia appear to be, at least in part, secondary to urinary Mg and Ca loss. The mean plasma TBARs concentration was found to be and no sex related difference was observed. In addition, TBARs levels were significantly correlated with age (r = 0.26 p < 0.01), glycemia (r = 0.31 p < 0.01) (r = 0.31 p < 0.01) total cholesterol (r = 0.26 p < 0.01) cholesterol: cholesterol HDL ratio (0.31 p < 0.01), apoB (r = 0.39 P < 0.001) triglycerides (r = 0.39 p < 0.001) but it was not correlated to trace elements. The mean GPX and SOD erythrocyte concentrations in diabetic subjects were found respectively 39.72 ± 9.44U/gHb and 1.23 ± 0.20 U/gHb. Statistically significant differences in GPX concentrations were observed between men and women (37.59 ± 9.25 for men and for women). The results confirm the disturbances of trace elements and antioxidants status and an increase in peroxidation markers in diabetic subjects.

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ZINC DEFICIENCY INCREASES THE DELETERIOUS EFFECT OF HIGH FRUCTOSE DIET ON FETAL DEVELOPMENT AND FREE RADICAL ACTIVITY IN RATS B. Lachili1,2, P. Faure2, C. Ribuot3, J. Arnaud2, M. J. Richard2, M. Sève2, and A. Favier2 1

Faculté de Médecine Université de Batna 05000 Batna, Algérie 2 Laboratoire de Biologie du Stress Oxydant Faculté de Pharmacie, Grenoble I France 3 Laboratoire de Physio-pharmacologie Faculté de Pharmacie, Grenoble I France

We have previously observed that high fructose diet leads to insulin resistance and increased free radical activity in rats.1 As this diet can modify mineral metabolism, and knowing the effect of insulin resistance on fetal development, the aim of this work was to determine whether or not zinc could interplay with the deleterious effects of high fructose diet in pregnant rats. Four groups of pregnant female rats were designed: control (C) receiving a standard diet (n = 7, Zn 64ppm), control zinc deficient (CZD) receiving a control diet but zinc deprived (n = 12, Zn: 6ppm), high fructose fed groups (n = 7, F, standard diet but 58% of sugars were fructose, Zn: 64ppm) and fructose zinc deprived group receiving the F diet but zinc deprived (n = 09, Zn: 6ppm, FZD). The animals were sacrificed after 20 days pregnancy. Metabolic parameters (plasma glucose, plasma triacylglycerol, cholesterol), zinc status and free radical activity were measured as well as morphologic data of the rats of the fetus. As expected, plasma zinc was significantly lower in both zinc deficient groups. Plasma glucose was not different among the groups as observed previously in this model,2 but triacylglycerol was increased in groups fed with fructose. Lipid peroxidation, measured by TBARS, was significantly increased in group FZD (p < 0.009) [2.52 ± 0.26 vs but surprisingly plasma protein thiol groups were increased (p < 0.01) [3.21 ± 0.22 vs Red cell Cu, Zn, SOD activity was significantly decreased in F rats and more decreased in FZD group (p < 0.03) 536

Zinc Deficiency Increases the Deleterious Effect of High Fructose Diet on Fetal Development

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[1.81 ± 0.36 vs 1.43 ± 0.15U/mgHb] in comparison with the remaining groups. Conversely, red cell Se GPX activity was increased group FZD (p < 0.026) [270.1 ± 35.1 vs 317.7 ± 37.1U/gHb]. Concerning weight gain of the rats, it was significantly lower in group F, this effect being aggravated in group FZD (p < 0.001) [145.7 ± 19.7 vs 107.8 ± 9.4 g]. Fetal resumption was raised in group FZD in comparison with group F. Fetus weight was increased in group F but decreased by zinc deficiency. In conclusion, high fructose diet leads to fetal development modifications. A synergistic effect of zinc deficiency of the effects of fructose diet diet is observed. In parallel zinc deficiency majors the effect of fructose on lipid peroxidation and Cu, Zn, SOD activity. More works are necessary to determine if the effect of fructose diet on fetal development is due to insulin resistance or the increased lipid peroxidation. REFERENCES 1. P. Faure, E. Rossini, N. Wiernsperger, M.J. Richard, A. Favier, and S. Halimi, Diabetes, In press, February 1999. 2. P. Faure, E. Rossini, J.L. Lafond, M.J. Richard, A. Favier, and S. Halimi, 1997, J. Nutr. 123:103–107.

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ZINC 100 PPM DOES NOT IMPROVE INSULIN SENSITIVITY OF HIGH FRUCTOSE FED RATS LEADING TO INSULIN RESISTANCE

E. Rossini, S. Bouvard, M. J. Richard, S. Halimi, and A. Favier LBSO, Grenoble, France

We have previously observed that high amount of vitamin E leads to an improvement of insulin sensitivity in high fructose fed rats (1). The aim of this work was to evaluate the effect of zinc as we previously observed that zinc depletion leads to an insulin resistance. The rats (post weaning, 50g) were divided into 3 groups: the control group (C, n = 16) which received a purified diet containing 60g/100g carbohydrates, the highfructose-fed group (FT, n = 16) fed a diet in which 56.8% of the carbohydrates were fructose, and a high-fructose and zinc supplemented group (FZn, n = 16) fed the FT diet supplemented with Zinc 100ppm (vs 10ppm in C and FT groups). The duration of the treatment was 6wk. Insulin sensitivity was determined in half of the rats in each group using the euglycemic hyperinsulinic glucose clamp technique. The remaining rats were investigated for metabolic and free radical parameters, and for plasma, liver and muscle (soleus muscle) copper and zinc concentrations. The group FT had a significantly lower insulin sensitivity than the C group. The group FZn exhibited a higher insulin resistance than the FT group, parallel to a significant weight increase (more than 20% in comparison with C and FT groups). In comparison with group FT, Cu Zn SOD and Se GSHPx activities were also improved in FZn group. Therefore the question arises to know if the deleterious effect of zinc on insulin sensitivity is linked to its effect on the animal weight. More studies are running using lower zinc supplementations (40ppm) associated with the pair feeding technique as the zinc supplementation leads to a significant increase of food intake.

REFERENCE P. Faure., E. Rossini., J.L. Lafond, M.J. Richard, A. Favier, and S. Halimi. J Nutr 123:103–107, 1997.

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DESIGN OF NEW OXOVANADIUM (IV) COMPLEXES FOR TREATMENT OF DIABETES. BIOVAILABILITY, SPECIATION, AND TISSUE TARGETING CONSIDERATIONS

K. H. Thompson, J. H. McNeill, and C. Orvig Medicinal Inorganic Chemistry Group Chemistry Department and Faculty of Pharmaceutical Sciences University of British Columbia Vancouver, B. C., Canada V6T 1Z1

Vanadium compounds are being widely investigated for their orally available insulin-mimetic potential, both in experimentally-diabetic animals and in humans. To date, the only apparent drawback to this potential therapy has been gastrointestinal distress requiring diminution of dose in short-term trials. Very low absorption and tissue uptake of inorganic vanadyl and vanadate salts require high intakes to achieve therapeutic effect; therefore, current pharmacochemical investigations are focused on increasing absorption and uptake, thus minimizing dose required to achieve glucose-lowering. Modification by organic ligand chelation must also take into account thermodynamic and hydrolytic stability, strength of complexation, and requirements of neutral charge and hydrophilic/lipophilic balance to maintain cell membrane permeability. Comparison of several new oxovanadium(IV) complexes showed wide variation in specific tissue uptake, and considerable improvement in tissue retention with lower doses required for glucose-lowering response. These design principles have general applicability to considerations of bioavailability of other metal ions, whether used as nutritional supplements or as therapeutic agents.

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THE METALLIC COMPONENT OF THE GLUCOSE TOLERANCE FACTOR, G.T.F. Cobalt Against Chromium

F. Silió1, A. Santos2, and B. Ribas3 1

Fundación Entorno Y Salud 28010 Madrid Spain 2 Centro Ciencia de Materiales CSIC 28049 Madrid Spain 3 Instituto de Salud “CARLOS III” 28220 Madrid Spain

Glucose Tolerance Factor (G.T.F.) is a unique widespread, basic substance limiting it highly specific role to the glucose overload of obese, hyperinsulinism diabetes; that means the removal of excess ketone bodies (Ac-Ac) and NADH allowing to glucose intolerance (GI), GTF exerce it-substrate driven-specific activity at optimum point within the nM concentration. GTF was found in pork kidney extracts as a brown substance by Mertz and Schwarz giving the specific action of regulation of glucose tolerance (specific removal of elevated glucose concentrations. The failure to isolate GTF from kidney extracts allow to Mertz to employ bulk yeast cultures as a source of GTF. Yeast extract behave as biologically active and a purified extract was isolated but in the (brick red) inactive form. From the analysis of such substance Mertz conclude that: Nicotinic acid, chromium and the aminoacids glycine, cysteine and glutamic acid were the GTF components. We though in a more physiological species: Nicotin AMIDE, Cobalt and glutathione (G-SH) and with these data, we undertake successful synthesis of GTF (Silio et al., 1996) Mineralization of Kidney GTF and further ICPAAS give 6.63% cobalt, while chromium was not detectable. The synthetic preparation of GTF permitted us the study of its physicochemical features allowing us to carry on further studies of kidney and yeast GTF preparations. Spectral U.V. data and common 2nd derivative spectrum was the decisive fingerprint for physicochemical identity of kidney, yeast and synthetic GTF. About its metallic component Schwarz affirm that “the presence of chromium was ascertained by benzidine blue test” (Schwarz and Mertz, 1959) in such extracts and in the pancreatic GTF discharge, but this proof is 540

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highly unespecific; manganese, cobalt, vanadium, iron give positive test and—nitrogen coordinated compounds- (cobalt GTF, vitamin B12, Hemin) give very sensitive benzidine reaction. From yeast culture extracts, GTF activity and containing fractions, were separated as two well defined peaks. Recent evidence shows that NAA from human kidney give 0.06p.p.m. CrIII while 20.0p.p.m. cobalt and 27.8 p.p.m. in pancreas. The mechanism of NADH reoxidation by GTF residues in the driving of ketone bodies through non-ketone pathways. Rat liver mitochondria and NADH oxidation by FMN luciferase were our choice system, Ac-Ac metabolization, malate-coupled, undergoes an 800% stimulus in the presence of GTF. All three sources of GTF (synthetic, kidney and yeast) behave as very active within the isolated mitochondria-luciferase test. Therefore, we can conclude that GTF is an unique, cationic substance containing nicotinamide, glutathione and cobalt. Its role is NADH reoxidation by Ac-Ac metabolization coupled to Krebs cycle.

REFERENCES Silió et al. Cobalt the metallic component of “Glucose Tolerante Factor” GTF. In: Metall Ions in Biology and Medicine, Vol. IV 133–136. Ph. Collery ed. John Libbey, Paris 1996. Schwarz, K. and Mertz, W. (1959) Chromium and the Glucose Tolerance Factor. In: Arch. Bioch. Biophys. 85, 293.

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173

THE INTERACTION BETWEEN DIETARY COPPER AND EXCESS IRON—INCREASES THE RISK OF HEART DISEASE

Meira Fields and Charles G. Lewis Nutrient Requirements and Functions Laboratory Beltsville Human Nutrition Research Center U.S. Department of Agriculture, ARS Beltsville, Maryland 20705 USA

As interest in oxygen free radical mechanisms of pathogenesis has intensified, it has become apparent that transition metal ions, especially iron, play a key role in initiating tissue damage. Iron has been reported to contribute to coronary artery disease, the leading cause of death in western countries. However, studies conducted at Beltsville Human Nutrition Research Center have repeatedly shown that iron alone could not increase the risk of heart disease unless it is accompanied by copper deficiency (Fields and Lewis, 1997). Copper and iron are essential trace elements required for numerous critical functions and maintenance of a large number of body systems (Ettinger, 1984). It is therefore not surprising that their deficiency is responsible for numerous diseases affecting circulatory, respiratory, nervous, endocrine, immune and reproductive systems. Physiological states including growth, aging, pregnancy, lactation and exercise may result in altered needs and utilization of both copper and iron. It was known since 1928 (Hart et al., 1928) that copper and iron metabolism are interrelated. Under conditions of copper deficiency caused by either dietary, environmental, or genetic factors, excess iron accumulates particularly in the liver. When copper is given, liver iron is reduced due to mobilization of iron from the liver to extrahepatic tissues. Although iron is essential to all living species, iron in excess of cellular needs is extremely toxic and is likely to participate in iron-stimulated free radical reactions (McCord, 1991; Halliwell, 1982). Oxidative stress is a condition where an imbalance exists Address all correspondence to: Dr. Meira Fields; USDA, ARS, BHNRC, NRFL; Bldg. 307, Rm. 330, BARCEast; Beltsville, Maryland 20705-2350 USA; telephone: 301-504-9412; fax: 301-504-9062; email: [email protected] Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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in the oxidant/antioxidant equilibrium in favor of the oxidants. During oxidative stress, formation and release of reactive oxygen species are increased inducing structural and functional alterations of cellular membranes. These alterations are promoted by the attack of free radicals on membrane polyunsaturated fatty acids, proteins and DNA. Iron, a strong oxidant, under certain redox environment has the potential to generate free radicals. The speciation of iron which causes molecular alterations to cell DNA during oxidative stress, is of much current interest, inasmuch as such damages are believed responsible for induction of chronic degenerative diseases such as cancer, diabetes and atherosclerosis (Araujo et al., 1995; Salonen, 1993; Halliwell and Chirico, 1993). Oxidative modifications of low-density lipoproteins (LDL) have been suggested to play a role in the pathogenesis of atherosclerosis (Witztum and Steinberg, 1991; Halliwell and Chirico, 1993). This process is dependent on media containing metal ions such as copper or iron. Elevation of blood lipids such as cholesterol and triglycerides also play a crucial role in the development of atherosclerosis and coronary heart disease. We hypothesize that oxidative stress brought about by the combination of excess liver iron and copper deficiency is a potential inducer of hyperlipidemia and coronary heart disease. This hypothesis was based on data derived from our laboratory which clearly show that copper deficiency and excess liver iron are prerequisite for hyperlipidemia. We also reported that if one of these factors is prevented, blood lipids remain unchanged (Fields and Lewis, 1997). Reasons for this phenomenon could be due to oxidative stress brought about by the combination of inadequate antioxidant protection and generation of free radicals. Inadequate antioxidant protection is a consequence of copper deficiency due to a reduction of activity of the copper containing enzyme, superoxide dismutase, the first defense line against oxidative insult. Other antioxidant enzymes such as glutathione peroxidase are also suppressed by copper deficiency. However, inadequate antioxidant protection by itself is not sufficient to cause generation of reactive oxygen species and oxidative stress. The presence of high concentrations of a transition metal ion such as iron in the absence of antioxidant protection can initiate oxidative stress. This hypothesis is supported by our findings which show that the reduction of liver iron either by chelation therapy or by reduction of iron consumption prevented both free radical formation and hyperlipidemia (Fields et al., 1991; Fields et al., 1993). During the last 15 years we repeatedly reported that the type of dietary carbohydrate, simple versus complex, was responsible for numerous pathologies and hyperlipidemia associated with copper deficiency (Fields et al., 1984). The consumption of simple sugars such as fructose and sucrose caused severe pathologies and premature mortality of the laboratory rat, and was instrumental in elevating blood levels of cholesterol and triglycerides. No such increases were noted when the diets consumed contained starch. Furthermore, the combination of fructose with copper deficiency resulted in generation of free radicals associated with excess liver iron. Excess liver iron occurred when the animals consumed a diet containing normal levels of iron recommended for optimal growth of rodents. No free radicals were detected when starch was fed, although livers of copper-deficient rats contained high levels of stored iron. When liver iron was reduced in copper-deficient rats fed fructose, no free radicals were generated, and blood lipids were not elevated. These data illustrate the role of iron in inducing oxidative stress and hyperlipidemia of copper deficiency. It should be borne in mind that the consumption of fructose even in copperadequate rats induces hyperlipidemia compared with the consumption of starch. In addition, the metabolism of fructose in the liver generates reduced nucleotides, providing

The Interaction between Dietary Copper and Excess Iron

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optimal redox environment for iron to generate free radicals. In order to demonstrate that the combination of excess liver iron and copper deficiency and not fructose are potential inducers of hyperlipidemia, fructose was omitted from the diet. In a recent study, hyperlipidemic properties of iron and copper deficiency were determined in the absence of fructose. Copper-deficient diets containing starch were fed. Levels of dietary iron were either adequate (50. g Fe/g) or elevated (500. g Fe/g). The diets were either deficient (0.6.gCu/g) or adequate copper (6.0.gCu/g). Within 5 weeks of feeding these copper-deficient diets, rats which consumed the high levels of dietary iron developed severe pathological changes and some died prematurely due to heart rupture. In addition, the combination of high-iron with low copper induced hyperlipidemia. In the past, we could never raise blood lipids in rats fed starch. However, the consumption of high levels of dietary iron was able to induce hyperlipidemia in copper-deficient rats fed starch. This is the first time that we were able to demonstrate that regardless of the type of dietary carbohydrate, excess liver iron is a potential inducer of hyperlipidemia when antioxidant protection is compromised. It is well established that saturated fat is atherogenic, and its consumption results in elevated blood lipids. Since unsaturated fat does not contribute to increases in blood lipids as does saturated fat, the National Academy of Sciences, the United States Department of Agriculture (USDA) and Health and Human Services (HHS) have recommended that dietary saturated fat be reduced or substituted with unsaturated fat. One of the mechanisms responsible for hyperlipidemic properties of saturated fat may involve iron. It has been reported that the consumption of saturated fat leads to elevations of liver iron, but unsaturated fat does not (Johnson et al., 1992). If indeed high liver iron has the potential to raise blood lipids when saturated fat is consumed, higher levels of dietary iron may be needed to negate the protective effect of unsaturated fat. In a recent study, we fed rats either a copper-deficient (0.6. gCu/g) or a copper-adequate (6.0. gCu/g) diet containing either saturated or unsaturated fat. The diets also contained either adequate (50. g Fe/g) or high iron (500. g Fe/g). The diets contained starch as the sole source of dietary carbohydrates. No fructose was fed because the consumption of fructose may mask hyperlipidemic properties of iron, and causes elevation of blood lipids by the synergistic effects of fructose and saturated animal fat. Unfortunately, this study had to be terminated prematurely due to untimely deaths of three copperdeficient rats fed the high levels of dietary iron. Regardless of the type of dietary fat, all copper-deficient rats belonging to the high-iron dietary groups exhibited the highest levels of liver iron. These levels of excess liver iron were associated with the highest levels of blood cholesterol and triglycerides. Collectively, the numerous studies conducted in our laboratory clearly show that under conditions of oxidative stress, neither the type of dietary carbohydrate nor the type of dietary fat but levels of dietary iron are potential inducers of hyperlipidemia and coronary heart disease.

REFERENCES Araujo, J.A., Romano, E.L., Brito, B.E., Parthe, V., Romano, M., Bracho, M., Montano, R.F., and Cardier, J., 1995, Iron overload augments the development of atherosclerosis lesions in rabbits. Arterioscler. Thromb. Vase. Biol. 15:1172–1180. Ettinger, M.J., 1984, Copper metabolism and diseases of copper metabolism, in: Copper Proteins and Copper Enzymes, Volume III (R. Lontie, ed), pp. 175–229, CRC Press, Inc., Boca Raton, Florida.

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Fields, M., Ferretti, R.J., Smith, J.C., and Reiser, S., 1984, The interaction of type of dietary carbohydrates with copper deficiency, Am. J. Clin. Nutr. 39:389–395. Fields, M. and Lewis, C.G., 1997, Hepatic iron overload may contribute to hypertriglyceridemia and hypercholesterolemia in copper-deficient rats, Metabolism 46:377–381. Fields, M., Lewis, C.G., Lure, M.D., Burns, W.A., and Antholine, W.E., 1991, The severity of copper deficiency can be ameliorated by deferoxamine, Metabolism 40:105–109. Fields, M., Lewis, C.G., Lure, M.D., Burns, W.A., and Antholine, W.E., 1993, Low dietary iron prevents free radical formation and heart pathology of copper-deficient rats fed fructose, Proc. Soc. Exp. Biol. Med. 202:225–232. Halliwell, B., 1982, Superoxide-dependent formation of hydroxyl radicals in the presence of iron salts is a feasible source of hydroxyl radicals in vivo, Biochem. J. 205:461–462. Halliwell, B. and Chirico, S., 1993, Lipid peroxidation: its mechanism, measurement, and significance, Am. J. Clin. Nutr. 57(Suppl):715S–725S. Hart, E.B., Steenbock, H., Waddell, J., and Elvehjem, C.A., 1928, Iron in nutrition. VII Copper as a supplement to iron for hemoglobin building in the rat, J. Biol. Chem. 77:797–812. Johnson, P.E., Lukaski, H.C., and Korynta, E.D., 1992, Effects of stearic acid and beef tallow on iron utilization by the rat, Proc. Soc. Exp. Biol. Med. 200:480–486. McCord, J.M., 1991, Is iron sufficiency a risk factor in ischemic heart disease? Circulation 83:1112–1114. Salonen, J.T., 1993, Role of iron as cardiovascular risk factor, Curr. Opin. Lipidol. 4:277–282. Witztum, J.L. and Steinberg, D., 1991, Role of oxidized low density lipoproteins in atherogenesis, J. Clin. Invest. 88:1785–1802.

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SELENIUM AND CHRONIC HEART FAILURE

Michel de Lorgeril, Patricia Salen, and Michèle Accominotti Explorations Fonctionnelles Cardiorespiratoires et Métaboliques Niveau 6, CHU Nord de Saint-Etienne 42055 Saint-Etienne, France Laboratoire de Biochimie Pharmaco-Toxicologie et Analyse de Traces Hôpital Ed. Herriot, 69003 Lyon France

1. INTRODUCTION The incidence of chronic heart failure (CHF), the common end-result of most cardiopathies, is increasing steadily (Cowie, 1997). At the same time, evidence is accumulating that bioavailability and dietary intakes of selenium are falling in the community leading to declining serum selenium levels (Rayman, 1997). Selenium deficiency has been identified as a possible factor in the etiology of non-ischemic CHF syndromes, especially in areas of very low selenium intakes such as China (Keshan disease) and Western Africa (Ge, 1993). The main aim of this project was to investigate whether selenium (and certain other trace-elements) may be implicated in the occurrence and/or severity of CHF in our community.

2. METHODS AND PATIENTS We examined the relationships between serum selenium and exercise capacity in CHF patients. We also studied the relationships between dietary and serum selenium in these patients. We retained only clinically stable patients with recent measurements of left ventricular ejection fraction (LVEF), a marker of the severity of the myocardial disease, and of peak exercise oxygen consumption (peak an indicator of the functional tolerance of the disease and of the prognosis. All patients underwent symptom-limited upright bicycle exercise testing and respiratory gas analysis was performed with a breathby-breath apparatus. Dietary habits were evaluated using 24-hour recalls (de Lorgeril, 1994) using specific trace-element tables for selenium, zinc and copper intakes. We also studied sex- and age-matched apparently healthy subjects. Atomic emission spectroscopy Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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was used to measure copper and zinc and electrothermal atomic-absorption spectroscopy for selenium (Nève, 1987). Quality control for trace-element analysis was performed by comparison with reference standards and participation in an interlaboratory comparison study (Nève, 1992).

3. RESULTS No correlation was found between the trace-elements (see Table 1 for mean values) and LVEF (mean ± SE, 29 ± 6%) and between zinc and copper in one side and peak (15 ± 3ml/min/kg) on the other side. However, serum selenium was strongly correlated with peak by univariate analysis (polynomial regression) and r = 0.87 in a multilinear model in which selenium, age, sex and LVEF were entered as independent covariates and peak as the dependent variable. The relation between peak and LVEF was significant (partial r = 0.42, p < 0.05) only after adjustment for age, sex and selenium. In terms of nutrients (Table 2), there was a linear correlation between selenium intakes and serum levels: r = 0.65, p = 0.001. The lower selenium intakes in CHF patients resulted from lower intakes in grains and vegetables (confirmed by the significant difference in fiber intake) and in fish: selenium from fish represented (mean ± SE) in CHF versus 17.9 ± 5.6 in controls (p < 0.05), confirmed by the significantly lower intake in long-chain omega-3 fatty acids). It was not compensated by higher intakes of selenium from meat versus 2.9 ± 1.2, p < 0.05).

4. DISCUSSION This study strongly suggests that selenium is a major determinant of exercise tolerance in CHF patients. As we found a significant correlation between dietary and serum selenium (indicating that the low serum levels actually resulted from low intakes), the data are very suggestive of a causal relationship between low selenium and low exercise capacity. Trials are however needed to definitely prove that, by increasing selenium intake, the incidence and severity of CHF would be reduced. It has been proposed that antioxidants might be important in the genesis of CHF (Singal, 1998). Our discovery provides additional evidence in favor of this thesis and may contribute to explain the low cardiovascular mortality rate in Mediterranean populations, whose traditional diets are naturally rich in selenium, and in cardiac patients who adopt a Mediterranean diet (de Lorgeril, 1994) or increase their consumption of fish (Burr, 1989). The main dietary sources of selenium are actually grains, vegetables and marine foods, often used by the Mediterraneans. Worthy of mention, for instance, is that 100gr of cooked octopus, the Greek National dish, provides about of selenium.

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Selenium has a variety of functions. The main one is its role as an antioxidant in the enzyme selenium-glutathion peroxidase (GP). Recognition that several metabolic effects of selenium are not associated with GP has forced re-evaluation of its function. Another selenoprotein, designated selenoprotein P, has been discovered which is postulated to serve as extracellular antioxidant defense (Burk, 1994). More recently, the selenium-dependent thioredoxin reductase system has been proposed to contribute to ascorbate regeneration (May, 1992). CHF is associated with peripheral vasoconstriction and impaired skeletal muscle metabolism, both attributed to endothelial dysfunction (Drexler, 1998). There is an increased oxidative stress in CHF patients (Keith, 1998) and endothelial dysfunction is improved by ascorbate (Hornig, 1998). The seleno-dependent systems probably act synergistically with ascorbate18 to neutralize oxygen radicals and preserve endothelium function, which in turn helps to maintain exercise capacity. This may be a realistic interpretation of the close correlation we found between exercise capacity and selenium in this study. However, randomized trials are warranted to examine whether selenium supplementation may reduce the incidence and severity of CHF.

REFERENCES Burr M.L., Fehily A.M., and Gilbert J.F. 1989, Effects of changes in fat, fish, and fibre intakes on death and myocardial reinfarction: the Diet and Reinfarction trial (DART). Lancet; 334:757–761. Burk R.F. and Hill K.E. 1994, Selenoprotein P. A selenium-rich extracellular glycoprotein. J Nutr; 124:191–197. Cowie M.R., Mostred A., and Wood D.A. 1997, The epidemiology of heart failure. Eur Heart J; 18:208–225. De Lorgeril M., Renaud S., Mammelle N., Salen P., Monjaud I., Martin J.L., Touboul P., and Delaye J. 1994, Mediterranean alpha-linolenic acid-rich diet in secondary prevention of coronary heart disease. Lancet; 343:1454–1459. Drexler H. 1998, Endothelium as a therapeutic target in heart failure. Circulation; 98:2652–2655. Ge K. and Yang G. 1993, The epidemiology of selenium deficiency in the etiological study of endemic diseases in China. Am J Clin Nutr (Suppl); 57:259S–263S. Hornig B., Arakawa N., Kohler C., and Drexler H. 1998, Vitamin C improves endothelial function of conduit arteries in patients with chronic heart failure. Circulation; 97:363–368. May J.M., Mendiratta S., Hill K.E., and Burk R.F. 1997, Reduction of dehydroascorbate to ascorbate by the selenoenzyme thioredoxin reductase. J Biol Chem; 272:22607–22610. Keith M., Geranmayegan A., and Sole M.J. 1998, Increased oxidative stress in patients with congestive heart failure. J Am Coll Cardiol; 31:1352–1356. Nève J., Chamart S., and Molle L. 1987, Optimization of a direct procedure for the determination of selenium in plasma and erythrocytes using Zeeman effect atomic absorption spectroscopy. In: Brätter P. and Schramel P. eds. Trace element-analytical chemistry in medecine and biology. Vol 4. Berlin, Germany: Walter de Gruyter; 349–358.

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Nève J., Thomassen Y., and Van Damme M. 1992, Cooperative study on measurements of concentrations of selenium in freeze-dried blood. Pure Appl Chem; 64:765–780. Rayman M.P. 1997, Dietary selenium: time to act. BMJ; 314:387–388. Singal P.K., Khaper N., Palace V., and Kumar D. 1998, The role of oxidative stress in the genesis of heart disease. Cardiovasc Res; 40:426–432.

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ANTIOXIDANT STATUS OF PATIENTS WITH UNSTABLE ANGINA PECTORIS AND ACUTE MYOCARDIAL INFARCTION Momcilo B. Mihailovic1, Zorana Vasiljevic2, Sladjana Sobajic3, Ivan B. Jovanovic1, Olivera Pesut1, and Gordana Matic2 1

Department of Morphology and Physiology Faculty of Veterinary Medicine Belgrade, Yugoslavia 2 Coronary Care Unit Medical Clinical Center Belgrade, Yugoslavia 3 Department of Food Chemistry Faculty of Pharmacy Belgrade, Yugoslavia

1. INTRODUCTION Dietary selenium deficiency has been implicated in the etiology of cardiovascular diseases (Salonen, 1987; Huttunen, 1997). Epidemiological studies have provided some evidence for the role of selenium deficiency in etiology of atherosclerotic disease (Huttunen, 1997) and it was found that selenium status was lower in patients with coronary atherosclerosis (Yegin et al., 1997). The atherosclerotic process is followed by increased levels of lipid peroxides (Steinberg and Witztum, 1990). Selenoenzyme glutathione peroxidase, as an antioxidant enzyme, catalyses the reduction of a large range of lipid peroxides and, along with other cellular defenses, may play a role in preventing lipid peroxidation damage. Free radicals have been implicated as possible cause of reperfusion-arrhythmias and protective effect of selenium against ischemia-reperfusion injury was found (Tanguy et al., 1998). Malondialdehyde (MDA) is a major breakdown product split off from lipid peroxides and is accepted as an indirect marker of lipid peroxidation (Maseki et al., 1981). The aim of this study was to examine the antioxidant status of patients with unstable angina pectoris (USAP) and acute myocardial infarction (AMI) determining the blood and plasma selenium concentrations, plasma MDA and blood and plasma Se-dependent glutathione peroxidase (Se-GSHPX) activity. Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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2. PATIENTS AND METHODS Venous blood samples were taken from 83 randomly selected patients (USAP =13; men, 6; women, 7; AMI = 70; men, 50; women, 20) aged 36 to 86 years (mean 60.8) and 20 healthy volunteers (men 12, women 8) aged 39 to 79 years (mean 60.1) at the Coronary Care Unit of the Clinical Center in Belgrade, 1, 2, 3, 4 and 5 days after admission to the Unit. USAP and AMI were diagnosed on the basis of prolonged chest pain, ECG changes and activity of phosphokinase. Healthy volunteers had normal blood pressure, but most of the patients (USAP and AMI) were hypertensive over a long period before the incidence. Se concentration was determined using hydride generation atomic absorption spectrometry after wet ashing of the samples (Welz et al, 1987). Se-GSHPX was estimated spectrofotometricaly at 37°C by coupled test system (Sankari, 1985). The low concentration of butylhydroperoxide (<2.32nM) used in this method only determines the activity of Se-GSHPX (Burk et al, 1978). MDA was analyzed using the colorimetric method using thiobarbituric acid (Andreeva et al., 1988). Statistical evaluation of the results was performed using the Student’s t-test and linear regression analysis.

3. RESULTS None of the examined parameters were dependent on sex or time passed after the incidence. Whole blood and plasma Se concentrations in the control group were 81.9 and 71.8ng/ml, respectively (Fig. 1). Blood Se levels in USAP and AMI patients were signif-

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icantly lower (65.0 and 62.7ng/ml, respectively) as well as plasma Se levels (48.5 and 49.1 ng/ml, respectively). Blood and plasma Se-GSHPX activities in healthy subjects were 155.8 and respectively (Fig. 1). In comparison to the control group, significantly lower blood and plasma Se-GSHPX activities were found in USAP patients (128.9 and respectively) and AMI patients (123.1 and respectively). Mean plasma MDA level in control group was (Fig. 1). USAP patients had significantly higher MDA level Plasma MDA level of AMI patients was significantly higher than in both healthy controls and USAP patients About 30% of all AMI patients at the Coronary Care Unit were undergone to the thrombolitic therapy (streptokinase). Those had significantly higher levels of plasma MDA in comparison to AMI patients without thrombolytic therapy There was an inverse correlation between plasma MDA levels and plasma Se-GSHPX activities (r = 0.459, p < 0.005) (Fig. 2).

4. DISCUSSION AND CONCLUSION In comparison to the group of 20 clinically healthy individuals, USAP and AMI patients had significantly lower blood and plasma selenium levels and Se-GSHPX activities. These data are in agreement to those obtained by Salonen (1987) and Suadicani et al. (1992) who found an inverse correlation between selenium levels and the incidence of ischemic heart disease. These results could be interpreted by suppression of atherogenesis with selenium. Also, Pietinen et al. (1996) found that changes in diet in Finland from 1972 to 1992, which included three time increased selenium intake, have contributed to the dramatic decline in coronary heart disease mortality in Finland. Yegin et al. (1997) found that the decrease in both the activity of Se-GSHPX and selenium levels in erythrocytes parallel to the increase in the severity of coronary disease. A marked decrease of the antioxidant enzyme activities found in USAP and AMI patients suggest a possible drop in antioxidant status of the patients. In our previous study (Mihailovic et al., 1998) we found that hypertensive patients had decreased blood and plasma Se level and plasma Se-GSHPX activity and that arterial hypertension was the major risk factor for

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ischemic heart disease. The increased plasma levels of MDA is evidence of intensification of lipid peroxidation processes in the patients and might be regarded as a decreased ability of the organism to abolish free radicals. The higher level of plasma MDA in those AMI patient who were on thrombolitic therapy in comparison to those who were not, indicate that the increased lipid peroxidation is a possible cause of reperfusion syndrome. This is in agreement to results obtained by Tanguy et al. (1998). Decreased blood and plasma Se levels and GSHPX activities, and increased levels of plasma MDA in both USAP and AMI patients suggest that peroxides might play a key role in the genesis of ischemic heart disease. The higher level of plasma MDA in AMI patients who had undergone thrombolitic therapy than those who had not, indicate that peroxides might be included in the occurrence of reperfusion syndrome.

REFERENCES Andreeva, L.I., Kozhemyakin, L.A., and Kishkun, A.A., 1988, A modification of thiobarbituric test for measuring lipid peroxidation product, Labor. Delo. 11:48–58. Huttunen, J.K., 1997, Selenium and cardiovascular diseases—an update, Biomed. Environ. Sci. 10:220–226. Maseki, M., Nishigaki, I., Hagihara, M., Tomoda, Y., and Yagi, K., 1981, Lipid peroxide levels and lipid serum content of serum lipoprotein fractions of pregnant subjects with and without pre-eclampsia, Clin. Chim. Acta 155:155–161. Mihailovic, M.B., Avramovic, D.M., Jovanovic, I.B., Pesut, O.J., Matic, D.P., and Stojanov, V.J., 1998, Blood and plasma selenium levels and GSH-Px activities in patients with arterial hypertension and chronic heart disease, J. Environ. Path. Toxicol. Oncol. 17:285–289. Pietinen, P., Vartiainen, E., Seppanen, R., Aro, A., and Puska, P., 1996, Changes in diet in Finland from 1972 to 1992: impact on coronary heart disease risk, Prev. Med. 25:243–250. Salonen, J.T., 1987, Selenium in ischemic heart disease, Int. J. Epidemiol. 16:323–328. Steinberg, D. and Witztum, J.L., 1990, Lipoproteins and atherosclerosis, JAMA 264:3047–3057. Suadicani, P., Hein, H.O., and Gintelberg, F., 1992, Serum selenium concentration and risk of ischemic heart disease in a prospective cohort study of 3,000 males, Atherosclerosis 96:33–42. Tanguy, S., Boucher, F., Besse, S., Ducros, V., Faviar, A., and de Leiris, J., 1998, Trace elements and cardioprotection: increasing endogenous glutathione peroxidase activity by oral selenium supplementation in rats limits perfusion-induced arrythmias, J. Trace. Elem. Med. Biol. 12:28–38. Wojcicki, J., Razewicka, L., and Barcew-Wizniewska, B., 1991, Effect of selenium and vitamin E on the development of experimental atherosclerosis in rabbits, Atherosclerosis 87:9–16. Yegin, A., Yegin, H., Aiciguzel, Y., Deger, N., and Semiz, E., 1997, Erythrocyte selenium-glutathione peroxidase activity is lower in patients with coronary atherosclerosis, Jpn. Heart J. 38:793–798.

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THE EFFECT OF ZINC DEFICIENCY ON PARAMETERS OF LIPOPROTEIN METABOLISM AND LIPOLYSIS IN RATS FED DIFFERENT FATS

K. Eder, S. Wild, and M. Kirchgessner Institute of Nutrition Physiology Technical University of Munich D-85350 Freising Germany

1. INTRODUCTION Previous studies demonstrated that zinc deficiency causes hyperlipidemia and fatty livers in rats (Eder and Kirchgessner, 1994a,b). The reason for those effects are unknown. The present study, therefore, investigated several parameters of lipid metabolism in zincdeficient rats which could be associated with those observations. Since our recent studies demonstrated that the effect of zinc deficiency on the lipid metabolism is modified by the type of dietary fat, two different dietary fats (coconut oil with high levels of saturated fatty acids and fish oil with high levels of polyunsaturated fatty acids) were used.

2. MATERIALS AND METHODS An experiment with 48 male Sprague-Dawley rats weighing 123g in average was carried out. The rats were divided into four groups and received zinc-adequate (40mg Zn/kg) or zinc-deficient (0.8mgZn/kg) diets with a mixture of coconut oil and soybean oil (85:15, w/w) or salmon oil and soybean oil (85:15, w/w) as dietary fats. In order to ensure an adequate food intake, all the rats were force-fed four times daily (800, 1,300, 1,800, 2,300) by gastric tube and received 11.0g food dry matter per day. The semisynthetic diet consisted of (g/kg) casein (200), corn starch (300), saccharose (258), fat mixture (150), vitamins and minerals (60), cellulose (30) and DL methionine (2). The experiment included 10 days of regular feeding. At the morning of the day, rats were fed, where then three hours after injected with heparin (to release hepatic lipase and lipoprotein lipase from the endothel) and were exactly 15 minutes thereafter killed by decapitation. Parameters measured were: Lipids in liver, plasma and individual lipoprotein fractions; Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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free fatty acids and insulin in plasma; distribution of apolipoproteins in triglyceride-rich lipoproteins and in high-density lipoproteins; activities of lipoprotein lipase in postheparin plasma, heart and adipose tissue and the activity of hormone sensitive lipase in adipose tissue.

3. RESULTS AND DISCUSSION Zinc-deficient rats fed both types of fat exhibited lower body weight gains and lower zinc concentrations in plasma than the equivalent zinc-adequate control rats (see Table 1). This proves the zinc-deficient state of those rats. Zinc-deficient rats fed the coconut oil diet exhibited a fatty liver with markedly increased concentrations of triglycerides whereas zinc-deficient rats fed fish oil had only slightly increased concentrations of triglycerides in the liver compared with control rats. The activities of hepatic lipogenic enzymes as well as concentrations of free fatty acids in plasma were also stronger increased by zinc deficiency in the rats fed coconut oil than in the rats fed fish oil. Thus, the study suggests that the fatty liver in zinc-deficient rats

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fed the coconut oil diet is caused by enhanced de-novo fatty acid synthesis and by increased uptake of free fatty acids from the blood. Increased concentrations of free fatty acids in blood might to be due to an increased activity of hormone-sensitive lipase in adipose tissue. The activity of that enzyme was generally increased by zinc-deficiency; however, in disagreement with the effects on free fatty acids in plasma, this effect was more pronounced in rats fed fish oil than in rats fed coconut oil. Insulin is known to suppress the lipolysis in adipose tissue, and thus an impaired secretion of insulin could be the reason for enhanced lipolysis. However, plasma concentrations of insulin were not reduced, but rather increased by zinc-deficiency. Zinc-deficient rats fed coconut oil also exhibited markedly increased concentrations of triglycerides in plasma and triglyceriderich lipoproteins (chylomicrons and VLDL) whereas zinc-deficient rats fed fish oil did not. This result indicates an impaired plasma triglyceride clearance in zinc-deficient rats fed coconut oil. This suggestion is confirmed by a reduced activity of lipoprotein lipase in postheparin-serum and adipose tissue in those animals. Zinc-deficient rats fed fish oil, in contrast, did not exhibit altered activities of post-heparin plasma lipoprotein lipase. It is known that lipoprotein lipase is regulated by apolipoproteins in lipoproteins. Zinc deficiency reduced the percentage of apo C in triglyceride-rich lipoproteins and in HDL. In the fraction of chylomicrons plus VLDL, this effect caused by zinc-deficiency was stronger in the rats fed coconut oil than in the rats fed fish oil. This suggests that the reduced activity of lipoprotein lipase and the increased concentrations of triglycerides in plasma and triglyceride-rich lipoproteins in zinc-deficient rats fed coconut oil are caused by an altered rate of apolipoprotein synthesis. In conclusion, this study shows that zinc deficiency has profound effects on lipogenesis, release of fatty acids from adipose tissue and the composition of apolipoproteins. In general, those effects were more pronounced in rats fed diets with a high level of saturated fatty acids than in rats fed diets with a high level of polyunsaturated fatty acids. Hence, this study in agreement with previous demonstrates that the effect of zinc deficiency on the lipid metabolism is modified by dietary fatty acids.

REFERENCES Eder, K. and Kirchgessner, M. 1994a: The effect of isolated zinc deficiency on parameters of lipid and protein metabolism in rats fed a diet with coconut oil or fish oil. Trace Elem. Electrolytes 11:55–60. Eder, K. and Kirchgessner, M. 1994b: Dietary fat influences the effect of zinc deficiency on liver lipids and fatty acids in rats force-fed equal quantities of diet. J. Nutr. 124:1917–1924.

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PLASMA LEVEL OF ANTIOXIDANT MINERALS (Cu, Zn, Mn, AND Se) AND Fe A Comparison of Patients with Cerebrovascular Disease and Healthy Adults in Korea

I. S. Kwun, H. S. Jang*, and C. S. Kwon Department of Food and Nutrition Andong National University Andong *Department of Home Economics Education Kyungpook National University Taegu, South Korea

1. INTRODUCTION Attention has been focused on antioxidant micronutrients, since the disorder of free radical events may be involved in a number of diseases. It has been reported that oxidative stress may play a role in the pathogenesis of cerebral ischemia (Siesjo, 1992), and Alzheimer’s disease and vascular dementia (Ihara et al., 1997). Several trace elements protect the cell from oxidative cell damage by incorporating into antioxidant enzymes. Zinc, copper and manganese are required for superoxide dismutases in both cytosol and mitochondria. Selenium is an essential component of glutathione peroxidase. Iron is a constituent of catalase, a hemeprotein, which catalyzes the decomposition of hydrogen peroxide (Machlin and Bendich, 1987). In this study, the level of plasma antioxidant minerals (Zn, Cu, Mn, and Se) and Fe in patients with cerebrovascular disease (CVD) was compared with those of healthy adults to investigate the implication of antioxidant trace elements in this chronic disease.

2. MATERIALS AND METHODS Subjects Fifty patients, aged 32–79 yrs, with cerebrovascular disease and 117 healthy adults, aged 21–73, were selected for this study. Among the 50 patients (male 25, female 25), 23 Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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were from cerebral infarction, 12 from cerebral hemorrhage, and 15 from cerebral ischemia.

Plasma Trace Elements (Cu, Zn, Mn, Se, and Mn) Determination For the plasma trace element assay, venous blood samples were collected in heparinized, trace metal-free polyethylene tubes after overnight fasting. Plasma samples were diluted 1:10 with 0.125 mol HCl for trace element analysis. Plasma Cu, Zn, Mn, and Se levels were determined by Inductively Coupled Plasma Spectrometer-Mass Spectrometer (VG Elemental, PQIII STE, England) and Fe measured by Atomic Absorption Spectrophotometer (Perkin Elmer 5100; Perkin Elmer, Norwalk, CT).

TBARS (Thiobarbituric Acid-Reactive Substances) Determination Plasma TBARS for lipid peroxide determination were assayed by the spectrophotometric Yagi’s method, as described by Ohkawa, Ohishi, and Yagi (1979), using malonaldehyde bis (diethylacetal) as the standard and expressing the results as nmol/mL.

Statistical Analysis Differences between the patients with CVD group and the healthy subjects group, male and female within each patients or healthy subjects group, and age groups were tested by one-way analysis of variance. Duncan’s multiple range test was used for multiple comparison between age groups.

3. RESULTS AND DISCUSSION Cu, Zn, Mn, Se, and Fe Concentrations in Plasma The concentrations of plasma antioxidant minerals in the patients with CVD and the healthy subjects are shown in Table 1. Plasma Cu, Zn, Mn, Fe, and Se concentrations in the patients with CVD were significantly lower than those of the antioxidant trace minerals in the healthy subjects (at p < 0.05 except Se at p < 0.01). Interestingly, the plasma Mn level in both the patients with CVD (350.5 ± 26.2ng/dl) and the healthy subjects

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(469.9 ± 35.0ng/dl) was much higher compared to normal plasma Mn ranges (35.9 ~ 95:9 ng/dl) (Aggett, 1991). Most of the antioxidant mineral concentrations, except Se in the patients with CVD, were in the normal ranges in both the patients with CVD and the healthy subjects.

Plasma TBARS Concentration Plasma TBARS concentrations in the patients with CVD and the healthy subjects are also shown in Table 1. Plasma TBARS concentration was higher in the patients with CVD group even without statistical significance.

Plasma Trace Elements Concentration by Sex and Age Group Sex did not affect the plasma antioxidant mineral levels in either group, except the Fe level in the healthy subjects group. Fe concentration was higher for males (207.0 ± 86.3mg/dl) than for females (121.5 ± 50.5mg/dl) (p < 0.05).

4. CONCLUSION The results of this study suggest that the incidence of cerebrovascular disease was related with lower plasma antioxidant mineral concentrations (Cu, Fe, Mn, Se, and Zn). Specifically, Se was the major depleted antioxidant trace element in the patients with CVD.

REFERENCES Ihara, Y., Hayabara, T., Sasaki, K., Fujisawa, Y., Dawada, R., Yamamoto, T., Nakashima, Y., Yoshimune, S., Kawai, M., Kibata, M., and Kuroda, S., 1997, Free radicals and superoxide dismutase in blood of patients with Alzheimer’s disease and vascular dementia. J. Neural. Sci. 153:76–81. Machlin, L.J. and Bendich, A.A., 1987, Free radical tissue damage: protective role of antioxidant nutrients. FASEB J. 1:441–445. Ohkawa, H., Ohishi, N., and Yagi, K., 1979, Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal. Bioc. 95:351–358. Aggett, P.J., 1996, Essential mineral and trace element nutriture methodology, in: Nutritional Status Assessment, (F. Fidanza, ed.), 410–412, Chapman & Hall, New York. Siesjo, B.K., 1992, Pathophysiology and treatment of focal cerebral ischemia. Part II: Mechanisms of damage and treatment. J. Neurosurg. 77:337–354.

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TRACE ELEMENTS AS DETERMINED BY ICP-MS ANALYSIS IN PATIENTS WITH CORONARY ARTERY DISEASE

Ellen Burgess, Robert Audette*, Merril Knudtson, and George Wyse Foothills Hospital/University of Calgary 1403—29th Street, Calgary Alberta, T2N 2T9 and *University of Alberta Hospital 8440—112th Street, Edmonton, Alberta T6G 2B7

This baseline study examined a broad distribution of trace elements employing ICP-MS analysis from patients being actively treated for coronary artery disease. The biological specimens were collected using a stringent trace element protocol and a suite of trace elements were analysed in serum (Al, Ba, Be, Cu, Mn, Ni, Se, V, Zn), whole blood (Cd, Co, Mo, Pb, Tl) and urine (Al, As, Ba, Be, Bi, Cd, Cu, Hg, Mn, Sb, Se, Pb, Tl, V, Zn) employing a Perkin Elmer Elan 6000 ICP-MS. These patients (n = 14) ranged in age from 44–77 years with a mean age of 63 ± 10 years. Most trace elements were within the normal range, while some were either low normal (Zn) or elevated (Mn, Ni, Mo). Values were:

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Selenium levels are inversely related to age (r = –0.592, p = 0.03). Serum Mn levels correlated to serum Ni levels (r = 0.777, p = 0.001) and selenium levels correlated to zinc levels (r = –0.646, p – 0.013). Urinary Mn levels were closely correlated to serum Mn levels (r = 0.884, p < 0.001). Mn levels were high even with elevated urinary excretion whereas V levels were within normal range with elevated urinary excretion. In summary, the levels of most trace elements were within normal range with urinary excretions being high or normal. The mean serum level of Zn was low-normal, but levels of Mn, Ni and Mo were high. High serum levels of Mn with high urinary excretion suggests a high level of exposure in these adults.

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THE EFFECT OF ZINC DEFICIENCY ON SOME KEY ENZYMES OF LIPID METABOLISM IN RATS FED OLIVE OIL OR LINSEED OIL

K. Eder, K. Waldhauser, and M. Kirchgessner

Institute of Nutrition Physiology Technical University of Munich Freising, Germany

Previous studies demonstrated that zinc deficiency increases the levels of n-3 polyunsaturated fatty acids (PUFA) at the expense of n-6 PUFA in tissue phospholipids (PL). The reason for this is unknown. Altered activities of phospholipase A2 (PL A2) and lyso-phospholipid acyl transferases (AT) could be possible reasons for this phenomenon. Therefore, the present study investigated the activities of those enzymes in zinc-deficient rats fed diets containing linseed oil (rich in n-3 PUFA) or olive oil (rich in monounsaturated fatty acids). In order to control for the food intake, all the rats were fed by gastric tube. Semisynthetic diets used contained 0.5 (zinc-deficient diets) or 45 mg zinc per kg (control diets). The rats fed the zinc-deficient diets became severely zincdeficient after 13 days as proved by their zinc concentrations and activities of alkaline phosphatase in plasma. Zinc-deficient rats fed linseed oil diets exhibited increased levels of n-3 PUFA, particularly of eicosapentaenoic acid in hepatic PL at the expense of n-6 PUFA compared to their equivalent controls. In contrast, zinc-deficient rats fed olive oil exhibited only slight alterations of fatty acid levels compared to their controls. The concentration of phosphatidylcholine, the major PL in the liver was reduced by zinc deficiency in the rats fed linseed oil but not in the rats fed olive oil. The activity of hepatic PL A2 was not influenced by zinc deficiency, regardless of the dietary fat. In contrast, the activities of AT with various substrates were reduced by zinc deficiency in the rats fed linseed oil, but not in the rats fed olive oil. This suggests that an altered balance between n-6 and n-3 PUFA by zinc deficiency in rats fed diets rich in n-3 might be rather due to a disturbed incorporation of PUFA into PL than to a disturbed PL degradation. Additionally, the study shows that the effects of zinc deficiency on fatty acid metabolism are more severe in rats fed diets rich in n-3 PUFA than in rats fed diets with low levels of n-3 PUFA.

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LIPID CONCENTRATIONS OF LOW-DENSITY LIPOPROTEINS AND THEIR OXIDATIVE SUSCEPTIBILITY IN ZINC-DEFICIENT RATS

K. Eder and M. Kirchgessner Institute of Nutrition Physiology Technical University of Munich Freising Germany

It is known that the concentrations of lipids in low-density lipoproteins (LDL) as well as their susceptibility to lipid peroxidation in humans influence the risk for coronary heart disease (CHD). This study was performed to investigate whether zinc deficiency influences those parameters in rats fed diets rich in polyunsaturated fatty acids (PUFA) or monounsaturated fatty acids (MUFA). Therefore, rats were fed zinc-adequate (45 mg Zn/kg) or zinc-deficient (0.5 mg Zn/kg) semisynthetic diets containing either olive oil or linseed oil, using a bifactorial design. To ensure an adequate food intake, all the rats were force-fed by gastric tube. After 13 days of feeding, rats fed the zinc-deficient diets became severely zinc-deficient as proved by their zinc concentrations and activities of alkaline phosphatase in plasma. Lipid concentrations in plasma and LDL as well as the oxidative susceptibility of LDL to lipid peroxidation were mainly influenced by the type of fat. Rats fed olive oil had markedly higher lipid concentrations in plasma and LDL but a markedly lower susceptibility of LDL to copper induced in vitro lipid peroxidation than rats fed linseed oil. Zinc deficiency also affected those parameters; however, the direction of those effects was different for rats fed linseed oil and rats fed olive oil. In the rats fed linseed oil, zinc deficiency increased the concentration of cholesterol and total lipids in plasma and LDL and increased the copper-induced in-vitro peroxidation of LDL. The increased susceptibility to oxidation was due to an increased incorporation of long-chain n-3 PUFA into LDL, particularly of eicosapentaenoic acid; the concentrations of tocopherols in LDL as most important antioxidants were not altered by zinc deficiency. In rats fed olive oil, zinc deficiency did not considerably alter plasma and LDL lipid concentrations. The susceptibility of LDL to lipid peroxidation was even reduced by zinc deficiency in the rats fed olive oil. The reason for this effect could not be explained on the base of LDL fatty acids and tocopherols because zinc-deficiency did not influence those parameters in the rats fed olive oil. In conclusion, the study suggests that zinc 566

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deficiency could influence parameters which are known to be associated with the development of CHD in humans. However, the effects of zinc deficiency were distinctive in rats fed different fats. In rats fed olive oil, the effects of zinc deficiency in this respect were favourable; in rats fed linseed oil, the effects of zinc deficiency in this respect were unfavourable.

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COMPARATIVE EVALUATION OF TRACE ELEMENTS CONTENTS IN AORTIC WALL AND IN HAIR SAMPLES OF YOUNG MALES A. V. Skalny**, A. A. Zhavoronkov*, A. L. Chernyaev*, and A. V. Koudrine*** Laboratory of Geographical Pathology Research Institute of Human Morphology Russian Acad. Med. Scie., Tsurupa Street 3, 117418, Moscow, Russia* International Center of Biotic Medicine Lesnaya., 59, 103055, Moscow, Russia** Imperial College School of Medicine Hammersmith Hospital Du Cane Road, London W12 0NN U.K***

Levels of 12 trace elements were investigated in aortic wall normal aorta and fatty streaks// and in hair samples from 35 men, 20–40 years of age, who died in automobile accidents or due to suicides /hanging/—23 persons /group 1/; 12 chronic alcohol abusers /group 2/ who died from alcoholic poisoning /concentration of alcohol in blood exceeded 3 ppm/. Trace elements contents in tissues were evaluated with plasma spectrometer ICAP-9000 Thermo Jarrell Ash /USA/. In group 1 no significant differences were found between trace elements levels in normal aorta and in fatty streaks. In alcohol abusers decrease of Fe, Co, Si concentrations was found, comparing with their levels in normal aorta, as well as decrease of Fe, Co, Mn, K and Pb levels, comparing with group 1. Elevation of Si and Ca concentrations and significant decrease of Pb level were found in normal aorta. In hair samples significant differences between the groups were seen only for Na levels. But a trend to increase of Fe, Mn, Mg, Zn, K and Pb contents was found in group of alcohol abusers. Correlative analysis demonstrated close relationships both between elements concentrations in aorta and hair samples, mostly in alcohol abusers and between levels of trace elements and volume of atherosclerotic lesions in aorta.

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HEART DISEASE RISK IN THE UK AS INFLUENCED BY ENVIRONMENTAL FACTORS AND DEFINED BY HAIR CALCIUM CONCENTRATION A. MacPherson1 and J. Bacsó2 1

SAC, Auchincruive, Ayr KA6 5HW Scotland 2 Institute of Nuclear Research H-4001 Debrecen, P.O. Box 51 Hungary

Maps showing the distribution of coronary heart disease (CHD) across the UK were produced and revealed the well recognised decreasing severity with decreasing latitude. Hair calcium concentration is inversely related to its concentration in the intima of the aorta such that high values are never associated with high aorta calcium or damage to the vessel walls. The reverse is also true and >90% of admissions to hospital of patients with acute myocardial infarction had low hair calcium concentrations. Thus the measurement of hair calcium concentration might prove to be a good diagnostic marker of CHD risk. To evaluate its suitability for this purpose a UK-wide survey of hair calcium concentrations was undertaken and the results related to the recorded incidence of CHD in the 40 different health districts sampled. Highly significant inverse relationships were found between hair calcium concentration and the standardised mortality ratios for coronary heart disease on both a district and county basis. Thus the measurement of hair calcium reliably reflected the known pattern of heart disease risk in the UK. This paper considers which factors may have helped to produce these highly significant regional differences in hair calcium concentration and their importance in the aetiology of CHD. The availability of hard drinking water, the amount of exposure to uv light, diet and stress might all be involved. This paper attempts to evaluate the first two of these parameters. Water hardness data were obtained from the Water Authorities and data on sunshine hours from the Metereological Office. Statistical analysis was by regression and multivariate regression techniques. In simple regression hair calcium, water hardness and sunshine hours accounted for 58, 41 and 49% respectively of the variance in CHD incidence. 569

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When mean CHD is regressed against these different parameters highly significant relationships are found as shown in the following equations: Mean CHD = 119 – 0.285 water hardness Mean CHD = 334 – 0.152 water hardness – 80.6 log hair Ca Mean CHD = 234 – 0.0827 Sunhrs Mean CHD = 389 – 0.0427 Sunhrs – 79.1 log hair Ca Mean CHD = 205 – 0.0598 Sunhrs – 0.131 water hardness Mean CHD = 334 – 0.0339 Sunhrs – 0.096 water hardness – 62.1 loghair Ca The combination of water hardness and sunshine hours accounted for 54% of the variance in CHD and for some 65% of the variance in hair calcium: log hair Ca = 2.22 + 0.0003 Sunhrs + 0.0015 water hardness Thus these two environmental factors account for two thirds of the variance in hair calcium concentration which in turn accounts for 58% of the inter-district variance in CHD. As hair calcium is inversely related to CHD incidence water hardness presumably exerts its effect by increasing the available dietary supply of the element while increased sunshine hours will reflect in increased uv light and vitamin D synthesis with consequent improved calcium absorption and metabolism. Southeast England benefits uniquely from both hard water and the highest sunshine hours in the UK which results in its comparatively low incidence of CHD.

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REGULATION OF HEPATIC CHOLESTEROL AND LIPOPROTEIN METABOLISM IN COPPER-DEFICIENT RATS

A. Mazur, C. Sérougne, C. Moundras, D. Bayle, F. Millat, and Y. Rayssiguier Centre de Recherches en Nutrition Humaine Unité Maladies Métaboliques et Micronutriments INRA, Theix, 63122 St Genès Champanelle and Laboratoire de Physiologie de la Nutrition Université Paris Sud, 91405 Orsay France

The hypercholesterolemic effect of copper deficiency in various experimental animal models is well documented, and it has been suggested that it arises from an increased rate of hepatic cholesterol synthesis. We have shown that copper-deficiencyinduced hypercholesterolemia is associated with changes in lipoprotein distribution and results mainly from an increase in the HDL and LDL fractions in conjunction with increased plasma apo E and apo B concentrations. In particular, the abundance of apo B100 in the plasma and its hepatic synthesis were increased in copper-deficient rats. Previously, it has been proposed that the increased cholesterol synthesis may be due to the elevated hepatic glutathione and in turn elevated 3-hydroxy-3-methyl-glutaryl CoA reductase activity, a key enzyme involved in cholesterogenesis. In the present work, we have studied the abundance and the activity of selected proteins involved in cholesterol and lipoprotein metabolism. For this purpose rats were fed a copper-deficient diet for 8 weeks and compared to pair-fed and ad libitum fed controls. The activity and protein level of HMG CoA reductase and hydroxylase were assessed in isolated liver microsomes radiochemically and by immunoblot. In isolated hepatic membranes, LDL binding was assessed using labeled lipoproteins and HDL binding protein (HB) levels were evaluated by using specific antibodies. The most important results of this study is that copper deficiency produced a marked increase in the activity of HMG CoA reductase and hydroxylase. The increase in the activity of HMG CoA reductase (assessed with and without addition of dithiothreitol) occurred without significant increase in the enzyme (protein) abundance. Our results demonstrate that the increased activity of HMG CoA reductase in copper deficiency is due to alteration in the regulation of enzyme 571

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activity and not of its protein mass. Since copper deficiency leads to a marked decrease in the antioxidant defense system further studies will be performed to establish the relationship between consequences of the oxidative stress and alteration in cholesterol metabolism.

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COPPER BODY STATUS AND CARDIOVASCULAR DISEASES

G. Mielcarz Department of General Chemistry Karol Marcinkowski University School of Medical Sciences 60-780 Poznan Poland

Copper deficiency is associated with a severe cardiomyopathy and EKG ST-T abnormalities. In rats and swine prolonged copper deficiency can provoked hemopericardium, hemothorax and heart rupture. However, the significance of these animal studies in humans is little known. Copper contains superoxide dismutase, an intracellular enzyme which prevents oxidation of lipoproteins. However it is difficult to assess copper body status by plasma copper determination, because copper concentration in plasma has large individual variations. In this study we investigated copper concentration in post-mortem material from hospitalised patients following sudden death. We determined copper concentration in heart, liver and kidney in 15 males and 18 females aged 35–85. The degree of atherosclerosis in the thoracic, abdominal and coronary arteries was measured by determining an atherosclerosis index on scale of 0–100%. It was two groups: 1—with low atherosclerosis (decreased intimal surface of artery below 70%) and 2—with high atherosclerosis (decreased intimal surface of artery above 70%). Samples were digested of 6mol/l nitric acid in 7ml Teflon vessels in a microwave oven and the resulting digests were made up to 2 ml with de-ionized water. Copper was determined by graphite furnace atomic absorption spectroscopy. There was a statistically significant difference (P < 0.05) for the mean liver copper concentration between high and low atherosclerosis. In high atherosclerosis degree in both male and female copper levels was decreased. Also in heart tissue copper concentration was lower (P < 0.05), but only in male. In kidney there was no significant difference in copper concentration in both male and female. Our findings indicated that decreased body copper status in humans might be associated with developing of atherosclerosis.

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INCREASED PLASMA GLUTATHIONE PEROXIDASE ACTIVITY IN PATIENTS WITH ACUTE MYOCARDIAL INFARCTION

B. A. Zachara1, M. Ukleja-Adamowicz2, J. Lecka1, and E. Nartowicz2 1 2

Department of Biochemistry and Department of Cardiology and Internal Medicine Medical University, Bydgoszcz Poland

Glutathione peroxidase (GSH-Px), a selenium-dependent enzyme, is one of the most important enzymes involved in the protection of the cell against peroxidative processes, particularly in the heart muscle. The aim of the study was to determine the concentration of selenium (Se) and the activity of GSH-Px in patients with acute myocardial infarction (AMI) and to observe the behaviour of these parameters during the thrombolysis therapy. The study comprised two groups of AMI patients and a control group. The first group consisted of 49 patients from whom blood samples were taken after admission to the intensive care unit and subsequently after 3, 7, 14 and 30 days of hospitalization. In the second group of patients (n = 18) blood was taken for measuring only the GSH-Px activity in plasma. In this group blood samples were collected after admission to the hospital, 6, 12, 24, 48 hours, 3, 7, 14 and 30 days later. Control group comprised 58 healthy subjects. Se levels in whole blood and plasma were measured spectrofluorometrically with 2,3-diaminonaphthalene as a complexing reagent. GSH-Px activity in red cells and plasma was measured spectrofotometrically with t-butylhydroperoxide as substrate. In the first group of patients Se concentrations in whole blood and plasma as well as GSH-Px activities in red cells and plasma did not differ significantly from healthy subjects. Both Se levels and GSH-Px activities were stable during the entire period of the study. In the second group of patients, however, plasma GSH-Px activity increased after admission to the hospital and reached the highest value after 48 hours. This activity was significantly higher compared to healthy subjects (p < 0.01) and to the mean initial activity of this group (p < 0.02). In the later period the activity decreased to the values of healthy subjects. We suggest that the increased activity of GSH-Px in plasma of AMI patients is the response of the organism to the increased levels of reactive oxygen species produced during reperfusion and thrombolysis. 574

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SELENIUM IN CANCER PREVENTION M. P. Rayman* and L. C. Clark† *

School of Biological Sciences University of Surrey Guildford GU2 5XH, UK † Arizona Cancer Center College of Medicine University of Arizona, Tucson USA

There is a considerable body of evidence showing a protective effect of selenium (Se) against cancer which has recently been reinforced by the results of a number of important studies in the US and Finland (Clark et al., 1996; Clark et al., 1998; Knekt et al., 1998;Yoshizawa et al., 1998).

1. SELENIUM AND CANCER INCIDENCE The extensive experimental evidence, reviewed by Medina and Morrison (1988), and Combs and Clark (1997), indicates that Se supplementation reduces the incidence of cancer in animals There are over 100 published studies in 20 different experimental animal models of spontaneous, viral, and chemically induced tumours. In two thirds of these studies, a significant reduction in tumour incidence was reported and in one third of these studies, a reduction of over 50% in tumour incidence was observed. This literature indicates that Se supplementation in animals can reduce the incidence of skin, colorectal, breast, stomach, hepatic, oesophageal, oral, tracheal, pancreatic, kidney and lung cancers. Geographic studies have shown a consistent trend for populations with low Se levels to have a higher cancer mortality rate. Epidemiologic cancer studies in groups of individuals have produced more variable results, but the trend in most studies is for individuals with lower Se levels to have a higher incidence of cancer. A number of cohort and nested case-control studies have shown a lower risk of internal malignancies for individuals with higher plasma Se levels. A recent meta-analysis of cohort studies comparing the effect of serum Se, retinol, beta-carotene, and vitamin E suggests that Se has a remarkably consistent protective effect (Comstock, Bush, and Helzlsouer, 1992). Other Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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recent epidemiologic studies showing the protective effect of Se have involved cancers of the bladder and ovaries (Helzlsouer, Comstock, and Morris, 1989), pancreas (Burney, Comstock, and Morris, 1989), thyroid (Glattre et al., 1989), lung and stomach (Knekt et al., 1990). Additional epidemiologic designs have shown protective effects of Se for cancers of the esophagus (Jaskiewicz et al., 1988) lung (Gerhardsson et al., 1985; Miyamoto, Araya et al., 1987), skin (melanoma) (Reinhold et al., 1989), head and neck (Westin et al., 1989), brain (Philipov, and Tzatchev, 1988), stomach (Caygill et al., 1989) and a suggested effect for prostate cancer (Criqui et al., 1991; Hardell et al., 1995). Two studies published recently have reinforced these findings. In a nested casecontrol study within a cohort of 9,000 Finnish individuals, the adjusted relative risk of lung cancer between the highest and lowest tertiles of serum Se was found to be 0.41 (Knekt et al., 1998), while the Health Professionals’ Cohort Study demonstrated a strong inverse dose-response association between toenail Se and prostate cancer (Yoshizawa et al., 1998). This study showed a 65% reduction in the risk of advanced prostate cancer among men with the highest, as compared to the lowest, Se status (by quintile of toenail Se). Although not all epidemiologic studies have demonstrated a protective effect for high plasma Se levels and cancer, contradictory results may relate to the intricacies of epidemiologic research and the complexities involved in evaluating Se status (Willett, 1988; Clark and Alberts, 1995). For instance, a recently published Italian study (Vinceti et al., 1998) showed a small excess of malignant melanoma in subjects exposed to inorganic Se from tap water. However the excess number of cases was small (n = 6) suggesting the possibility of a random effect and there was no data on the Se status of the subjects. An earlier case-control study of Se and melanoma in Germany observed a significant inverse association with serum Se levels (Reinhold et al., 1989) while the NPC trial (Clark et al., 1996) showed no effect on melanoma incidence of a supplement of organic Se yeast.

2. SELENIUM AND CANCER PREVENTION Intervention Trials in China NCI sponsored trials in China for the prevention of oesophageal cancer observed a reduction in total cancer mortality in the intervention arm containing selenium, and vitamin E (Blot, Li, Taylor, Guo, Dawsey, and Li, 1995). Other trials in China reported a reduced incidence of liver cancer but methodological issues cloud a clear interpretation of the results (Yu, Zhu, and Li, 1997). Recently, in a double-blind randomised clinical trial in Italy, a 44% reduction in the incidence of recurrent neoplastic polyps was observed for subjects in the group randomised to of Se plus other vitamins and minerals versus a placebo (Bonelli, Conio, and Massa, 1998).

The Nutritional Prevention of Cancer (NPC) Trial The Nutritional Prevention of Cancer (NPC) trial (Clark et al., 1996; Clark et al., 1998) was the first trial conducted in a Western population, to show that a nutritional supplement could reduce the incidence of cancer. No known cancer strategy to date has produced results comparable with those of the NPC trial which used organic selenium as the preventative agent. This was a randomised, double-blind, placebo-controlled clin-

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ical intervention study, limited to patients (n = 1312) with a previous history of nonmelanoma skin cancer who were randomised from 1983–1991. The primary end points for the trial were the incidences of squamous cell carcinomas and basal cell carcinomas. The secondary end points, established in 1990, were all-cause mortality and total-cancer mortality, total cancer incidence (excluding non-melanoma skin cancer), and the incidences of lung, prostate and colorectal cancers. Patients received a daily dose of of Se or a placebo for up to 10 years. While there was no effect on the primary end points, there was a statistically significant reduction of 37% (RR = 0.63, 95% CI, 0.47–0.85) in total cancer incidence with the supplement of selenium (Clark et al., 1996; Clark et al., 1998). There were 77 cancers in the treatment group versus 119 cases in the placebo group during the first ten-years of follow-up between 1983 and 1993. A significant reduction in the incidence of lung (46%), colorectal (58%), and prostate cancer (63%) was observed. A statistically significant 50% reduction in total cancer mortality was also observed in the Se-supplemented group, with 29 deaths in the treatment group and 57 deaths among controls (RR = 0.50, 95% CI, 0.31–0.80). Sub-group analysis for the blinded phase, 1983–96, led to some interesting findings. Analysis of total cancer incidence by gender showed a 32% reduction in total cancer incidence for males in the Se group versus the placebo group, and a 9% reduction for females. Males appear to have a much larger and more precisely estimated treatment effect than females. The low proportion of women randomised in the NPC trial does not provide an adequate sample size for a precise estimate of the treatment effect. Analysis of total cancer incidence by age showed a more significant treatment effect in those over 65 (RR 0.53; 95% CI 0.36, 0.78; p < 0.001) compared to those under 65 (RR 0.77; 95% CI 0.47, 1.25; p = 0.25). Analysis of treatment effect by plasma Se level shows that the strongest treatment effect was observed in subjects who were classified at baseline as being in the bottom tertile of plasma (Table 1).

3. IMPLICATIONS OF LINK BETWEEN MAGNITUDE OF TREATMENT EFFECT IN NPC TRIAL AND SELENIUM STATUS Plasma Se levels below are now the norm in much of northern Europe (with the exception of Finland where Se is added to fertilizers). Part of the reason for this is that the use of high Se wheat from North America for bread making in some of these areas has largely been replaced by low-Se European and UK varieties (Rayman, 1997). For instance, in the UK, levies imposed on foreign imports when Britain

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joined the European Union (EU), coupled with changes in bread-making technology, resulted in an increased use of low-selenium, lower-protein European and UK varieties of wheat. Subjects in the NPC trial had a baseline Se intake of around (Clark et al., 1996). Current dietary Se intake in the UK is around (Ministry of Agriculture, Fisheries and Food, 1997), much lower than the RDA of for men and for women. The estimated mean intake of Se of adults in Sweden and Denmark is similarly low (Johnsson and Akesson, 1997; Suadicani, Hein, and Gyntelberg, 1997). In the early 1980s, intake was in the Dutch population. Given the increased market share of European versus American wheat in the Netherlands, Se intake is likely to have decreased since then. Correspondingly low serum/plasma selenium levels have been found in these countries, particularly in the UK, where values of were measured recently (Barrington et al., 1997; Shortt et al., 1997; Scott et al., 1998). Literature values of serum/plasma Se measured in various European locations [France (4), Italy (3), Spain, Austria, Denmark (2), Germany, Sweden, Poland, UK(4)] since 1990 have been collected together in Figure 1. In these 18 locations, the highest value of serum/plasma selenium measured was (Veneto, Italy). Thus many subjects from European locations would now fall into the bottom tertile of the NPC trial Such European populations would therefore be ideal for testing the effect of Se supplementation on cancer risk, as the strongest treatment effect for cancer in the NPC trial was observed in those participants who had the lowest baseline Se levels.

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4. THE PRECISE TRIAL One of the principal conclusions of the NPC trial and the editorial comment on the trial (JAMA, JNCI, Lancet, DNA Biomarkers & Prevention) was the need to confirm the results in additional independent trials, prior to making public health recommendations regarding Se supplementation (Clark et al., 1996). This conclusion provides a strong rationale for conducting a cancer prevention trial in the general population, in order to replicate and extend the results of the NPC trial. The PRECISE trial has been planned to provide more generalisable results, as 40,000 subjects will be recruited from the general population of 60 to 74-year olds in several different countries—Denmark, Finland, Sweden, the UK and the US. The data from the low-Se countries can be compared to those study cohorts from the US and Finland where Se intakes, and therefore status, are higher but still moderate. In addition, the PRECISE trial will both replicate and extend the design of the NPC trial, by using supplementation doses of and of Se per day. Confirmation of the viability of this mode of preventing cancer by extending this study to other population groups as planned in PRECISE, would have tremendous potential for reducing cancer incidence and mortality for many of the commonest forms of the disease at relatively low cost and should not be delayed, particularly in the light of declining Se status in many of the participating countries, notably the UK.

ACKNOWLEDGMENTS M.P.R. gratefully acknowledges the support of the Cancer Research Campaign for the UK pilot of the PRECISE Clinical Trial.

REFERENCES Barrington, J.W., Taylor, M., Smith, S., and Bowen-Simpkins, P.J., 1997, Selenium and recurrent miscarriage, J. Obs. Gyn. 17:199–200. Blot, W., Li, J.Y., Taylor, P.R., Guo, W., Dawsey, S.M., and Li, B., 1995, The Linxian Trials: Mortality rates by vitamin-mineral intervention group, Am. J. Clin. Nutr. 62:1424S–1426S. Bonelli, L., Conio, M., and Massa, P., 1998, Chemoprevention with Antioxidants of Metachronus Adenomas of the Large Bowel, Cancer Prevention and Control 100:A351. Burney, P.G.J., Comstock, G.W., and Morris, J.S., 1989, Serologic Precursors of Cancer: Serum Micronutrients and the Subsequent Risk of Pancreatic Cancer, Am. J. Clin. Nutr., 49:895–900. Caygill, C.P., Lavery, K., Judd, P.A., Hill, M.J., and Diplock, A.T., 1989, Serum selenium and gastric cancer in two regions of Norfolk, Food Additives & Contaminants, 6:359–363. Clark, L.C. and Alberts, D.S., 1995, Selenium and Cancer: Risk or Protection? J. N. C. I. 87:473–475. Clark, L.C., Combs, G.F.Jr., Turnbull, B.W., Slate, E.H., Chalker, D.K., Chow, J., Davis, L.S., Glover, R.A., Graham, G.F., Gross, E.G., Kongrad, A., Lesher, Jr, J.L., Kim Park, H., Sanders, Jr, B.B., Smith, C.L., and Taylor, R., for the Nutritional Prevention of Cancer Study Group, 1996, Effects of selenium supplementation for cancer prevention in patients with carcinoma of the skin: A randomized controlled trial, J. A. M. A. 276:1957–1963, [published erratum appears in J. A. M. A. (1997) 277(19): 1520]. Comment in: J. A. M. A., 1996, 276(24):1984–1985. Comment in: J. A. M. A., 1997, 277(11):880–1. Clark, L.C., Dalkin, B., Krongrad, A., Combs Jr, G.F., Turnbull, B.W., Slate, E.H., Witherington, R., Herlong, J.H., Janosko, E., Carpenter, D., and Borosso, C., 1998, Decreased Incidence of Prostate Cancer with Selenium Supplementation: Results of a Double-Blind Cancer Prevention Trial, Br. J. Urology 81:730–734.

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Combs, G.F. and Clark, L., 1997, Selenium and Cancer Prevention, in: Antioxidant Nutrients and Disease Prevention, (Garewal, ed.), CRC Press, Boca Raton, F1. Comstock, G., Bush, T., and Helzlsouer, K., 1992, Serum Retinol, Beta-Carotene, Vitamin E, and Selenium as Related to Subsequent Cancer of Specific Sites, Am. J. Epidemiol. 135:115–120. Criqui, M.H., Bangdiwala, S., Goodman, D.S., Blanar, W.S., Morris, J.S., Kritchevsky, S., Lippel, K., Mebane, I., and Tyroler, H.A., 1991, Selenium, retinol, retinol-binding protein and uric acid: Associations with cancer mortality in a population-based prospective case-control study, Ann. Epidemiol 1:385–393. Gerhardsson, L., Brune, D., Nordberg, I.G.F., and Wester, P.O., 1985, Protective Effect of Selenium on Lung Cancer in Smelter Workers, Brit. J. Industrial Med. 42: 617–626. Glattre, E., Thomassen, Y., Thoresen, S.O., Haldorsen, T., Lund-Larsen, P.G., Theodorsen, L., and Aaseth, J., 1989, Prediagnostic Serum Selenium in a Case-Control Study of Thyroid Cancer, Int. J. Epidemiol. 18:45–49. Hardell, L., Degerman, A., Tomic, R., Marklund, S.L., and Bergfors, M., 1995, Levels of selenium in plasma and glutathione peroxidase in erythrocytes in patients with prostate cancer or benign hyperplasia, Eur. J. Cancer Prevention 4:91–95. Helzlsouer, K.J., Comstock, G.W., and Morris, J., 1989, Selenium, lycopene, alpha-tocopherol, beta-Carotene, retinol, and subsequent bladder cancer, Cancer Research, 49:6144–6148. Jaskiewicz, K., Marasas, F.O., Rossouw, J.E., Van Niekerk, F.E., and Heine Tech, E.W., 1988 Selenium and Other Mineral Elements in Populations at Risk for Esophageal Cancer, Cancer 62:2635–2639. Johnsson, L. and Akesson, B.A., 1997, Availability of selenium from soils in relation to human nutritional requirements in Sweden, Swedish Environmental Protection Agency. Stockholm, Sweden. Knekt, P., Aromaa, A., Maatela, J., Alfthan, G., Aaran, R.K., Hakama, M., Hakulinen, T., Peto, R., and Teppo, L., 1990, Serum Selenium and Subsequent Risk of Cancer Among Finnish Men and Women, J. N. C. I. 82:864–868. Knekt, P., Marniemi, J., Teppo, L., Heliovaara, M., and Aromaa, A., 1998, Is low selenium status a risk factor for lung cancer? Am. J. Epidemiol. 148, 975–982. Medina, D. and Morrison, D.G., 1988, Current Ideas on Selenium as a Chemopreventive Agent, Pathol. Immunopathology Research 7:187–199. Ministry of Agriculture, Fisheries and Food, Joint Food Safety and Standards Group, Food Surveillance Information Sheet No .126 (Oct. 1997), London, UK. Miyamoto, H., Araya, Y., Ito, M., Isobe, H., Dosaka, H., Shimizu T., Kishi, F., Yamamoto, I., Honma, H., and Kawakami, Y., 1987, Serum Selenium and Vitamin E Concentrations in Families of Lung Cancer Patients, Cancer 60:1159–1162. Philipov, P. and Tzatchev, K., 1988, Selenium concentrations in serum of patients with cerebral and extrecerebral tumors, Zenralblatt. Fur Neurchirurgie 49:344–347. Rayman, M.P., 1997, Dietary selenium: time to act, B. M. J. 314:387–388. Reinhold, U., Blitz, H., Bayer, W., and Schmidt, K.H., 1989, Serum selenium levels in patients with malignant melanoma, Acta Dermato-Venereol. 69:132–136. Scott, R., MacPherson, A., Yates, R.W.S., Hussain, B., and Dixon, J., 1998, The effect of oral selenium supplementation on human sperm motility, Br. J. Urology 82:76–80. Shortt, C.T., Duthie, G.G., Robertson, J.D., Morrice, P.C., Nicol, F., and Arthur, J.R., 1997, Selenium status of a group of Scottish Adults, Eur. J. Clin. Nutr. 51:400–404. Suadicani, P., Hein, H.O., and Gyntelberg. F., 1997, Strong mediators of social inequalities in risk of ischaemic • heart disease: a six-year follow-up in the Copenhagen Male Study, Int. J. Epidemiol. 26(3):516–522. Vinceti, M., Rothman, K.J., Bergomi, M., Borciani, N., Serra, L., and Vivoli, G., 1998, Excess melanoma incidence in a cohort exposed to high levels of environmental selenium, Cancer Epidemiol. Biomarkers Prevent. 7:853–856, Editorial: Clark, L.C. and Jacobs, E.T., 1998, Cancer Epidemiol Biomark. Prevent. 7:847–848, Editorial: Kristal, A.R. and Moe, G., 1998, Cancer Epidemiol. Biomarkers Prevent. 7:849–850, Reply: Vinceti et al., 1998, Cancer Epidemiol. Biomarkers Prevent. 7:853–856. Westin, T., Ahlbom, E., Johansson, E., Sandstrom, B., Karlberg, I., and Edstrom, S., 1989, Circulating Levels of Selenium and Zinc in Relation to Nutritional Status in Patients With Head and Neck Cancer, Arch. Otolaryngol.—Head Neck and Surg. 115:1079–1082. Willett, W.C., 1988, Selenium and Cancer: Whether selenium protects against cancer is still unknown, B. M. J. 297:573–574. Yoshizawa, K., Willett, W.C., Morris, S.J., Stampfer, M.J., Spiegelman, D., Rimm, E.B., and Giovannucci, E., 1998, Toenail selenium as a predictor for prostate cancer incidence, J. Natl. Cancer Inst. 90:1219–1224. Yu, Shu Yu, Zhu, Ya Jun, and Li, Wen Gang, 1997, Protective role of selenium against hepatitis B virus and primary liver cancer in Qidong, J. Biol. Trace Elem. Research 56:117–124.

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TRACE ELEMENTS IN HYPERTENSION

Gianfranco Vivoli, Margherita Bergomi, Paola Borella, and Sergio Rovesti Department of Hygiene Microbiology and Biostatistics University of Modena and Reggio Emilia Modena, Italy

As suggested by a large body of experimental and observational studies, some trace elements seem to be involved in blood pressure regulation. In this review we briefly summarize the controversial data reported in literature, as well as the results of our studies on the role played by some toxic (lead and cadmium) and essential (zinc and copper) trace elements in the pathogenesis and development of hypertension. Studies carried out on different animal species have shown that low doses of lead may induce a slight elevation of blood pressure (Perry et al., 1988). Most of the epidemiologic studies carried out since 1980, both population- and occupation-based, reviewed by Hertz–Picciotto and Croft (1993), have shown a positive relation between low-moderate lead exposure and blood pressure levels. In the general population, for increases in blood lead levels from below to over increases in blood pressure have been reported ranging from 1.2–4 to 1.4–8 mmHg, for diastolic and systolic blood pressure respectively. A recent meta-analysis has estimated that in men a decrease in blood lead levels from is related to a decrease in systolic blood pressure of 1.25 mmHg (Schwartz, 1995). Even the most recent studies have confirmed the influence of lead on blood pressure across a wide range of ages (Menditto et al., 1994; Proctor et al., 1996; Wolf et al., 1995). It has been observed that the cumulative lead burden, evaluated by lead content in cortical bone, is a significant risk factor for developing hypertension in the general male population (Hu et al., 1996). These data might support the temporal relation between exposure and outcome and suggest, in the general population, a cumulative effect of lead exposure. Although the relation between lead and hypertension is not universally accepted (Staessen et al., 1994), the evidence of a causal association is supported by the consistency of results across epidemiological studies carried out

Address all correspondence to: Dr. Sergio Rovesti, Department of Hygiene, Microbiology and Biostatistics, University of Modena and Reggio Emilia, Via Campi 287, 41100 Modena, Italy Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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with different methods on subjects at different levels of exposure, particularly as regards the direction of the effect and the appropriate temporality observed in longitudinal studies. Regarding the dose-response relation, it has not been possible to establish a threshold value, and the more constant pressure effect has been observed for low lead exposure. Furthermore, the biological plausibility, provided by the effects observed in different animal species fed with low–moderate doses of lead, strengthens the hypothesis of a causal association. Among the possible mechanisms reported by which lead may affect blood pressure are the nephrotoxicity of lead and a lead-effect on the calcium-mediated control of vascular smooth muscle contraction (Chai and Webb, 1988; Loghman-Adham, 1997). Changes in blood vessel permeability and catecholamine content of myocardium and blood vessels have been suggested by animal experiments. Furthermore lead may affect the renin-angiotensin system through neurogenic mediators (Hertz-Picciotto and Croft, 1993; Goyer, 1993; Schwartz, 1995). Recent data suggest that the rise in blood pressure linked to lead exposure may be mediated by increased serum levels of parathyroid hormone and calcitriol (Kristal-Boneh et al., 1998). Finally results of laboratory experiments (Ding et al., 1998) are consistent with the view that hypertension in lead-exposed animals is related to both decreased nitric oxide (NO) and increased reactive oxygen species. Regarding cadmium, many experimental data show that long-term exposure to this metal at environmental levels can induce a mild increase of blood pressure. However, the effect of cadmium on blood pressure has not been confirmed by other studies, as reviewed by Friberg et al. (Friberg et al., 1986). Possible explanations of these inconsistencies are the different experimental conditions (animal species, strain, sex, dose, and route of administration). As suggested by some animal studies, the relation between cadmium and blood pressure seems not to be linear but biphasic, and the effects can be observed at lower experimental doses or for the interaction with other cations (Boscolo and Carmignani, 1986). More conflicting results have been obtained from human studies, as recently reviewed (Houtman, 1996; Nakagawa and Nishijo, 1996). Some authors found higher levels of cadmium in different biological matrixes (blood, urine, hair, autoptic specimens) of hypertensives than in normotensives, while other studies did not detect any significant difference between cases and controls. The controversial results on this topic may be partially accountable to the different characteristics of the examined subjects as regards the levels of exposure, and to a not always adequate control of the potential confounding variables (age, sex, ethnic group, body mass index, smoking habits and pharmacological treatment). Our early studies on the relation between cadmium and blood pressure, carried out in the 1970s, showed that cadmium levels and cadmium/zinc ratio in kidney cortex of hypertensive subjects were significantly higher than in controls (Vivoli et al., 1975). More recently we have found higher levels of cadmium in blood of mild hypertensives compared to controls matched for the main confounding variables, with a greater difference in non-smokers (Vivoli et al., 1989). A significant relationship between blood pressure values and hair cadmium levels has also been observed in male children aged 6–7 years (Bergomi et al., 1989). In a cross-sectional population study carried out on Belgian urban and rural districts at low and high cadmium exposure, no statistical association was found between cadmium exposure and blood pressure levels. However, several markers of renal tubular dysfunction, urinary calcium, and serum alkaline phosphatase activity were positively associated to urinary cadmium levels (Staessen et al., 1996). In the National Health and Nutritional Examination Survey (NHANES II), carried out from 1976 to 1988, a positive relationship was observed between urinary cadmium and

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both systolic and diastolic blood pressure after checking for the main confounding variables. However, after exclusion of treated hypertensives, the regression coefficients between blood pressure and urinary cadmium were not significant (Whittemore et al., 1991). The mechanism by which cadmium can affect blood pressure regulation is not clearly defined. However, animal experiments have suggested an inhibitory effect of cadmium on contractile properties of vascular smooth muscle and cardiac muscle, which is probably accountable to a depletion of intracellular pool of calcium (Boscolo and Carmignani, 1986; Giles et al., 1983). Low levels of cadmium seem to inhibit the activity of monoamine oxidase, catechol-O-methyl transferase and the production of prostacyclin (Revis, 1977; Caprino et al., 1982). The renal damage induced by cadmium can cause hypertension through the retention of salts and water and through the production of angiotensin. It has been hypothesized that the cardiovascular effect of cadmium may be partly accountable to interaction with homeostasis of some essential elements. In rats treated with cadmium, a relevant increase of zinc and copper content was found in kidney cortex (Boscolo and Carmignani, 1986). In our studies on humans, we have observed a significant increase of the cadmium/zinc ratio in different biological matrixes (Vivoli et al., 1989). It has been hypothesized that an imbalance of zinc and copper may also be involved in blood pressure regulation, even if the literature data on this topic are scarce and conflicting (Klevay, 1989; Salonen et al., 1991; Houtman, 1996). Epidemiologic studies have shown significant differences in seric and urinary levels of zinc and copper between hypertensives and controls, whereas no difference was revealed in other reports (Bergomi et al., 1997; Vivoli et al., 1987; Vivoli et al., 1995). These conflicting data may be attributed to the difficulties in the assessment of zinc and copper status since the measure of these trace elements in serum and urine is not suitable to detecting marginal deficiencies. In a case-control study in which we evaluated the zinc and copper content of serum, urine, and hair of untreated hypertensives and matched controls, no significant difference was found in zinc levels of the biological matrixes investigated. We observed that the activity of the zinc-dependent enzymes alkaline phosphatase (AP) and lactic dehydrogenase (LDH) was significantly lower in hypertensives than in matched controls and negatively correlated to values of both diastolic and systolic blood pressure. Our most interesting observation concerns the finding of higher urinary copper excretion in hypertensives than in normotensives (Vivoli et al., 1987). These data were not observed in studies carried out in young populations by other authors (Taittonen, Nuutinen, Rasanen, MussaloRauhamaa, Turtinen, and Uhari, 1997). In order to confirm our previous results and better elucidate the mechanism by which zinc and copper may be involved in blood pressure regulation we have carried out another case-control study in mild untreated hypertensives (Vivoli et al., 1995; Bergomi et al., 1997). No significant difference was found between hypertensives and normotensives in the mean levels of the metals in the biological matrixes examined (serum, red blood cells and urine) and of the activity of AP, LDH, copper-zinc superoxide dismutase (Cu-Zn SOD) and lysyl oxidase (LOX) assayed in serum and/or red blood cells. Interesting results were observed in the analysis of the correlations, which showed a very different pattern according to blood pressure condition. A significant relationship between serum and urinary levels of both zinc and copper was found only in normotensives. The lack of any association in hypertensives suggests that in the hypertensive condition a modification of the homeostasis of zinc and copper might occur. It was also found that in hypertensives the activities of AP, LDH and Cu-Zn SOD were negatively related to diastolic blood pressure values. We also observed that in hypertensives, serum zinc was inversely correlated to blood pressure, while no significant

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relationship was found in normotensives. Furthermore, both diastolic and systolic blood pressure levels were negatively associated with LOX activity only in hypertensives. A rise of serum copper content, a decrease of Cu-Zn SOD activity in erythrocytes and a significant inverse relationship between LOX activity and blood pressure values in hypertensives were observed in our most recent investigation. These results are of particular interest since impaired activity of some copper-dependent enzymes might have adverse effects on the cardiovascular system. Experimental studies in copper-deprived animals have shown a reduction of endothelium control of vascular tone (Lacka et al., 1997; Lynch et al., 1997; Saari, 1992; Schuschke, 1997). Among the mechanisms by which copper deficiency may cause impairment of endothelial function, a reduced activity of copper-dependent enzymes Cu-Zn SOD and LOX has been hypothesized. An increased activity of free radicals, associated with the reduced activity of the antioxidant enzyme Cu-Zn SOD, may interfere with the normal NO-mediated relaxation through two mechanisms, a direct inactivation of NO and increased lipid peroxidation. Since the subendothelial collagen structure is known to have a role in vessel relaxation, a reduced activity of the copper-dependent enzyme LOX, with consequent impaired cross-linking of elastin and collagen, may also be involved in the alteration of endothelial function (Lacka et al., 1997; Saari, 1992; Schuschke, 1997). Although the mechanism by which zinc may interfere with blood pressure regulation is not clearly defined, there is considerable evidence that zinc deficiency can increase the tissue oxidative damage and that adequate zinc levels may be necessary in order to maintain the integrity of vascular endothelial cells. It has been suggested that lowered zinc concentrations in hypertensives could be indicative of increased lipid peroxidation levels (McClain et al., 1995; Lacka et al., 1997).

REFERENCES Bergomi, M., Borella, P., and Fantuzzi, G., 1989, Sangue, denti e capelli: tre diverse matrici utilizzate per valutare l’esposizione al piombo e al cadmio in bambini residenti in zona industriale, Ann. Ig. 1:1185–1196. Bergomi, M., Rovesti, S., Vinceti, M., Vivoli, R., Caselgrandi, E., and Vivoli, G., 1997, Zinc and copper status and blood pressure, J. Trace Elem. Med. Biol. 11:166–169. Boscolo, P. and Carmignani, M., 1986, Mechanisms of cardiovascular regulation in male rabbits chronically exposed to cadmium, Br. J. Ind. Med. 43:605–610. Caprino, L., Dolci, N., Togna, G., Villa, P., Bucci, R., and Carunchio, V., 1982, Effects of cadmium on platelet thromboxane and vascular prostacyclin production, Toxicol. Appl. Pharmacol. 65:185–188. Chai, S.S. and Webb, R.C., 1988, Effects of lead on vascular reactivity, Environ. Health Perspect. 78:86–89. Ding, Y., Vaziri, N.D., and Gonick, H.C., 1998, Lead-induced hypertension. II. Response to sequential infusions of L-arginine, superoxide dismutase, and nitroprusside, Environ. Res. 76:107–113. Friberg, L., Elinder, C.G., Kjellstrom, T., and Nordberg, G., 1986, In: Cadmium and health. A toxicological and epidemiological appraisal. Exposure dose and metabolism, CRC Press, Boca Raton, FL. Giles, W., Hume, J.R., and Shibata, E.F., 1983, Presynaptic and postsynaptic actions of cadmium in cardiac muscle, Fed. Proc. 42:2994–2997. Goyer, R.A., 1993, Lead toxicity: current concerns, Environ. Health Perspect. 100:177–187. Hertz-Picciotto, I. and Croft, J., 1993, Review of the relation between blood lead and blood pressure, Epidemiol. Rev. 15:352–373. Houtman, J.P., 1996, Trace elements and cardiovascular diseases, J. Cardiovasc. Risk 3:18–25. Hu, H., Aro, A., Payton, M., Korrick, S., Sparrow, D., Weiss, S.T., and Rotnitzky, A., 1996, The relationship of bone and blood lead to hypertension. The Normative Aging Study, JAMA 275:1171–1176. Klevay, L.M., 1989, Ischemic heart disease as copper deficiency, Adv. Exp. Med. Biol. 258:197–208. Kristal-Boneh, E., Froom, P., Yerushalmi, N., Harari, G., and Ribak, J., 1998, Calcitropic hormones and occupational lead exposure, Am. J. Epidemiol. 147:458–463.

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Lacka, B., Grzeszczak, W., Strojek, K., and Twardowska-Saucha, K., 1997, Lipid peroxidation, antioxidant enzymes activity, and trace element concentration in primary hypertension, Trace Elem. Electrolytes 14:61–65. Lynch, S.M., Frei, B., Morrow, J.D., Roberts, L.J., Xu, A., Jackson, T., Reyna, R., Klevay, L.M., Vita, J.A., and Keaney, J.F., 1997, Vascular superoxide dismutase deficiency impairs endothelial vasodilator function through direct inactivation of nitric oxide and increased lipid peroxidation, Arterioscler. Thromb. Vasc. Biol. 17:2975–2981. Loghman-Adham, M., 1997, Renal effects of environmental and occupational lead exposure, Environ. Health Perspect. 105:928–939. McClain, C., Morris, P., and Hennig, B., 1995, Zinc and endothelial function, Nutrition 11:117–120. Menditto, A., Morisi, G., Spagnolo, A., and Menotti, A., 1994, Association of blood lead to blood pressure in men aged 55 to 75 years: effect of selected social and biochemical confounders. NFR Study Group, Environ. Health Perspect. 102 Suppl 9:107–111. Nakagawa, H. and Nishijo, M., 1996, Environmental cadmium exposure, hypertension and cardiovascular risk, J. Cardiovasc. Risk 3:11–17. Perry, H.M.Jr, Erlanger, M.W., and Perry, E.F., 1988, Increase in the blood pressure of rats chronically fed low levels of lead, Environ. Health Perspect. 78:107–111. Proctor, S.P., Rotnitzky, A., Sparrow, D., Weiss, ST., and Hu, H., 1996, The relationship of blood lead and dietary calcium to blood pressure in the Normative Aging Study, Int. J. Epidemiol. 25:528–536. Revis, N., 1977, A possible mechanism for cadmium-induced hypertension in rats, Life Sci. 22:479–488. Saari, J.T.,1992, Dietary copper deficiency and endothelium-dependent relaxation of rat aorta, Proc. Soc. Exp. Biol. Med. 200:19–24. Salonen, J.T., Salonen, R., Korpela, H., Suntioinen, S., and Tuomilehto, J., 1991, Serum copper and the risk of acute myocardial infarction: a prospective population study in men in eastern Finland, Am. J. Epidemiol. 134:268–276. Schuschke, D.A., 1997, Dietary copper in the physiology of the microcirculation, J. Nutr. 127:2274–2281. Schwartz, J., 1995, Lead, blood pressure, and cardiovascular disease in men, Arch. Environ. Health 50:31–37. Staessen, J.A., Buchet, J.P., Ginucchio, G., Lauwerys, R.R., Lijnen, P., Roels, H., and Fagard, R., 1996, Public health implications of environmental exposure to cadmium and lead: an overview of epidemiological studies in Belgium. Working Groups, J. Cardiovasc. Risk 3:26–41. Staessen, J.A., Bulpitt, C.J., Fagard, R., Lauwerys, R.R., Roels, H., Thijs, L., and Amery, A., 1994, Hypertension caused by low-level lead exposure: myth or fact?, J. Cardiovasc. Risk 1:87–97. Taittonen, L., Nuutinen, M., Rasanen, L., Mussalo-Rauhamaa, H., Turtinen, J., and Uhari, M., 1997, Lack of association between copper, zinc, selenium and blood pressure among healthy children, J. Hum. Hypertens. 11:429–433. Vivoli, G., Barbolini, G., Vecchi, G., and Bergomi, M., 1975, Ricerche sull’accumulo renale ed aortico del cadmio e dello zinco e sull’eventuale rapporto con l’ipertensione arteriosa, Ig. Mod. 68:237–257. Vivoli, G., Bergomi, M., Borella, P., Fantuzzi, G., and Caselgrandi, E., 1989, Cadmium in blood, urine and hair related to human hypertension, J. Trace Elem. Electrolytes Health. Dis. 3:139–145. Vivoli, G., Bergomi, M., Rovesti, S., Pinotti, M., and Caselgrandi, E., 1995, Zinc, copper, and zinc- or copperdependent enzymes in human hypertension, Biol. Trace Elem. Res. 49:97–106. Vivoli, G., Borella, P., Bergomi, M., and Fantuzzi, G., 1987, Zinc and copper levels in serum, urine, and hair of humans in relation to blood pressure, Sci. Total Environ. 66:55–64. Whittemore, A.S., Di Ciccio, Y., and Provenzano, G., 1991, Urinary cadmium and blood pressure: results from the NHANES II survey, Environ. Health Perspect. 91:133–140. Wolf, C., Wallnofer, A., Waldhor, T., Vutuc, C., Meisinger, V., and Rudiger, H.W., 1995, Effect of lead on blood pressure in occupationally non exposed men, Am. J. Ind. Med. 27:897–903.

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PERSISTENCE OF GOITER DESPITE ORAL IODINE SUPPLEMENTATION IN GOITROUS CHILDREN WITH IRON-DEFICIENCY ANEMIA IN THE CÔTE D’IVOIRE

Michael Zimmermann, Pierre Adou, Toni Torresani, Christophe Zeder, and Richard Hurrell Human Nutrition Laboratory Swiss Federal Institute of Technology Zürich, Switzerland The Department of Pediatrics University of Zürich, Switzerland and The National Institute of Public Health Ministry of Health, Abidjan Côte d’Ivoire

1. INTRODUCTION There is a huge endemia of iodine deficiency disorders (IDD) in western and central Africa where it is estimated 250 million people are at risk for IDD and 50 million have goiter. Multiple nutritional and environmental influences contribute to the prevalence and severity of IDD in iodine-deficient areas. Deficiencies of selenium and vitamin A may modify thyroid hormone metabolism and potentially exacerbate IDD (Thilly et al., 1992; Wolde-Gabriel et al., 1993). Another micronutrient that could potentially influence IDD is iron. Animal and human studies have suggested iron deficiency impairs thyroid metabolism (Beard et al., 1990). In the Ivory Coast, more than half of schoolage children in the western and northern regions are iodine deficient and 23–25% suffer from iron-deficiency anemia (Latapie et al., 1981). The aim of this study was to determine whether iodine-deficient, goitrous children who were also suffering from iron deficiency anemia could synthesize thyroid hormones and reduce goiter size when given iodine.

2. MATERIALS AND METHODS The study was carried out in two isolated villages (total population = 1450) in an area of endemic goiter in the Danané Health District, a mountainous region in the Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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western Côte d’Ivoire. Children aged 6 to 15 years in the two villages (n = 419) were screened and divided into two groups. Group 1 (n = 53) consisted of goitrous children with a Hb > 120g/L; Group 2 (n = 51) consisted of goitrous children who were iron-deficient and anemic (Hb < 110 g/L). Baseline measurements on all children in the morning before administration of the iodized oil included: iodine in spot urines, TSH and serum on blood spotted onto filter paper, serum retinol and serum selenium. Thyroid gland volume was measured using an Aloka SSD-500 Echocamera (Aloka, Mure, Japan) with a high-resolution 7.5 MHz linear transducer. Each child in Groups 1 and 2 then received an oral dose of 0.4 ml iodized poppyseed oil (Lipiodol, Guerbet, France) containing 200 mg of iodine. At 1, 5, 10, 15 and 30 weeks post intervention, spot urines were collected for measurement of UI and dried blood spots for determination of TSH and At 10, 15 and 30 weeks, thyroid volume was measured using ultrasound. At 15 weeks, spot urines were collected for measurement of urinary thiocyanate. At 30 weeks a venous blood sample was collected for redetermination of Hb. Of the 109 children who began the study, 104 completed it. On completion of the study, the children in Group 2 were provided with supplemental iron. Normative values for thyroid volume in children aged 6–12 years according to sex, age and BSA were used to define the presence or absence of goiter (WHO/ICCIDD, 1997). Blood and urine samples were were aliquoted and frozen at –20 °C until analysis. UI, Hb, ferritin, TfR, urinary thiocyanate (SCN), retinol, selenium, whole blood TSH and serum were measured as described previously (Zimmermann et al., 1999). Iron deficiency anemia was considered present if: Hb < 110 g/L + serum ferritin.< or Hb < 11 g/dl + TfR < 8.5mg/L + ZPP > heme.

3. RESULTS Table 1 compares Groups 1 and 2 at baseline. Although the BMIs of the groups were similar, the means for height and weight in Group 2 were significantly less (p < 0.05) than Group 1. Table 2 shows the changes in thyroid volume in Groups 1 and 2 after receiving the iodized oil. Thyroid volume decreased significantly vs. baseline in both groups at 10 weeks (p < 0.001). At 15 and 30 weeks there was no further decrease in Group 2, while in Group 1, thyroid size continued to fall. At 15 and 30 weeks, thyroid volume was significantly reduced in Group 1 compared to Group 2 (p < 0.001). At 30 weeks the mean % change in thyroid volume from baseline was –45% in Group 1 and –22% in Group 2. These differences were reflected in the change in goiter prevalence at 10, 15, and 30 weeks. A sharp difference in prevalence was apparent at 15 and 30 weeks, when goiter rates were 62–64% in Group 2 but only 31% and 12% in Group 1. UI remained significantly increased above baseline at 30 weeks in both groups (p < 0.001). Median TSH values at 5, 10, 15 and 30 weeks were reduced significantly (p < 0.001) compared to baseline in Group 1, while there was no change in Group 2. To test for associations, multiple regression of % change in thyroid volume at 30 weeks on Hb, serum retinol, serum selenium, UI/SCN ratio was done. The regression of % change on Hb was highly significant (p < 0.001). Adding serum retinol, serum selenium or UI/SCN ratio did not improve the prediction. The strong correlation between Hb and % change in thyroid volume is shown in Figure 1.

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4. DISCUSSION Iron may be only one of many nutritional and environmental factors that influence the pathogenesis of IDD in iodine-deficient areas. Protein-energy malnutrition, food goitrogens such as thiocyanates (SCN) from cassava and deficiencies of selenium and vitamin A may aggravate goiter (Hurrell, 1997; Thilly et al., 1992; Wolde-Gabriel et al., 1993). However, using multiple regression, serum retinol, serum selenium, and UI/SCN ratio were not significantly correlated with % change in thyroid volume at 30 weeks after oral iodine, while regression of % change on Hb was highly significant (p < 0.001). The findings in this study suggest that the presence of iron deficiency anemia in children may

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limit the effectiveness of an iodine intervention program. If confirmed, this finding will have broad public health implications related to the control of IDD. More than 2 billion people—mainly young women and children, most in the developing countries—are iron deficient (WHO/UNICEF/UNU, 1998). Children and pregnant women are also highly vulnerable to iodine deficiency, and are the main target groups for iodine supplementation programs. If iron deficiency is a nutritional factor that influences the pathogenesis of IDD, it may have a greater impact on IDD than previously described goitrogens because of its high prevalence in vulnerable groups.

REFERENCES Beard, J.L., Borel, M.J., and Derr, J., 1990, Impaired thermoregulation and thyroid function in iron-deficiency anemia, Am. J. Clin. Nutr. 52:813. Hurrell, R.F., 1987, Bioavailability of iodine. Eur. J. Clin. Nutr. 51:S9. Latapie, J.L., Clerc, M., Beda, B., et al., 1981, Aspects cliniques et biologiques du goitre endémique dans la région de Man (Côte d’Ivoire). Ann. Endocrinol. 42:517–530. Thilly, C.H., Vanderpas, J.B., Bebe, N., et al., 1992, Iodine deficiency, other trace elements and goitrogenic factors in the etiopathology of iodine deficiency disorders. Biol. Trace Elem. Res. 32:229–243. Wolde-Gebriel, Z., West, C.E., Gebru, H., et al., 1993, Interrelationship between vitamin A, iodine and iron status in schoolchildren in Shoa Region, Central Ethiopia. Br. J. Nutr. 70:593–607. WHO/ICCIDD, 1997, Recommended normative values for thyroid volume in children aged 6–15 years. Bull. WHO 75(2):95–97. WHO/UNICEF/UNU, 1998, Iron Deficiency Anemia: Prevention, Assessment and Control. Report of a joint WHO/UNICEF/UNU consultation, WHO, Geneva. Zimmermann, M.B., Adou, P., Torresani, T., Zeder, C., and Hurrell, R.F., 1999, Persistence of goiter despite oral iodine supplementation in goitrous children with iron-deficiency anemia in the Côte d’Ivoire. Am. J. Clin. Nutr. (in press).

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TRACE ELEMENTS IN PATIENTS TREATED BY ON-LINE HEMODIAFILTRATION The Blood Chromium Behaviour A. Baj, G. Bregante, R. D’Angelo, G. Bonforte*, F. Toffoletto, and M. Surian* Medicina del lavoro e Servizio di Nefrologia e Dialisi* dell’Ospedale di Desio via Mazzini 1, 20033 DESIO (MI) Italia

1. INTRODUCTION The interest in trace elements in haemodialysed (HD) patients has been generated by the recognition that Al accumulation may cause toxicity. Anomalies of other trace elements in specific tissues are however commonly presents in HD patients (Gallieni, Brancaccio and Cozzolino, 1996) although the clinical relevance of these abnormalities remains unclear. Recent attention has been devoted to elevated level of Chromium in tissues of HD patients (Wallaeys et al., 1986) in contrast with very low essential levels in normal subjects (Minoia et al., 1990). A study by Wallaeys et al. described the pathways involved in transport of Cr across the dialysis membrane (Wallaeys, Cornelis, Sabbioni, 1988). Padovese et al. confirmed that fresh peritoneal dialysate contained a Cr level 17 fold above the indicative reference plasma Cr concentration (Padovese et al., 1992). Borquet et al. studied the kinetics of Cr in Continuous Ambulatory Peritoneal Dialysis patients (Borguet et al., 1996) and justified, with mathematical models, Cr accumulation in blood and tissues. In haemodiafiltration (HDF), the reinfusion fluids and large membrane pores, which allow the passage of larger protein molecules, are additional factors influencing the balance of trace elements (Van Renterghem, Cornelis and Vanholder, 1992). Accumulation or depletion of trace elements may be more relevant if we use large quantities of reinfusate, as in the case of on-line production of reinfusate (Canaud et al., 1998). For these reasons we studied Cr levels in patients treated by HDF with large amounts of on-line produced reinfusate and dialysate. We compared Cr whole blood concentrations with those of our HD patients and studied Cr concentrations in on-line HDF patients through an extended period. In order to guarantee safety of HDF patients, other trace elements, as well as Cr, were monitored both in blood and in dialysis fluids. Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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2. MATERIALS AND METHODS Seven trace elements (Al, Cd, Cr, Cu, Pb, Se and Zn) were repeatedly evaluated in 24 on-line HDF patients from July 1993 to March 1997, at the beginning and the end of the session, in a longitudinal study after 12 (16–18 patients) or 30 months (5 patients). Nine trace elements (Al, As, Cd, Cr, Cu, Hg, Pb, Se, Zn) were evaluated in tap water, water after double reverse osmosis (2RO) and in dialysate/reinfusate. Clinical, nutritional and biochemical parameters of the HDF patients are reported in Table 1. Basal trace element values were compared with 20 HD patients, 20 uremic patients who had not been on dialysis, and with control groups of normal subjects. HD patients were chosen among subjects treated in our dialysis unit with similar dialysis age as HDF patients. Normal values, from subjects resident in the same geographical area, were obtained in our laboratory with the same analytical procedures. On-line HDF patients were treated thrice weekly for 211 ± 21min per treatment using polysulfone dialysers. Reinfusate and dialysate were produced on-line using tap water after two passages through reverse osmosis. The composition of dialysate/reinfusate was 138mMol, 2mMol, l.5 mMol, 0.5mMol, 109mMol, 32mMol, 3 mMoli per liter. Reinfusate was obtained by two passages though a synthetic Fresenius Polysulfone membrane with a wall thickness of (Diasafe R). Blood samples (10 ml) were collected at the beginning and at the end of the dialysis session into acid decontaminated polystyrene heparinized tubes. Plasma was separated by gentle centrifugation. All tubes and laboratory ware were rinsed overnight with diluted hydrochloric acid and washed five time with double distilled water. Pre-analytical factors were checked by evaluating the metals release of every material used in the analysis routine. Trace elements were determined by atomic absorption spectroscopy with electrothermal atomisation (GFAAS), flame atomisation (FAAS) or hydride generation (HGAAS) depending on the element. The analytical performance was checked during every measuring trend by certified standard materials (CRM194, Community Bureau of Standards; SRM8419, National Bureau of Standards) and commercially available samples with declared amount of trace elements (Seronorm TM, Nycomed, Norway). The quality of the laboratory was verified regularly by external control programmes (TEQAS, Surrey University, UK).

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3. RESULTS Plasma or whole blood values of trace elements studied are reported in Table 2. Each group of patients was compared with normal subject (P-value calculated according to Student t-test). On-line HDF patients were also compared with HD patients but no significant differences were found except for Cu (P < 0.02) and Zn (P < 0.001). Concentrations of trace elements at the beginning and at the end of treatment sessions are reported in Table 3. A significant increase in Al, Cr, Cu and Zn was observed. In the longitudinal study at 12 months (Table 4), trace element concentration remained stable, except for Cu. At 30 months, an increase in Cu and Pb was observed despite the small number of patients studied during long-term follow up. Data regarding trace elements in water and dialysis fluids are reported in Table 5. There was a decrease in all trace elements after 2RO, statistically significant for Al, Cr and Zn. The concentrate salts added small quantities of trace element impurities as documented by greater values in dialysate/reinfusate. However, the final concentration of micropollutants in dialysate/reinfusate were always lower than recommended guideline by international associations (Surian et al., 1988). In Table 6, the simultaneous determinations of trace elements in dialysate coming into artificial kidney (D-in) and that going out (D-out) are reported. For all micropollutants there was a ratio D-out/D-in greater than unit.

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4. DISCUSSION As would be expected increased Cr blood levels were observed in uremic patients who had been on dialysis. According to previous and recent works of other authors blood Cr in haemodialised patients is, an average, twenty time the normal level. Uremic patients who had not been on dialysis don’t show difference in Cr blood levels from normal subjects. These findings suggest a major role played by exposition to Cr impurities during dialysis treatment rather than disturbed renal function with reduced excretion of the

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metal. The exposition of patients during dialysis treatment is confirmed by the presence of Cr in dialysis fluid. The concentrations of the metal are very low but the great volume of fluids employed during the treatment produces an abnormal exposition of the patients. The addition of commercially available salt concentrates, contributes to a little increase of Cr in dialysate, as well as of other trace elements, but, at the present time, it seems realistically impossible to improve the purity of these products. No differences were found between haemodialysis patients and on-line haemodiafiltration patients, so that the dialysis technique is not responsible for Cr blood levels found in patients. We did not observe a further accumulation of Cr in the longitudinal study; a steady state in on-line HDF patients is achieved at 14/16 fold the level of normal subjects. These findings for blood Cr as well as for other trace elements confirms the safety of the on-line HDF technique that can improve the overall quality of treatment in end stage renal failure patients. On the other hand the removal of trace elements during the HDF treatment is evident as their concentration in dialysate outflow is greater than at the inflow site. The increase in Al, Cd, Cu and Zn at the end of the treatment could be influenced by haemoconcentration and may be considered an expression of the increase of the fraction of trace elements linked to large proteins.

REFERENCES Borguet F., Wallaeys B., Cornelis R., and Lameire N., 1996, Transperitoneal absorption and kinetics of chromium in the continuous ambulatory peritoneal dialysis patient—an experimental and mathematical analysis, Nephron, 72:163–170. Canaud B., Bosc J.Y., Leray H., Stec F., Argiles A., Leblanc M., and Mion C., 1998, On-line haemodiafiltration: state of the art, Nephrol Dial Transplant, 13:3–11. Gallieni M., Brancaccio D., and Cozzolino M., 1996, Trace elements in renal failure: are they clinically important?, Nephron Dial Transplant, 11:1232–1235. Minoia C., Sabbioni E., Apostoli P., Pietra R., Pozzoli L., Gallorini M., Nicolau G., Alessio L., and Capodaglio E., 1990, Trace element reference values in tissues from inhabitants of the European community. I. a study of 46 elements in urine, blood and serum of Italian subjects, The Science of Total Environment, 95:89–105. Padovese P., Gallieni M., Brancaccio D., Pietra R., Fortaner S., Sabbioni E., Minoia C., Markakis K., and Berlin A., 1992, Trace elements in dialysis fluids and assessment of the exposure of patients on regular Hemodialysis, Hemofiltration and Continuous Ambulatory Petritoneal Dialysis, Nephron, 61:442–448. Surian M., Bonforte G., Scanziani R., Dozio B., Baj A., Dellavedova L., and Toffoletto F., 1998, Trace elements and micropollutant anions in the dialysis and reinfusion fluid prepared on-line for haemodiafiltration, Nephrol Dial Transplant, 13:24–28.

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Van Renterghem D., Cornelis R., and Vanholder R., 1992, Behaviour of 12 trace elements in serum of uremic patients on hemofiltration, J Trace Elements Electrolytes Health Dis, 6:169–174. Wallaeys B., Cornelis R., Mees L., and Lameire N., 1986, Trace elements in serum, packed cell and dialysate of CAPD patients, Kidney Int, 30:599–604. Wallaeys B., Cornelis R., and Sabbioni E., 1988, Kinetics of chromium during peritoneal dialysis, The Science of Total Environment, 71:401–410.

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RELATIONSHIPS BETWEEN TRACE ELEMENT NUTRITURE AND PROGRESSION OF HUMAN IMMUNODEFICIENCY VIRUS INFECTION

John D. Bogden, Joan H. Skurnick, Francis W. Kemp, Shenggao Han, Joan Lloyd, Herman Baker, and Donald B. Louria Department of Preventive Medicine and Community Health UMDNJ-New Jersey Medical School Newark, NJ 07103-2714 USA

Immune function is highly dependent on nutritional status, since its large mass and high rate of cellular turnover make the immune system a major user of nutrients. Furthermore, there is enhanced nutrient utilization during acute and chronic infections, including HIV-1 infection. Glutathione (GSH), magnesium (Mg), copper (Cu), zinc (Zn), and choline are among the nutrients required to support cellular immune function; we have found reduced intra and/or extracellular concentrations of GSH, Mg, and choline in patients with HIV-1 infection in a prior study (Skurnick et al., 1996). The current study assessed relationships among HIV-1 progression and several nutritional/biochemical variables including three essential elements—magnesium, copper and zinc.

METHODS Subjects were 104 HIV infected outpatients and 29 uninfected controls. Male (n = 88) and female (n = 45) subjects ranged in age from 35–57; the HIV infected subjects represented a broad range of disease progression. There were 34, 44, and 26 in CDC categories A, B, and C, respectively; their CD4 counts were 600 ± 43, 310 ± 23, 108 ±18 The range of CD4 counts was broad, 1–1,129 Trace element and mineral concentrations were determined by atomic absorption spectrophotometry, choline by a microbiological method, glutathione by spectrophotometry at 412 nm, and Address all correspondence to: Dr. John D. Bogden; Department of Preventive Medicine and Community Health; UMDNJ-New Jersey Medical School, 185 South Orange Avenue, Newark, NJ 07103-2714, U.S.A., telephone: 973-972-5432; fax: 973-972-7625; email: [email protected] Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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CD4 T lymphocyte counts by fluorescence activated cell sorting. Data were evaluated by analyses of variance, multiple regression analysis, and calculation of Pearson correlation coefficients.

RESULTS Blood concentrations for seronegative controls and at various stages of HIV infection are provided in Table 1. Hematocrits were decreased in HIV seropositive subjects with the most advanced disease (CDC stage C). Plasma copper concentrations were increased at CDC stage C. Plasma Mg and erythrocyte (RBC) Mg and GSH concentrations were decreased earlier in the course of infection. Seropositive subjects who reported a history of light to moderate ethanol ingestion had significantly lower RBC Mg concentrations than those who did not drink alcoholic beverages (4.23 ± 0.17 vs. 4.63 ± 0.10 mg/dl). Statistically significant (p < 0.05) associations were found between CD4 count and hematocrit (r = 0.40), plasma Mg (r = 0.25), and plasma Zn (r = 0.21). A multiple regression analysis using forward selection with CD4 count as the dependent variable was conducted; independent variables were hematocrit, erythrocyte Mg and GSH, ethanol consumption, age, sex, and plasma Mg, Cu, Zn and choline (total and free). Independent variables that were significant joint predictors of CD4 count were hematocrit (partial to enter model = 0.27), free plasma choline and plasma Zn Collectively, these 3 factors explained 43% of the variability in CD4 counts.

DISCUSSION The data show that RBC GSH and Mg concentrations decline early in the course of HIV infection, while plasma Mg and Zn concentrations decrease more slowly. The data also suggest that Mg nutriture may be further compromised in HIV infected patients who consume alcoholic beverages. The best predictors of CD4 count in univariate or multivariate models were hematocrit, plasma zinc, magnesium, and free choline. Limitations of the study are that the results describe associations that may not have a causal

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relationship, and that blood concentrations are only one measure of nutrient status. In addition, treatment of HIV-1 infection with drugs may contribute to compromised nutrient status. Nevertheless, the results provide additional evidence that changes in nutritional status may contribute to progression of HIV-1 infection. It is unlikely that compromised nutritional status functions in isolation to influence HIV-1 progression, but rather acts in concert with other factors such as viral load and genetics. Compromised nutrition that develops early in the course of infection may exert its most substantial adverse effects only after other factors result in further deterioration of host defenses. (Supported in part by NIH # N01-AI-95013 and Accuhealth, Inc.)

REFERENCE Skurnick, J.H., Bogden, J.D., Baker, H., Kemp, F.W., Sheffet, A., Quattrone, G., and Louria, D.B., 1996, Micronutrient profiles in HIV-1 infected heterosexual adults. J AIDS Human Retrovirol. 12:75–83.

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IRON AND MANGANESE HOMEOSTASIS IN CHRONIC LIVER DISEASE Relationship to Pallidal T1-Weighted Magnetic Resonance Signal Hyperintensity

Elise A. Malecki, Attila G. Devenyi, Todd F. Barron, Timothy J. Mosher, Paul Eslinger, Claire V. Flaherty-Craig, and Lorenzo Rossaro The Pennsylvania State University College of Medicine Hershey Pennsylvania, USA

1. INTRODUCTION Signal hyperintensities of the globus pallidus on T1-weighted magnetic resonance (MR) imaging have been described in cirrhotic patients (Devenyi et al., 1994; Hauser et al., 1996; Spahr et al., 1996; Thuluvath et al., 1995); these MR abnormalities have been correlated with the degree of portal systemic shunting, hyperammonemia, and severity of the liver disease. The correlation of these MR abnormalities with circulating Mn concentrations, together with the finding of increased Mn concentrations in the pallidi of cirrhotic patients, leads to the reasonable conclusion that these signal hyperintensities are due to the influence of paramagnetic Mn is excreted almost exclusively via the bile. Therefore, it is not surprising that a combination of cholestasis and portacaval shunting will contribute to Mn accumulation in extrahepatic tissues including brain. Similar MR abnormalities have been described in patients receiving Total Parenteral Nutrition with supplemental Mn (Mirowitz and Westrich, 1992; Quaghebeur et al., 1996). The competition between Mn and iron for intestinal absorption is well established in humans and rats. Given the interrelationships between Mn and iron absorption and transport, we elected to study the relationship between pallidal hyperintensity on T1weighted MRI and measures of iron and Mn status. Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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2. METHODS Ten female and eleven male patients, age 33–69y, with chronic liver disease were enrolled in the study after written consent was obtained. This study was approved by the Clinical Investigation Committee of The Pennsylvania State University College of Medicine. Diagnoses of the patients were as follows (n): alcoholic liver disease (5), primary biliary cirrhosis (9), primary sclerosing cholangitis (3), hepatitis B virus (2), hepatitis C virus (1), and deficiency (1). Patients were excluded if there was a history of transfusion therapy or parenteral nutrition within the past six months or if their occupational history included mining, smelting, or battery manufacture. Magnetic resonance images were obtained from patients on a Toshiba 1.5 T MRT 150A clinical scanner. Images were interpreted by a single radiologist masked to the clinical status of the patient and laboratory results.

3. RESULTS Figure 1 shows representative T1-weighted MR images. Eight subjects had normal MRI (MRI score = 1). Signal hyperintensity in the pallidum was observed in 13 of 21 subjects: four patients had mild hyperintensity (MRI score = 2), three moderate (MRI score = 3), and six exhibited marked hyperintensity (MRI score = 4). MRI score was associated with serum total bilirubin, alkaline phosphatase activities, and aspartate aminotransferase activities (tau-b = 0.36, P < 0.05; tau-b = 0.42, P < 0.05; tau-b = 0.42, P < 0.05; respectively). Erythrocyte Mn concentration was positively correlated with MRI score (tau-b = 0.52, P < 0.005). Four out of the six patients with the highest MRI score had abnormally elevated erythrocyte Mn concentrations Serum Mn concentrations were not as useful, as ten of the 21 patients had concentrations below the laboratory’s detection limit of Only two patients had abnormally high serum Mn concentrations both these patients had MRI scores of 2. The log of erythrocyte Mn concentration was inversely related to all measures of iron status tested: hemoglobin (R = –0.73, P < 0.0005); hematocrit (R = –0.62, P < 0.005); and serum Fe concentrations (R = –0.65, P < 0.005). The log of erythrocyte Mn concentration was also strongly inversely correlated with TIBC saturation (R = –0.62, P < 0.005). In one subject with PBC and iron deficiency, therapy with iron supplements reduced the erythrocyte Mn level from 170 to concommitant with an increase in the TIBC saturation from 7.1 to 28.2%. There was no appreciable attenuation of the MR signal hyperintensity one year later. The MRI signal hyperintensities did not present consistently with self-reported neurological symptoms in our sample of patients (Table 1), except for sleep disturbance. Tremor and weakness are hallmark symptoms of Mn neurotoxicity, and they tended to correlate with MRI score (P < 0.15).

4. DISCUSSION There is controversy over the role of the transferrin-receptor mediated uptake mechanism in the relationship between iron status and Mn accumulation in the brain.

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Transferrin’s normal function is to transport through the blood from enterocytes to target tissues. Under normal conditions transferrin is only about 30% saturated with leaving a potential binding capacity for other metal ions. Under acidic or oxidizing conditions, will be oxidized to and can bind transferrin (Davidsson et al., 1989). However, brain uptake of Mn is decreased if Mn is allowed to equilibrate with plasma (Rabin et al., 1993). We hypothesize that the inverse relationship between iron status and erythrocyte Mn concentrations is due to competition primarily at the level of intestinal absorption, and not at the level of transferrin-mediated uptake. It is probably the iron status within enterocytes which determines absorption of enteral manganese (Gunshin et al., 1997; Han et al., 1999). We had similar equivocal neuropsychological findings related to the MR T1weighted hyperintensities as other investigators (Thuluvath et al., 1995; Kulievsky et al., 1993). The tendency of correlation of tremor and muscle weakness to MR hyperintensities that we found is provocative, and suggests caution is warranted.

5. SUMMARY Iron deficiency resulted in increased erythrocyte Mn concentrations presumably due to increased Mn absorption. Erythrocyte Mn concentrations were further correlated with MR T1-weighted signal hyperintensities of the globus pallidus. We recommend that patients with chronic liver disease and poor iron status improve their iron status with increased dietary intake of lean meat, poultry and fish, and not consume Mn supplements without concurrent nonheme iron supplementation.

ACKNOWLEDGMENT Supported by PHS-NIH, General Clinical Research Center, CIC #93-023. Excerpted with permission from Neuro Toxicology 20: 647-652, InTox Press, Inc., 1999.

REFERENCES Butterworth, R.F., Spahr, L., Fontaine, S., and Pomier-Layrargues, G., 1995, Manganese toxicity, dopaminergic dysfunction and hepatic encephalopathy. Metab. Brain Dis. 10:259–67. Davidsson, L., Lönnerdal, B., Sandström, B., Kunz, C., and Keen, C.L., 1989, Identification of transferrin as the major plasma carrier protein for manganese introduced orally or intravenously or after in vitro addition in the rat. J. Nutr. 119:1461–1464. Devenyi, A.G., Barron, T.F., and Mamourian, A.C., Dystonia, hyperintense basal ganglia, and high whole blood manganese levels in Alagille’s syndrome. Gastroenterology 106:1068–1071. Gunshin, H., Mackenzie, B., Berger, U.V., Gunshin, Y., Romero, M.F., Boron, W.F., Nussberger, S., Gollan, J.L., and Hediger, M.A., 1997, Cloning and characterization of a mammalian proton-coupled metal-ion transporter. Nature 338:482–488. Han, O., Fleet, J.C., and Wood R.J., 1999, Reciprocal regulation of HFE and NRAMP2 gene expression by iron in human intestinal cells. J. Nutr. 129:98–104. Hauser, R., Zesiewicz, T., Martinez, C., Rosemurgy, A., and Olanow, C., 1996, Blood manganese correlates with brain magnetic resonance imaging changes in patients with liver disease. Can. J. Neurol Sci. 23:95–98.

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Kulievsky, J., Pujol, J., Junqué, C., Deus, J., Balanzó, J., and Capdevilla, A., 1993, MRI pallidal hyperintensity and brain atrophy in cirrhotic patients: two different MRI patterns of clinical deterioration? Neurology 43:2570–2573. Mirowitz, S.A. and Westrich, T.J., 1992, Basal ganglia signal intensity alterations: reversal after discontinuation of parenteral manganese administration. Radiology 185:535–536. Pomier-Layrargues, G., Spahr, L., and Butterworth, R.F., 1995, Increased manganese concentrations in pallidum of cirrhotic patients [letter]. Lancet 345(8951):735. Quaghebeur, G., Taylor, W., Kingsley, D., Fell, J., Reynolds, A., and Milla, P., 1996, MRI in children receiving total parenteral nutrition. Neuroradiology 38:680–683. Rabin, O., Hegedus, L., Bourre, J.M., and Smith, Q.R., 1993, Rapid uptake of manganese (II) across the bloodbrain barrier. J. Neurochem. 61:509–517. Spahr, L., Butterworth, R., Fontaine, S., Bui, L., Thierren, G., Milette, P., Lebrun, L., Zayed, J., Leblanc, A., and Pomier-Layrargues, G., 1996, Increased blood manganese in cirrhotic patients: relationship to pallidal magnitic resonance signal hyperintensity and neurological symptoms. Hepatology 24:1116– 1120. Thuluvath, P., Edwin, D., Yue, N., DeVilliers, C., Hochman, S., and Klein, A., 1995, Increased signals seen in globus pallidus in T1-weighted magnetic resonance imaging in cirrhotics are not suggestive of chronic hepatic encephalopathy. Hepatology 21:440–442.

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EFFECT OF DEPRESSION AND OF ANTIDEPRESSANT THERAPY ON SERUM ZINC LEVELS A Preliminary Clinical Study Malgorzata Schlegel-Zawadzka1, Andrzej Zieba4, Dominika Dudek4, Miroslaw Krosniak1, Maria Szymaczek4, and Gabriel Nowak2,3* 1

Department of Food Chemistry and Nutrition Collegium Medicum Jagiellonian University 9 Medyczna Str., 30-688 Kraków Poland 2 Laboratory of Radioligand Research Collegium Medicum Jagiellonian University 9 Medyczna Str., 30-688 Kraków Poland 3 Institute of Pharmacology Polish Academy of Sciences 12 Sm.tna Str., 31-343 Kraków Poland 4 Department of Psychiatry, Collegium Medicum Jagiellonian University 21b Kopernika Str., 31-501 Kraków, Poland

Depression is not a homogenous illness, in that it exhibits variety of symptoms, such as: primary (core) symptoms (lack of interest—anhedonia, mood depression) and secondary symptoms (loss of interest, disorders of appetite, disorders in attention concentration, disorders in sleep, thoughts and suicidal tendencies, feelings of weariness, psychomotor slowness/excitation, senses of guilt and of little value). Clinical depression is recognised when at least five symptoms have been observed for at least two weeks. The presence and intensity of symptoms may be assessed by different methods (e.g. Hamilton, 1960). Chronic antidepressant therapy is necessary for successful therapy in humans. In spite of almost 40-years of research the neurochemical mechanisms as well as the markers of depression have not been established. *email: [email protected] Address all correspondence to: Dr Malgorzata Schlegel-Zawadzka; Dept. Food & Chem., Collegium Medicum Jagiellonian University, 9 Medyczna Str., 30-688 Kraków, Poland, fax: 0048126543949, email: [email protected]. Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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Some clinical investigations have pointed to alterations of the blood zinc level as a potential marker of depression. Our previous results demonstrated that antidepressants drugs and electroconvulsive shock caused selective changes in the zinc level in the rat brain (Nowak and Schlegel-Zawadzka, 1999). Moreover, our data indicate a similarity between animal models of depression (chronic unpredictable stress) and human depression with regard to the serum zinc level (Nowak et al., 1999). In the present study we investigated the effect of depression (unipolar) and chronic antidepressant treatment on the serum zinc level in psychiatric patients.

1. MATERIAL AND METHODS The clinical trial consisted of two groups of 19 major depressed patients (unipolar) and 16 control healthy volunteers. Patients were treated from two to six weeks by amitriptyline, imipramine and clomipramine. Their blood was collected thrice—before, and two weeks and six weeks after antidepressant treatment. Blood was always drawn at 8.00 a.m., the serum separated, frozen and stored at –20°C for 1–2 months before assay. The severity of depression was measured by the Hamilton Depression Rating Scale (17 items; Hamilton, 1960). The zinc concentration in serum was determined using flame atomic absorption spectrometry (Perkin Elmer 5100 PC equipped with a 5100 ZL Zeeman Furnace Module). The details of the measurement procedure have been presented earlier (Nowak et al., 1999). The study was approved by the Ethical Committee for Experimental Clinical Investigation Collegium Medicum Jagiellonian University. Group differences were assessed using the unpaired t-Test or Fisher’s Exact Test. Relationships between variables were assessed using Spearman’s rank order correlation coefficient. Data were deemed significant when p < 0.05.

2. RESULTS The mean ± SD of the age of the depressed and the control groups were 42.2 ± 10.6 and 37.0 ± 9.1 years, respectively, and were not significantly different (p = 0.126, t-Test). There were no significant differences in the ratio male/female between depressed (7/12) and control (10/6) (Fisher’s Exact Test). The mean ± SD of the Hamilton Depression Rating Scale (HDRS) before treatment was 18.9 ± 5.3 in the depressed patients. After two weeks treatment the HDRS was significantly reduced to 9.7 ± 4.4 and slightly more to 8.0 ± 5.4 after six weeks. There was no significant correlation between the age and the serum zinc in the control group (r = 0.069, p = 0.801), in the depressed (r = –0.182, p = 0.4556) and in the pooled control + depressed (r = –0.024, p = 0.337). The serum zinc level after two or six weeks of antidepressant treatment was not significantly different from control zinc level (Table 1). However, there was a significant correlation between the serum zinc levels before, during and after treatments and HDRS (r = –0.353, p = 0.020, Fig. 1).

3. DISCUSSION The pathophysiology of depression as well as the mechanism of action of antidepressant drugs is unknown, although recent reports suggest the glutamatergic pathway as a common target for antidepressant therapy (Huang et al., 1997; Skolnick et al., 1996;

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Pilc et al., 1998). Zinc ion is a potent modulator of the ionotropic glutamatergic NMDA receptor which is itself involved in the mechanism of antidepressant drugs (Skolnick et al., 1996). In a previous study we reported details of the brain and serum zinc levels after chronic antidepressant treatment in rats. Whilst antidepressant drugs and electroconvulsive shock (ECS) induced an increased hippocampal zinc concentration, in their effect on this ion in the serum these treatments differ (Nowak and Schlegel-Zawadzka, 1999). We conclude that the blood zinc concentration does not directly reflect alterations in the concentrations of this ion in the brain. In the present study we have confirmed the notion that unipolar depression is associated with lower blood zinc levels (Maes et al., 1997). Moreover, the severity of the illness (HDRS) measured before, during and after antidepressant treatment is negative correlated with the serum level of this ion. The present results demonstrate that a lower serum zinc level accompanies depression and is normalized by a successful antidepressant therapy. These results indicate that serum zinc is a sensitive marker of unipolar depression.

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ACKNOWLEDGMENTS The authors thank Polish Pharmaceutical Society for financial support.

REFERENCES Hamilton, M., 1960, A rating scale for depression, J. Neurol. Neurosurg. Psychiatry, 23:56–61. Huang, N.-Y., Layer, R.T., and Skolnick, P., 1997, Is adaptation of NMDA receptors an obligatory step in antidepressant action? in: Antidepressants. New pharmacological strategies, (P. Skolnick, ed.), pp. 125–143, Humana, Totowa. Maes, M., Vandoolaeghe, E., Neels, H., Demedts, P., Wauters, A., Meltzer, H.Y., Altamura, C., and Desnyder, R., 1997, Lower serum zinc in major depression is a sensitive marker of treatment resistance and of the immune/inflammatory response in that illness. Biol. Psychiat., 42:349–358. Nowak, G. and Schlegel-Zawadzka, M., 1999, Alterations in serum and brain trace element levels after antidepressant treatment. Part I. Zinc, Biol. Trace Elem. Res., 67:85–92. Nowak, G., Zieba, A., Dudek, D., Krosniak, M., Szymaczek, M., and Schlegel-Zawadzka, M., 1999, Serum trace elements in animal models and human depression. Part I. Zinc, Hum. Psychopharmacol. Clin. Exp., 14:83–86. Pilc, A., Branski, P., Palucha, A., Tokarski, K., and Bijak, M., 1998, Antidepressant treatment influences group I of glutamate metobotropic receptors in slices from hippocampal CA1 region. Eur. J. Pharmacol., 349:83–87. Skolnick, P., Layer, R.T., Popik, P., Nowak, G., Paul, I.A., and Trullas, R., 1996, Adaptation of N-methyl-Daspartate (NMDA) receptors following antidepressant treatment: implications for the pharmacotherapy of depression, Pharmacopsychiatry, 29:23–26.

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ABNORMALITIES OF IRON HOMEOSTASIS IN THE PREGNANCY SYNDROME PRE-ECLAMPSIA M. P. Rayman1, J. Barlis1, S. Sokari1, R. W. Evans2, C. W. G. Redman3, and L. J. King1 1

School of Biological Sciences University of Surrey Guildford, UK 2 Division of Biomolecular Sciences King’s College London London, UK 3 Nuffield Department of Obstetrics and Gynaecology John Radcliffe Hospital University of Oxford Oxford, UK

Pre-eclampsia is a disorder of pregnancy that is believed to affect one in ten of all pregnancies to some degree. It is believed that blood-borne agents arising from the ischaemic placenta are the cause of the generalised endothelial-cell damage which gives rise to the symptoms of hypertension, proteinuria, and sudden oedema, characteristic of this condition (Roberts et al., 1989). The high levels of circulating lipid hydroperoxides believed to be present in pre-eclampsia (Wickens et al., 1981) are among the candidate agents capable of causing such damage to the vascular endothelium. Disturbances in iron homeostasis have been observed in pre-eclampsia (Entman, Richardson, and Killam, 1983; Entman et al., 1987; Hubel et al., 1996; Samuels et al., 1987). Iron entities could arise by destruction of red cells from necrotic and haemorrhagic areas of the ischaemic placenta or from intravascular haemolysis (Balla et al., 1993; Samuels et al., 1987) and may contribute to endothelial-cell injury through the initiation and propagation of lipid peroxidation. Plasma iron is usually almost entirely safely bound to the transport protein transferrin, which can bind two iron atoms per molecule. The greater the extent to which transferrin is saturated with iron, the lower the antioxidant capacity of the plasma. Released iron is normally safely sequestered in the intracellular iron-storage protein ferritin. Levels of ferritin measured in the serum reflect the amount of storage ferritin (Worwood, 1995). Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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This study compares a number of iron parameters, including serum ferritin and the extent to which transferrin is saturated with iron, in pre-eclamptic women and matched pregnant controls.

SUBJECTS AND METHODS Serum was prepared from blood collected at the John Radcliffe Hospital from 40 severely pre-eclamptic patients, of average gestation 33 weeks, and controls, matched for age, parity and gestation. Pre-eclampsia was defined using the criteria of gestational hypertension (diastolic pressure reading greater than 90 mm&Hg on two or more readings at least 4 h apart), proteinuria and reversal of these conditions after delivery (Perry and Beevers, 1994). Clinical characteristics of the patients recorded or measured, included aspartate aminotransferase (AST) concentration, an indicator of liver damage. Serum total iron (transferrin-bound) and unsaturated iron binding capacity (UIBC)—a measure of free or apotransferrin—were measured on a Cobas Mira Plus automatic analyser (Roche Diagnostic Systems, Hertfordshire, UK) at the University of Surrey using a colorimetric method based on the formation of a red-coloured chelate between divalent iron and ferrozine (Stoney, L. L., 1970) (Unimate 5 iron and Unimate 7 UIBC kits, Roche Products). Accuracy and precision were determined in the case of serum total iron, by analysis of control serum N (Roche Products) and a lyophilized second-generation human serum certified reference material supplied from the lab of Versieck and coworkers, Ghent, (Versieck et al., 1988) while for UIBC, control serum N was used. Total iron binding capacity (TIBC) was calculated as the sum of the serum iron and UIBC. The percentage saturation of iron binding capacity was calculated as: serum iron ×100/TIBC. Percent transferrin saturation was determined by urea polyacrylamide gel electrophoresis using a method originally developed by Makey and Seal12 and modified by Evans and Williams (Evans and Williams, 1980). This method separates transferrin into the apo-, monoferric- (of which there are two forms, C-terminal and N-terminal) and diferric-forms. Percent transferrin saturation was calculated as follows:—

Apotransferrin levels in the two patient groups were also obtained by this method allowing comparison with corresponding UIBC values, which also give some measure of apotransferrin. Serum ferritin was determined by a microparticle enzyme immunoassay method using the Abbott AxSYM system (Abbott Laboratories, Diagnostics Division, Maidenhead, UK). The Kolmogorov-Smirnov procedure was used to ascertain that the data was normally distributed and therefore the experimental variables in the two groups were compared by parametric analysis (two-tailed Student’s t-test).

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RESULTS Ferritin, percent-transferrin-saturation and apoferritin values were only determined on a subset of samples: –20 pairs. Five pre-eclamptic subjects and one control, with AST levels >42IU/L, were excluded from the ferritin analysis data owing to the likely inclusion of ferritin released from the damaged liver. One control sample was lost. Of the 40 pre-eclamptic subjects, four with very high values were excluded from the data analysis of serum iron, and three, again with very high values, from the data analysis of percent saturation of iron-binding capacity, since the Kolmogorov-Smirnov test suggested that they were from a different population. These were all subjects whose AST levels indicated liver damage. Means, numbers of subjects, standard deviations, ranges and significance levels of the various parameters measured are shown in Table 1. Mean total serum iron concentration and serum ferritin were significantly higher in patients with pre-eclampsia relative to matched pregnant controls (iron 21.8 vs ferritin 39.8 vs UIBC and TIBC were significantly lower in the preeclamptic group than in the matched controls (UIBC 48.1 vs TIBC 73.6 vs Percent saturation of iron-binding capacity in women with pre-eclampsia was significantly higher than that of matched pregnant controls, the ratio being about 1.7 (31.7 vs 18.2%). Similarly, percent saturation of transferrin (the equivalent measurement by the gel method), was significantly higher in the pre-eclamptic group relative to the normal-pregnancy group (29.7 vs 13.5%), the ratio being of the same order (2.2). There was a highly significant correlation between the two methods for both patient groups (R = 0.94 for combined data, p < 0.0001). Apotransferrin levels in pre-eclamptic patients, measured in arbitrary units, were significantly lower than in matched pregnant controls (321 vs 535).

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DISCUSSION Serum iron in this group of pre-eclamptic patients was 38% higher than in the matched control group. As transferrin is the most important iron-binding protein in serum, determination of the percent saturation of iron-binding capacity as measured by the colorimetric method, is to a good approximation, a determination of the percent saturation of transferrin, as obtained directly by gel electrophoresis. Both methods, which were very significantly correlated, indicated the extent of transferrin saturation with iron in the preeclamptic patients to be about double that of the controls. Five of the 40 pre-eclamptic subjects had percent saturation levels (50–74%) within the range associated with iron overload (Worwood, 1995). These subjects did not include the three referred to above who had been excluded from the data analysis, and whose percent saturation levels ranged from 83–100%. Apotransferrin and UIBC levels, which are more or less equivalent, suggest that pre-eclamptic women only have around two-thirds of the capacity to bind additional iron as do controls (pre-eclamptic: control ratios, 0.60 and 0.66 respectively). Furthermore, their total iron-binding capacity (TIBC) is only 83% of that of the controls. While these findings are in agreement with those of previous studies (Entman et al., 1983; Entman et al., 1987; Hubel et al., 1996; Samuels et al., 1987), there has been some disagreement about whether ferritin levels are raised in pre-eclampsia (Entman et al., 1983; Samuels et al., 1987). Our results show that ferritin levels are raised fourfold in pre-eclampsia, even when those patients exhibiting liver damage are excluded. The origin of the iron species in pre-eclampsia, if placental [from destruction of red cells, possibly in thrombosis and necrosis of the decidual-placental interface (Entman et al., 1987)], could be part of the aetiology of the disease. Whether or not this is the case, results indicate a decrease in the antioxidant capacity of serum by reduced serum-iron buffering in pre-eclampsia, which may exacerbate lipid peroxidation and endothelial-cell injury. Our findings suggest that raised serum iron and ferritin could be used diagnostically to warn of incipient pre-eclampsia and that iron supplements should not be given to pregnant women at high risk of pre-eclampsia without good reason.

ACKNOWLEDGMENTS The authors are grateful to Professor Luc Moens of the University of Ghent for supplying second-generation human serum certified reference material.

REFERENCES Balla, J., Jacob, H.S., Balla, G., Nath, K.A., Eaton, J.W., and Vercellotti, G.M., 1993, Endothelial-cell heme uptake from heme proteins: Induction of sensitisation and desensitization to oxidant damage, Proc. Natl. Acad. Sci. 90:9285–9289. Entman, S.S., Richardson, L.D., and Killam, A.P., 1983, Altered ferrokinetics in toxemia of pregnancy: a possible indicator of decreased red cell survival, Clin. and Exper. Hyper.—Hyper. in Pregnancy B2(l):171–178.

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Entman, S.S., Kambam, J.R., Bradley, C.A., and Cousar, J.B., 1987, Increased levels of carboxyhaemoglobin and serum iron as an indicator of increased red cell turnover in preeclampsia, Am. J. Obstet. Gynecol. 156(5):1169–1173. Evans, R.W. and Williams, J., 1980, The electrophoresis of transferrins in urea polyacrylamide gels, Biochem. J. 189:541–546. Hubel, C.A., Kozlov, A.V., Kagan, E.V., Evans, R.W., Davidge, S.T., McLaughin, M.K., and Roberts, J.M., 1996, Decreased transferrin and increased transferrin saturation in sera of women with preeclampsia. Implications for oxidative stress. Am. J. Obstet. Gynecol. 175:692–700. Makey, D.G. and Seal, U.S., 1976, The detection of four molecular forms of human transferrin during the iron binding process, Biochim. Biophys. Acta 453:250–256. Perry, I.J. and Beevers, D.G., 1994, The definition of pre-eclampsia, Brit. J. Obstet. Gynaecol. 101:587–591. Roberts, J.M., Taylor, R.N., Musci, T.J., Rodgers, G.M., Hubel, C.A., and McLaughlin, M.K., 1989, Pre-eclampsia: an endothelial cell disorder, Am. J. Obstet. Gynecol. 161:1200–1204. Samuels, P., Main E.K., Mennuti, M.T., and Gabbe, S.G., 1987, The origin of increased serum iron in pregnancy-induced hypertension. Am. J. Obstet. Gynecol. 157:721–725. Stoney, L.L., 1970, Ferrozine—a new spectrophotometric reagent for iron, Anal. Chem. 42:779–881. Versieck, J., Vanballenberghe, L., De Kesel, A., Hoste, J., Wallaeys, B., Vandenhaute, J., Baeck, N., Steyaert, H., Byrne, A.R., and Sunderman Jr., F.W., 1988, Certification of a second-generation biological reference material (freeze-dried human serum) for trace element determinations, Analytica Chimica Acta 204:63–75. Wickens, D., Wilkins, M.H., Lunec, J., Ball, G., and Dormandy, T.L., 1981, Free-radical oxidation (peroxidation) products in normal and abnormal pregnancy, Ann. Clin. Biochem. 18:158–162. Worwood, M., 1995, Measurement of iron status, in Iron: Nutritional and Physiological Significance, (British Nutrition Foundation), pp. 23–32, Chapman and Hall, London.

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LIPID PEROXIDATION IN PATIENTS WITH DISEASES KNOWN TO AFFECT TRACE ELEMENT STATUS Mariana Vlad1, P. J. Porr2, G. Uza3, and Maria Zirbo4 1

Institute of Public Health 3-rd Medical Clinic 3 Departmental Polyclinic II 4 University of Medicine and Pharmacy Cluj Napoca, Romania 2

1. INTRODUCTION Some investigators have shown higher serum copper (Cu) levels and lower serum zinc levels (Zn) in cancer, cardiovascular disease and diabetes mellitus patients (Andrews, 1979; Diaz et al., 1997; Klevay, 1980; Kok et al., 1998; Reunanen et al., 1996; Wolf et al., 1993). Causal interpretation of these data is difficult. A low plasma Zn level could be the result of a deficiency caused by a concentration of Zn near the tumor site, as occurring at wound margins (Savlov et al., 1962) and in these circumstances the Zn might be needed for normal tissue growth, repair, and defense mechanisms (Andrews, 1979). Published data show that Zn protects against free radical injury (Burke, 1985). Moreover, a low Zn status reduces the potential anticarcinogenetic effect of vitamin A (Prasad, 1983) and may affect immune response. Recently published data have shown that serum concentration is decreased in patients with malignant tumors (Gaziano et al., 1996; Gey, 1993; Mayne, 1996; Pietrzik, 1996), and have suggested that this provitamine has an important role in cancer prophylaxis. Starting from the above data, we studied the behaviour of the serum concentration of Cu, Zn, ceruloplasmin (Cp), glutathione (GHS), glutathione peroxidase activity (GHS-Px), vitamin A, and lipid peroxides, in patients with increased and prolonged oxidative stress.

Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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2. MATERIALS AND METHODS Materials The study was carried out on 535 subjects as follows: group A—100 healthy, aged 19–67 years, 57 males and 43 females; group B—150 patients suffering from cardiovascular diseases, aged 42–69 y, 62 m and 88 f; group C—110 patients suffering from various forms of cancer, aged 39–82 y, 47 m and 63 f; group D—50 patients with diabetes mellitus non-insulindependent type (NIDDM), aged 34–67 y, 25 m and 25 f; group E—65 patients with pulmonary diseases (chronic bronchitis, asthma, and acute pneumonia) other than malignancy, aged 19–65 y, 39 m and 26 f; group F—60 patients with liver cirrhosis, aged 28–68 y, 37 m and 23 f. Fasting venous blood samples were drawn from all subjects, using disposable plastic instruments to avoid metal contamination.

Methods Serum Cu and Zn concentrations were measured using Model 300 Perkin Elmer atomic absorption spectrophotometer, with flame atomization; length 213.9 nm were used for Zn and 324 nm for Cu. The standard solution contained Zn and Cu. The variation coefficient, calculated from duplicate, was 6.67 for Zn and 4.84 for Cu. Serum lipid peroxides were determined by a spectrofluorimetric method according to Yagi (1976). Antioxidant vitamins A and were evaluated by spectrophotometric detection by Moneger’s method that have been reported elsewhere (Tamas, Neamtu, 1986). Serum Cp was determined by Ravin’s procedure (Manta et al., 1976). Plasma GHS-Px activity was measured according to the method described by Günzler (1974).

Statistical Analysis Values are given as mean ± SD. The statistical significance (p values) of differences between means was estimated by computation of one tailed Student’s t-test.

3. RESULTS The serum Cu levels were found to be higher in patients with cancer, cardiovascular, pulmonary diseases and cirrhosis as compared to controls (p < 0.01) (Fig. 1). The highest values of serum Cu concentration were found in patients with lung and breast cancer (data not shown). In patients with cardiovascular, pulmonary diseases, diabetes mellitus and cirrhosis (considered to be one group) the mean of serum Cu values tend to grow up with age whereas serum Zn levels lowering with years (data not shown). In the patients with cancer, diabetes mellitus, and cirrhosis the serum Zn levels were lower (p < 0.01) as compared to controls (Fig. 1). Serum Zn concentration reached the lowest values in patients with lung and esophagus cancer (data not shown). The Cu/Zn ratio was significantly increased in patients with cancer, cardiovascular diseases, cirrhosis and pulmonary diseases as compared to the controls (p < 0.01); the highest value was noted in patients with cancer. In patients suffering from cancer and

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cardiovascular diseases the concentration of Cp is significantly higher than in the controls, as well as in the other groups (p < 0.01). (data not shown). The highest serum Cp level was found in patients with lung and esophagus cancer. The high levels of Cp were usually associated with raised serum Cu. However, a higher correlation could be calculated between Cu levels and Cp activities in all groups of patients (r = 0.87, p < 0.001). Blood GSH and plasma GSH-Px activity were significantly lower in patients with cancer and cirrhosis (p < 0.01) as compared to control (data not shown). The serum lipid peroxides levels were found to be significantly higher in cardiovascular, diabetes mellitus and cirrhosis patients as compared to the healthy subjects (p < 0.01) (Fig. 2). No significant difference between groups was observed regarding the vitamin A level (Fig. 2). Plasma was significantly decreased in cancer and cirrhosis patients as compared to the controls (p < 0.01) (Fig. 2). It was found a positive correlation between lipid peroxides and serum Cu (r = 0.81, p < 0.001) and Cp (r = 0.72, p < 0.001) levels and a negative correlation between Zn (r = 0.69, p < 0.001), GSH (r = 0.62, p < 0.001) and GSH-Px (r = 0.77, p < 0.001) concentrations.

4. DISCUSSION The average values of blood Cu concentration in group with cardiovascular diseases was one of the highest values after the levels found in patients with cancer. In a previous paper we have shown that the high blood Cu level could be considered as marker in establishing the diagnosis of thromboangiitis obliterans (Uza, 1989). The increase of blood Cu concentration in animals with experimental atherosclerosis was probably due to the mobilization of copper from arterial tissue, our study showing an important decrease concentration of Cu in the atheromatous tissue (Vlad et al., 1993, 1994, 1995).

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The highest blood Cu levels were found in patients with lung and breast cancer. Like in atherosclerosis, it should be taken in consideration the mobilization of Cu from the tumoral tissue and its shift into the blood. The increase of blood Cu concentration in patients with neoplasm’s indicates, according to some authors the appearance of inflammatory process in the body maybe in response to the tumor. The high blood Cu levels were often seen in the case of tumors with rapid development like lung and breast cancer and melanoma (Schrauzer, 1987). Some authors state that the blood Cu concentration in patients with malignant tumors can be normal or even lower (Bayer, 1982), according to the development and the stage of the disease (Bayer, 1982), its concentration decreases especially in the final stages of the disease (Schrauzer, 1987), and this is why the Cu levels should be monitored. In patients with high blood Cu levels an important decrease of blood Zn levels can be noted. A low Zn status reduces the potential anticarcinogenetic effect of vitamin A, decreases the protection against free radical injury and has an effect on the immune response (Prasad, 1983). Zn is an essential nonredox metal that has been regarded as having antioxidant properties. The decrease in serum Zn concentration suggested the idea of administering to those patients drugs with Zn as antioxidant. The high levels of serum lipid peroxides, the positive correlation with Cu and Cp levels and the negative correlation with Zn, GSH and GSH-Px levels are a consequence of chronic oxidative stress in the studied diseases.

REFERENCES Andrews, G.S., 1979, Studies of plasma zinc, copper, caeruloplasmin, and growth hormone. With special reference to carcinoma of the bronchus, J. of Clin. Pathol. 32:325–333.

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Bayer, W., 1982, in Mineralstoffwechsel und Abwehrsystem, (Schmidt, K. and Bayer, W. eds.), Verl. f. Medizin Dr. Ewald Fischer, Heidelberg, 17–32. Burke, J.P. and Fenton, M.R., 1985, Effect of a zinc-deficient diet on lipid peroxidation in liver and tumor subcellular membranes (42083), Proc. Soc. Exp. Biol. Med. 179:187–191. Diaz, M.N., Frei, B., Vita, J.A., and Keaney, J.F., 1997, Mechanisms of disease: Antioxidants and atherosclerotic heart disease, N. Engl J. Med. 337(6):408–416. Gaziano, L.M. and Hennekes, Ch.H., 1996, Update on dietary antioxidants and cancer, Path. Biol. 44(l):42–45. Gey, K.F., 1993, Prospects for the prevention of free radical disease, regarding cancer and cardiovascular disease, Br. Med. Bull. 49(3):679–699. Günzler, W.A., Kremers, H., and Flohe, L., 1974, Z. Klin. Chem. Klin. Biochem. 12:444–448. Klevay, L.M., 1980, Interaction of copper and zinc in cardiovascular disease, Ann. N. Y. Acad. Sci., 355:140–151 Kok, F.J., Van Duijn, C.M., Hofman, A., Van Der Voet, G.B., De Wolff, F.A., Paays, C.H.Ch., and Valkenburg, H.A., 1988, Serum copper and zinc and the risk of death from cancer and cardiovascular disease, Am. J. of Epidemiol. 128(2):352–359. Manta, I., Cucuianu, M., Benga, G., and Hodarnau, A., 1976, Metode biochimice in laboratorul clinic, Cluj-Napoca, Romania, Ed. Dacia. Mayne, S.T., 1996, Beta-carotene, carotenoids, and disease prevention in humans, FASEB J. 10:690–701. Pietrzik, K., 1996, Antioxidant vitamins, cancer, and cardiovascular disease, N. Engl. J. Med. 335(14), 3, 1065–1069. Prasad, A.S., 1983, Clinical, biochemical and nutritional spectrum of zinc deficiency in human subjects: an update, Nutr. Rev. 41:197–208. Reunanen, A., Knekt, P., Marniemi, J., Maki, J., Maatela, J., and Aromaa, A., 1996, Serum calcium, magnesium, copper and zinc and risk of cardiovascular death, Eur. J. of Clin. Nutr. 50:431–437. Savlov, E.D., Strain, W.H., and Huegin, F., 1962, Radiozinc studies in experimental wound healing, J. of Surgical Res. 2:209–212. Schrauzer, G.N., 1987, Trace elements in diagnosis and therapy. A review, Trace Element Analytical Chemistry in Medicine and Biology, 4, Schramel, P. and Bratter, P., eds, Walter de Gruyter, Berlin, NY. Tamas, V. and Neamtu, G., 1986, Pigmenti carotenoidici si metaboliti—Chimie si biochimie, Ed. Ceres, Bucuresti, vol. I. Uza, G. and Vlaicu, R., 1989, Serum zinc and copper in patients with atheroclerosis and trombangitis obliterans. Biol. Trace Elem. Res. 2:197–206. Vlad, M., Bordas, E., Tomus, R., Sava, D., and Farcas, E., 1993, Effect of copper sulfate on experimental atherosclerosis, Biol. Tr. Elem. Res. 38:47–54. Vlad, M., Caseanu, E., Uza, G., and Petrescu, M., 1994, Concentration of copper, zinc, chromium, iron, and nickel in the abdominal aorta of patients deceased with coronary heart disease, J. Tr. Elem. Electrolytes Health Dis., 8(2):111–114. Vlad, M., Uza, G., Zirbo, M., and Olteanu, Doina, 1995, Free radicals, ceruloplasmin, and copper concentration in serum and aortic tissue in experimental atherosclerosis, Supplement to Nutrition 11(5):588–591. Wolff, S.P., 1993, Diabetes mellitus and free radicals. Free radicals, transition metals and oxidative stress in the etiology of diabetes mellitus and complications, Br. Med. Bull. 49(3):642–652. Yagi, K., 1976, A simple fluorometric assay for lipoperoxide in blood plasma, Biochem. Med., 15:212–216.

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SELENIUM STATUS OF BREAST AND GASTROINTESTINAL CANCER PATIENTS IN TURKEY

B. Avsar and I. G. Gökmen Middle East Technical University Department of Chemistry 06531, Ankara, Turkey

Serum selenium concentrations of control subjects, breast cancer patients, gastric and colorectal cancer patients were determined using spectorofluorimetry. Serum samples were digested in aluminum digestion furnace using nitric, sulfuric and perchloric acids. Selenium was reduced to +4 state using hydrochloric acid. Then selenium-2,3-diaminonapthalene (Se-DAN) complex was formed and its fluoresence was measured using Perkin Elmer LS-50B spectrofluorometer, with excitation at 375 nm and emission at 520 nm. The detection limit of the method was found as and the precision for Se concentration as 3.60%. The accuracy of the method was found as 82 and 90% using two standard reference materials and selenium recovery was 103.9%. A questionnaire was filled with each subject which included personal information, disease state, habits and medication usage of the subjects. The answers for all the groups were compared with each other and correlated with serum selenium concentrations using SPSS 7.0 software package. Serum selenium concentrations of the healthy control subjects were found to be the highest of all, 69.5 ± While the serum selenium concentrations of the breast cancer patients were not significantly different than that of control subjects (65.2 ± gastric (53.9 ± and colorectal cancer patients (56.6 ± had similar serum selenium concentrations which were much less than that of control subjects and breast cancer patients. For the whole population, average serum selenium concentrations of females (65.0 ± was found to be significantly higher than that of males (58.7 ± Similar relationship was observed when only the average selenium concentrations of males and females in the control group were compared. The serum selenium concentrations of the females was found to be decreasing with age, yet no age dependency was seen within the male group. The serum selenium concentrations were 623

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decreasing with increasing the stage of the cancer among the breast cancer patients. In all the groups, non-smokers have higher serum selenium concentrations. Within the cancer patient groups, family history of cancer, effect of personality, occupation, effect of stress, economic status, education were some of the other parameters which were also investigated.

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ZINC AND SELENIUM INTAKE IN NONDIALYSED PATIENTS WITH CHRONIC RENAL FAILURE D. Mafra1, D. I. T. Favaro, and S. M. F. Cozzolino1 1

Faculdade de Ciências Farmacêuticas (school of pharmacy) USP São Paulo, Brazil 2 Instituto de Pesquisas Energéticas E Nucleares-Cnen (nuclear and energy research institute-nuclear energy national committee) São Paulo, Brazil

Changes in zinc and selenium metabolisms are described in patients with chronic renal failure. The reasons for the deficiencies of the two elements are not clear; however, the decrease in zinc and selenium dietary intake may contribute to alter the metabolism of these minerals. The present study measured zinc and selenium content in the diets of 14 patients with renal failure. The mean age of the patients was 52.3 ± 12 years and the mean serum creatinine was 3.4 ± 1.7 mg/dL. The duplicate portion technique was used for sample collection. These samples were collected and prepared for analysis at USP. The trace elements were qualitatively identified in the diets by neutron activation analysis. Solutions prepared with each mineral and with both of them were used as primary element standards. Reference materials for total diet (NIST SRM 1548) and Citrus Leaves (NIST SRM 1572) were used for checking precision and accuracy of the method. 200 mg of reference materials and diet samples were weighted in pre-cleaned polyethylene bags, placed together with the synthetic standard into polyethylene vials and irradiated in the research nuclear reactor IEA-R1 of the IPEN/CNEN-SP. Samples and standards were submitted to an 8 hour long-term irradiation, at a thermal neutron flow of Dietary fat, carbohydrate and protein were estimated by AOAC methods. Mean daily intakes were 4.4 ± 3.7 mg/day and 21 + for Zn and Se, respectively. Zinc concentration and protein intake show a high correlation (p < 0.001 and r : 0.91). The mean energy intake was 21.2 ± 8.9 kcal/kg/day, with a mean protein intake of 0.8 ± 0.4 kg/day.

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ZINC NUTRITIONAL STATUS IN PATIENTS WITH CHRONIC RENAL FAILURE, RESIDENTS IN SÃO PAULO, BRAZIL D. Mafra1, L. Cuppari2, and S. M. F. Cozzolino1 1

Faculdade de Ciências Farmacêuticas (school of pharmacy) USP-São Paulo, Brazil 2 Nefrologia (nephrology) Universidade Federal de São Paulo, UNIFESP (São Paulo Federal University) São Paulo, Brazil

Abnormalities in zinc metabolism in patients with chronic renal failure have been reported, especially in those undergoing hemodialysis. However, more studies are necessary with patients under conservative treatment, before dialysis. Zinc is an essential trace element with the greatest interest in nephrology, since there is good evidences for the correlation between zinc deficiency and some of the abnormalities commonly observed in chronic renal failure (CRF). However, it remains unclear if hypozincemia in CRF represents a true total body zinc depletion or an exchange from extra-cellular to intracellular compartments. The purpose of this study was to determine zinc levels in plasma, erythrocytes and urine of patients not under dialysis, with chronic renal failure. Nineteen patients, with a mean age of 50.0 ± 11.8 years and serum creatinine of 2.6 ± 1.6 mg/dL were studied. The control group was made up of 18 healthy volunteers with mean age of 46.5 ± 6.9 years. Plasma and urine were previously diluted and determined by direct aspiration in atomic absorption spectrophotometer, whereas erythrocytes had to be lysed prior to the determination in the same apparatus. Zinc levels in erythrocytes were increased whereas those in plasma were decreased when compared to normal controls, and for erythrocyte and plasma, respectively (p < 0.05). Urinary zinc excretions are normal in both groups. Based on our findings, we can conclude that there is an abnormal zinc distribution in patients with chronic renal failure, just under conservative treatment. This shows that zinc is altered before dialysis and not because of it. At present, it is not clear if low concentrations of plasmatic zinc give an indication of zinc deficiency in uremia, since erythrocyte values were increased. 626

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IRON, ZINC, COPPER LEVELS OF THALASSEMIA PATIENTS OF NORTHERN CYPRUS

R. Oktekin and G. Gökmen Middle East Technical University Department of Chemistry 06531, Ankara, Turkey

Thalassemia is a genetic blood disease, with a defect in the synthesis of hemoglobin. It has high occurence rate among the people of Mediterranean, Middle East and Asia. Being an island it is isolated and several marriages between close relatives are taking place in Cyprus and this results in high occurance of thalassemia among the islanders. If not cured thalassemia patients may die within a short period after birth due to iron accumulation in some organs. At the present bone marrow transplantation, blood transfusions and iron chelation therapy are the main cures for the disease. There are 168 thalassemia patients in the Turkish Republic of Northern Cyprus and 64% of these patients are older than 18 years of age. There is a Thalassemia Center in Northern Cyprus where these patients are followed and the necessary remedies are provided to them free of charge. In this study the blood serum samples of 40 thalassemia patients and 44 control subjects were collected by the Thalassemia Center of Northern Cyprus. The personal information of the patients and their treatment regimen were also gathered with the samples. The iron, zinc and copper concentrations of serum samples were determined by atomic absorption spectrometry, using micro injection technique. The detection limits for iron, zinc and copper were found as 0.039 mg/L, 0.015 mg/L and 0.030 mg/L, respectively and the recoveries for these elements were 121 ± 8%, 95 ± 5%, 97 ± 3%, respectively. The precision at 1 mg/L concentration of iron, zinc and copper were 1.9, 0.6 and 0.5%, respectively. The average serum iron concentration of thalessemia patients (3.33 ± 0.88 mg/L) was found significantly higher than that of control subjects (1.78 ± 0.65 mg/L). However, the opposite was observed for the average serum zinc concentrations, it was 0.93 ± 0.17 mg/L for the patients and 1.01 ± 0.15 mg/L for the control group. Since zinc is an important element for the growth and development, administration of zinc supplementation to the patients might be considered. The average serum copper concentrations of patients 627

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and the control group were found similar, 0.96 ± 0.13 mg/L and 0.88 ± 0.14 mg/L, respectively. No significant difference was observed between males and females for these elements in the patient and control group with exception of zinc, which was significantly higher for the control group male subjects compared to the control group females. Serum ferritin levels were correlated with serum iron concentrations, yet it was not a strong correlation, r = 0.37.

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BLOOD MARKERS OF OXIDATIVE STRESS IN PATIENTS WITH AMYOTROPHIC LATERAL SCLEROSIS D. Bonnefont-Rousselot1, M. C. Jaudon1, L. Lacomblez2,3, B. Bourely1, V. Doppler2, C. Bizard1, F. Salachas3, G. Bensimon2, J. Delattre1, and V. Meininger3 1

Laboratoire de Biochimie Service de Pharmacologie 3 Service de Neurologie Hôpital de la Salpêtrière 47, bd de l’Hôpital, 75651 Paris Cedex 13 France

2

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder resulting in motoneuron death. 90% of ALS cases are sporadic, whereas 10% are familial forms. In this latter case, a mutation in the SOD1 gene that encodes for Cu,Zn-superoxide dismutase (SOD) has been found in 20% of the patients, which could lead to an altered Cu,ZnSOD activity. Familial and sporadic forms of ALS are clinically and pathologically very similar. The pathogenesis of the disease is unknown, but it has been hypothesized that ALS would be associated to oxidative damages induced by free radicals. The aim of our study was to have a comprehensive assessment of the blood oxidative stress in patients with ALS. Therefore, we determined the level of plasma lipid peroxidation (thiobarbituric acid-reactive substances (TBARS) assayed by a spectrofluorimetric method), and the status of the antioxidant defenses: vitamin E, vitamin A and (lipophilic antioxidants determined by HPLC), and activities of erythrocyte Cu,Zn-SOD and of plasma and erythrocyte glutathione peroxidase (GSH-Px). Plasma selenium (assayed by electrothermal atomic absorption spectrophotometry at 196 nm) was also determined since this trace element is essential to the activity of the GSH-Px. These eight markers were determined in 36 patients with ALS before treatment (aged 54 ± 14) and in 25 patients with ALS treated with riluzole (aged 60 ± 13). Indeed, one of the other hypotheses proposed to explain the pathogenesis of ALS involves an excessive activation of glutamate receptors, and riluzole has been shown to decrease glutamate release. Moreover, no data was available on the oxidative stress status in patients treated with riluzole. These two groups of ALS patients were compared to a group of 20 age-matched controls (aged 629

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59 ± 11). Our results clearly showed significantly higher TBARS values in ALS patients from both groups before treatment and in patients given riluzole) than in control subjects (means ± SD). Similarly, erythrocyte SOD activity was significantly enhanced in ALS patients (737 ± 127 U/gHb before treatment and 718 ± 111 U/gHb in patients treated with riluzole) when compared to controls (629 ± 71 U/gHb). Plasma selenium, vitamin E, vitamin A and concentrations were not different in ALS patients when compared with controls, except for concentration which was lower in patients treated with riluzole than in controls vs. Plasma and erythrocyte GSH-Px activities were similar in ALS subjects as in controls. The high lipid peroxidation level assessed by TBARS concentration in plasma was in favor of the presence of an oxidative process in the disease. The enhancement of the erythrocyte SOD activity could involve, at least partly, an induction of this enzyme by oxidative stress. Moreover, treatment with riluzole did not lead to any major modification of the oxidative stress status of the ALS patients in comparison with patients before treatment.

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GLUTATHIONE PEROXIDASE ACTIVITY IS REDUCED IN HAEMODIALYSIS PATIENTS

H. E. Roxborough, C. M. Loughrey, C. Mercer, D. McMaster, and I. S. Young Departments of Clinical Biochemistry and Medicine TheQueen’s University of Belfast and the Regional Nephrology Unit Belfast City Hospital, Belfast Northern Ireland

Cardiovascular disease is the major cause of morbidity and mortality in patients with end-stage renal failure receiving renal replacement therapy. Increased free radical production and antioxidant depletion may contribute to the greatly increased risk of atherosclerosis in these patients. Se deficiency may contribute to reduce availability of the antioxidant enzyme glutathion peroxidase (GPx), the plasma form of which is synthesized in the kidney. The aim of this study was therefore to assess Se status and GPx activity in patients with end-stage renal failure on haemodialysis. Venous blood was collected from 87 haemodialysis patients immediately prior to and after dialysis and from 70 healthy controls. Serum GPx activity and Se levels were measured as described by McMaster et al.1 and immunoreactivivity determined using a commercially available kit. A significant reduction in pre-dialysis selenium was found when compared with control subjects vs. p< 0.001). Selenium concentration rose significantly after dialysis p = 0.02) but still remained significantly lower than control values (p < 0.001). Immunoreactive GPx was found to be similar in the two groups (dialysis patients controls 15.2 ± 1.6 µg/ml; p = 0.14) but activity was significantly reduced in dialysis patients (dialysis patients 106 ± 2.7 U/L, controls 281 ± 3.6 U/L: p < 0.001). Following a single episode of haemodialysis, GPx activity rose from 106 ± 2.7 to 146 ± 3.8 U/L (p < 0.001) although remaining significantly below control values (p < 0.005). Immunoreactive GPx, however, remained unchanged following dialysis (14.1 ± 1.3 to 14.6 ± p = 0.10), resulting in an overall increase in the ratio of GPx activity: GPx protein (pre-dialysis 8.8 ± 1.1 U/mg, post-dialysis 10.7 ± 1.5 U/mg, control 22.0 ± 1.7 U/mg: p < 0.001). Selenium concentration correlated significantly with GPx avtivity in post-dialysis, but not pre-dialysis samples (post dialysis, r = 0.36, p < 0.01; pre-dialysis, r = 0.15, p = 0.21). Reduced Se and GPx activity may contribute to atherogenesis in haemodialysis patients. 631

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REFERENCE 1. McMaster D., Bell N., Anderson P., Love A.H., Automated measurement of two indicators of human selemium status, and applicability to population studies. Cli Chem 1990, 36:211– 216.

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COPPER AND ZINC LEVELS IN PATIENTS WITH COLORECTAL POLYPS K. Linke†, G. Mielcarz‡, and K. Zietek† †

Department of Gastroenterology and Department of General Chemistry Karol Marcinkowski University School of Medical Sciences 60-780 Poznan, Poland

‡

Little is known about the involvement of copper and zinc in the colorectal polyps process via pro-oxidant or antioxidant effects. Although copper is used to induce oxidation in vitro, there is no experimental evidence that copper might be involved in oxidation in vivo. Copper and zinc contain superoxide dismutase, which dismutates the highly reactive superoxide anion to less reactive hydrogen peroxide. Recent studies indicate that copper deficiency enhanced the development of 1,2-dimetylhydrazine-induced colon tumours in rats. In this study we investigated copper and zinc concentration in plasma and in colon polyps among 25 patients and 15 healthy age and gender matched controls. Copper and zinc in plasma were determined by flame atomic absorption spectroscopy (Varian GTA-96). Polyps were removed at surgery. Control materials were taken from grossly normal areas. Samples were digested of 6 mol/l nitric acid in 7 ml Teflon vessels in a microwave oven and the resulting digest were made up to 2 ml with de-ionized water. Copper and zinc were determined by graphite furnace atomic absorption spectroscopy. There was a statistically significant difference (P < 0.05) for the mean plasma copper and zinc concentration between controls and patients with colorectal polyps. Zinc has lower but copper higher level in plasma. In polyps tissue was no significant difference in zinc concentration, but copper was significantly decreased (P < 0.05). These results are consisted with the other investigation which have demonstrated elevated serum copper levels in patients with a variety of maligancies. Serum copper associated with ceruloplasmin might increase as a part of the acute-phase process in inflammatory conditions. Decreased level of copper in the polyps tissue indicated that it is also possible that copper deficiency will also influence the development of cancer.

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TOTAL SUPEROXIDE DISMUTASE ACTIVITY AND GLUTATHIONE PEROXIDASE ACTIVITY IN PLASMA OF PHENYLKETONURIC SUBJECTS SUPPLEMENTED WITH SELENIUM M. Calomme1, P. Cos1, V. Ramaekers2, B. François3, M. Van Caillie-Bertrand1, and D. Vanden Berghe1 1

Department of Pharmaceutical Sciences University of Antwerp (U.I.A.) Belgium 2 Department of Paediatric Neurology University of Aachen Germany 3 Dr. Willems Institute, Belgium

The restriction of whole food proteins in the diet of dietetically treated phenylketonuric (PKU) subjects was reported to cause a low selenium intake (1) and is reflected in a profound biochemical selenium deficiency (2). The effect of selenium (Se) supplementation on the activity of total superoxide dismutase (SOD) and glutathione peroxidase (GSHPx) was investigated in plasma of ten dietetically treated phenylketonuric children (mean age ± SD: 12.0 ± 4.1). Non-parametric tests were used to analyze the statistical significance of the results obtained. As generally accepted, the GSHPx activity was correlated (R = 0.97, p < 0.001) with the selenium concentration in plasma and the mean baseline values of both parameters were lower GSHPx: 152 U/l, p < 0.0001) compared to age-matched controls GSHPx: 345 U/l). After ten weeks Se supplementation both the mean Se concentration and the mean GSHPx activity increased but remained slightly lower than age-matched controls. Interestingly, the mean total baseline SOD activity was higher (SOD: 62 NU/ml, p < 0.0001) compared to age-matched controls (SOD: 43 NU/ml) and decreased (p < 0.005) after ten weeks of Se supplementation.

E-mail: [email protected]

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These preliminary results suggest that an increased plasma SOD activity combined with a biochemical deficient Se status could promote oxidative stress and justify Se supplementation in dietetically treated PKU patients.

REFERENCES Selenium intakes in phenylketonuric children on a phenylalanine restricted diet. B. François, et al. Pediatric Research 1991, 30, 651. Thyroid function parameters during a selenium repletion/depletion study in phenylketonuric subjects. M. Calomme et al. Experientia 1995, 51, 1209–1215.

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THE CONTENT OF SELECTED BIOELEMENTS IN HAIR OF CHILDREN WITH INFANTILE CEREBRAL PALSY (PARALYSIS CEREBRALIS INFANTILIS, DIPLEGIA)

R. W. Wójciak and Z. Krejpcio Department of Food Hygiene and Human Nutrition Agricultural University Wojska Polskiego str. 31 60-624 Poznan, Poland

Macro- and trace elements have an important function in the physiology of animal and human organisms. Deficiencies of the bioelements in the organism may be the result of undernutrition or metabolic disorders and may lead to growth retardation, weakening of the immune, cardioviscular and nervous systems. For assessment of the mineral status of the body, blood, serum or plasma and urine are mostly used as biological materials. However, their value is limited and often questioned. Some authors are of the opinion that hair analysis can give more accurate data concerning some bioelement content in the organism therefore it is used in some cases as an alternative to blood tests. The purpose of this study was to assess the content of calcium, magnesium, zinc and copper in hair of children with infantile cerebral palsy. The study was carried out on 12 children (boys), aged 7–15, residents of the Care and Health Center of Gniezno, Poland (Palsy Group—P.G.). The children with infantile cerebral palsy showed bad psychosomatic status as a result of genetic disorder. They could not move on their own and had to stay under continuous hospital care. The reference group (R.G.) consisted of 26 healthy children (21 boys, 5 girls), aged 9–15, from the Elementary School in Gniezno. The material studied were 3-cm hair samples (0.5–1.0 g) taken from six—points of the occipital scalp from both group of children. Hair samples were prepared according to the method advised by the International Atomic Energy Authority (IAEA) and mineralized with the mixture of concentrated spectra—pure acids (HNO3 : HClO4, 2 : 1, v/v). The content of metals in diluted mineral solutions was determined by the flame atomic absorption spectrometry (Zeiss AAS—3 with BC). For statistical evaluation of data Chisquare and Student’s tests were applied using computer programme Statgraphics ver. 7.0. The mean content of Ca in hair was significantly lower (p < 0.001) in the P.G. (582 vs. the R.G The mean content of Mg in hair did not differ between 636

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the studied populations in the P.G. vs. in the R.G.). The mean content of Zn in hair was significantly higher (p < 0.001) in the P.G. vs. the R.G. while the content of Cu in hair of the P.G. was significantly (p < 0.01) lower vs. the R.G. respectively). Moreover, Chi—Square test showed a significant difference in the distribution of hair Ca, Mg and Cu values between these groups of children. The calculated Zn/Cu ratio in hair of the P.G. (34) was significantly higher (p < 0.01) in comparison to the R.G (18). The results obtained in this preliminary study indicate some changes in the mineral status of the body of children with infantile cerebral palsy that may be a result of both chronic disease, and/or various nutritional and environmental factors.

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SERUM COPPER IN JUVENILE RHEUMATOID ARTHRITIS

O. M. S. Amancio, D. M. A. Chaud, and M. O. E. Hilário Department of Pediatrics Federal University of São Paulo Paulista Medicine School São Paulo, Brazil

Juvenile Rheumatoid Arthritis (JRA) is a chronic inflammatory disease, which may result in an increase of serum copper. There are a few studies refering to children and adolescents with JRA. The objective of this work is to evaluate serum copper in JRA patients and relate this to the characteristics of the disease and to the use of corticosteroids. 46 patients whith JRA were assessed. They were diagnosed according to the American College of Rheumatology; 23 boys, with a mean of age of 10 years and 2 months; 26 of these had active disease, 9 with pauciarticular evolutive type (PAA); 13 with polyarticular (POA) and 4 with systemic one (SIA). Of the patients in remission, 11 made up the pauciarticular group (PAR) and 9 polyarticular (POR). These patients were regrouped for analysis of the following variables: disease activity and remission, age at onset < and >5 years old, period of evolution < and >3 years, POA < and >6 inflamed joints and JRA with and without actual use of corticosteroids. 24 healthy children and adolescents were assessed; 14 girls, with mean age of age 9 years and 6 months which constituted the control group. The biochemical determinations were carried out by atomic absoption spectrophotometry. For statistical analysis we used the Kruskall-Wallis and Mann-Whitney tests and differences were considered significant if values were found to be less than or equal to 5% The results showed that serum copper increased in POA group, mainly when more than 6 joints were involved, during active periods, in JRA < 3 years of evolution and in JRA with actual use of corticosteroids. We wish to emphasize that the majority of patients with JRA < 3 years of evolution show active disease, as do all patients using corticosteroids, so that, it is not possible to attribute the increase of serum copper to these variables. We concluded that disease activity and the number of inflamed joints are directly related to the increase of serum copper.

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SERUM COPPER IN JUVENILE RHEUMATOID ARTHRITIS Influence of Dietary Copper

D. M. A. Chaud, O. M. S. Amancio, and Hilário, M. O. E.

Department of Pediatrics Federal University of São Paulo Paulista Medicine School São Paulo, Brazil

A few studies related increased serum copper in effect of inflammatory diseases, however, the relationship to intake and serum level of this element deserves attention. Copper is an essential nutrient that plays important roles in inflammatory diseases, and for this reason, the knowledge of the intake for patients with Juvenile Rheumatoid Arthritis (JRA) is important, as well as the prevention and the correction of its deficiency. To correlate the intake of copper with serum levels of this element, in different groups of patients with JRA was the objective of this study. 42 patients with JRA were assessed, 22 girls, with mean of age of 9 years and 1 month, diagnosed according to the American College of Rheumatology. 22 showed active disease, 9 of these with pauciarticular (PAA), 13 with polyarticular (POA) and 20 showed JRA in remission, 11 of which with pauciarticular (PAR) and 9 with polyarticular (POR). The dietetic assessment was done by Food Register method, applied during 4 days (1 of these on weekend). Dietary copper intake was estimated through the software “Virtual Nutri” (USP, 1996), together with list of composition of foods of Holland et al. (1992). The results are showed in percentage of the minimum limit of “Estimated and Safe Adequate Daily Intake—ESADDI—RDA, 1989)”. The biochemical assessment was done by atomic absorption spectrophotometry. To verify the influence of dietetic copper over serum one, the groups were redistributed according to the intake of copper, it means, intake < and > median of intake in each group. The statistical analysis was done by Mann-Whitney test, and differences were considered significant if a values were found to be less than or equal to 5% (α < 5%). In PAA group, the serum copper was significantly increased when intake was >74%. In PAR and POA, serum copper was increased, in absolute numbers, when intake was major that, respectively 121.53% and 78.2%. In POR group we did not find difference in serum copper when the intake was < and >5.2%, suggesting that hepatic reserves are sufficient for the increase of serum copper, even when intake is low. There is direct relationship among intake of copper and increase of serum copper in PAA JRA and, probably in PAR and POA. 639

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GALLIUM NITRATE AND ZINC CONTENT IN PERIPHERAL BLOOD LYMPHOCYTES OF PATIENTS WITH LUNG CANCER

A. V. Koudrine* and A. V. Skalny** *Imperial College School of Medicine Hammersmith Hospital Du Cane Road, London W12 0NN U.K. **Center for Biotic Medicine Lesnaya, 59, 103055, Moscow Russia

Zinc is an essential trace element required for normal function of the immune system. Although numerous (more than 500) proteins require zinc as a cofactor, its precise functions remain unknown. Zinc is involved in prevention of tumor cells apoptosis. Gallium nitrate exerts its antitumor effect on certain lines of small cell lung carcinoma, bladder cancer and malignant lymphomas via a transferrin binding mechanism. Gallium could affect the ionic balance in tumor cells that result in hypocalcemia, hypomagnesemia and disturbance of divalent cations exchange (P. Collery et al., 1998). The aim of this study was to evaluate the effect of gallium nitrate as an antitumor agent on zinc content in lymphocytes. We studied 20 patients with lung cancer (III–IV stages) and a mean age of 65.1 years (62–67 years). Lymphocytes were obtained by sedimentation of blood in ficollverografin gradient and cultured in presence of gallium nitrate (equivalent to concentration of gallium in blood of treated patients). Zinc content was examined by an atomic absorption spectroscopy before and after incubation. A significant reduction of zinc content in lymphocytes of patients (0.061 ± 0.018 cells and cells respectively). The content of intracellular zinc was in high correlation with appearance of secondary immunodeficiency syndrome (bacterial, viral and fungi infections). The apoptosis of lymphocytes in gallium exposed culture was observed in comparison to non-treated culture of cells. We conclude that gallium nitrate may augment zinc outflow from lymphocytes of patients with lung cancer in co-culture.

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ANTIOXIDANT SYSTEMS IN NORMAL AND PREECLAMPTIC ALGERIAN PREGNANT WOMEN Brahim Lachili 1,2, Josiane Arnaud2, Henri Faure2, Marie Jeanne Richard2, Anwar Bouabsal, Anne Marie Roussel2, and Alain Favier2 1

Laboratoire Central de Biochimie CHU BENFLIS Touhami Bd Mohamed BOUDIA 05000 Batna, Algerie 2 Laboratoire de Biologie du Stress Oxydant (LBSO) Faculté de Pharmacie Domaine de la Merci, 38700 La Tronche France

Preeclampsia affects 5 to 7% of all pregnancies and is one of the most common, yet least understood, disorder of pregnancy. It is also a leading cause of maternal and perinatal mortality worldwide. Damage from free radicals has been implicated in preeclampsia. This study was undertaken to investigate the relationship between the oxidative stress linked to preeclampsia and the possible antioxidant status modifications. We measured plasma thiobarbituric acid reactant (TBARs), Zn, Se, Vit A, E, and Beta-Carotenes and CuZnSOD, SeGPx erythrocytes, in 2 groups of pregnant Algerian women [40 preeclamptic = 27 moderate and 17 severe preeclampsia] compared to 40 normal pregnant women. We observed an increased in plasma TBARs which was significantly higher in women with preeclampsia but there were no significant differences into the preeclamptic group When adjusting to cholesterol, the peroxidation remained significantly increased [TBARs/Cholesterol ratio: The plasma cholesterol was unchanged in the two groups [2.59 ± 0.52 vs 2.59 ± 0.43 g/l]. Plasma triglycerides were higher in women with preeclampsia [3.33 ± 1.00 vs 2.70 ± 0.84 g/l], especially in the severe preeclampsia [3.14 ± 1.06 vs 3.71 ± 0.79]. We observed no difference in status concerning antioxidant [Zn, Se, Vit A, E, and Beta-Carotenes] or in enzymatic antioxidants (CuZnSOD, SeGPX). 641

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Our results confirm the importance of oxidative stress in preeclamptic women. Since this stress is not induced by a depletion in antioxidants, it might result from an inflammatory sundrom or the placental ischemia and exacerbated by the increase in plasma lipids.

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EFFECT OF ERYTHROPOIETIN THERAPY AND SELENIUM SUPPLEMENTATION ON SOME ANTIOXIDANT PARAMETERS IN BLOOD OF UREMIC PATIENTS ON LONG-TERM HEMODIALYSIS B. A. Zachara1, A. Adamowicz2, U. Trafikowska1, A. Pilecki1, A. Trafikowska1, E. Nartowicz2, and J. Manitius2 1 2

Department of Biochemistry and Department of Nephrology Medical University Bydgoszcz, Poland

The objective of the study was to evaluate the effect of erythropoietin (EPO) therapy and selenium (Se) supplementation to chronic renal failure (CRF) patients undergoing chronic hemodialysis (HD) on blood Se, red cell glutathione (GSH), and blood lipid peroxidation products [measured as thiobarbituric acid reactive substances (TBARS)] levels. The activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were also evaluated. We have studied 22 adult patients on HD receiving EPO with (group I; n = 11) or without Se (group II; n = 11) and the results were compared with 18 healthy persons (controls). The patients were regularly receiving subcutaneous EPO (Eprex, Janssen-Cilag, France) in a dose of 2,000 U, 3 times a week. One group of EPO patients was supplied, after each HD session, with Se in the amount of 300 mg Se (Se-rich yeast) 3 times a week for 3 months. Blood samples from patients were taken into tubes containing lithium heparin at the beginning of the study and after 1, 2 and 3 months of Se supply, and 6 weeks after the termination of supplementation. A portion of blood was centrifuged, plasma was harvested and red cells were washed in saline solution and hemolyzed. Before HD the Se level in whole blood of group I (74.3 ng/ml) and II (76.4 ng/ml) was significantly lower (p < 0.01) compared with the control group (99.8 ng/ml). In the plasma of both treated groups the Se level (62.5 and 61.0 ng/ml) was also lower than that in the control group (73.5 ng/ml). Red cell and plasma GSH-Px activities of group I and II (10.3 and 15.0 U/g Hb in red cells, and 106 and 110 U/l plasma, respectively) were significantly (0.0001 < p < 0.005) lower than those in the control group (18.8 U/g Hb and 643

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201 U/l). By contrast, the GSH level in red cells of group I and II was significantly (p < 0.01) higher compared with the control group (2.76 and 2.68 vs 2.33 mmol/l cells). No significant differences were found in SOD activity and TBARS levels between the patients and the control group. During EPO and Se therapy the Se levels both in whole blood and plasma increased linearly and significantly and after 3 months they were 2.2 times higher than before treatment. Red cell GSH-Px activity increased also significantly and after 3 months it was 1.9 times higher than the initial value. During the entire period of the study, however, no significant changes in plasma GSH-Px activity was observed. In EPO group the Se levels both in whole blood and plasma decreased within 3 months by 14% (p, NS). In both groups GSH concentration in red cells showed a tendency to increment but the differences were statistically not significant. Other parameters studied did not change in either of the two groups. In conclusion, we would like to emphasize that the results indicate that due to renal failure, plasma GSH-Px (which is synthesized mainly in kidneys) did not change despite the fact that the Se level in plasma increased significantly. Treatment with EPO causes an increase in red cell GSH-Px activity.

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ARSENIC GROUNDWATER CONTAMINATION AND SUFFERINGS OF PEOPLE IN WEST BENGAL-INDIA AND BANGLADESH U. K. Chowdhury1, B. K. Biswas1, T. Roy Chowdhury1, B. K. Mandal1, G. Samanta1, G. K. Basu1, C. R. Chanda1, D. Lodh1, K. C. Saha1, D. Chakraborti1, S. C. Mukherjee2, S. Roy3, S. Kabir3, and Q. Quamruzzaman3 1

School of Environmental Studies Jadavpur University Calcutta-700032, India 2 Department of Neurology Calcutta Medical College Calcutta 3 Dhaka Community Hospital Dhaka-1217, Bangladesh

There are approximately 20 countries in the world where incidents of arsenic contamination of groundwater have become known. However, worlds two biggest cases of groundwater contamination & worst sufferings of people have been in Bangladesh (Dhar et al., 1997; Biswas et al., 1998) & West Bengal-India (Mandal et al., 1998). The magnitude of arsenic calamity in Bangladesh & West Bengal-India have surfaced only recently. In a report (Pearce, 1998) while narrating the magnitude of arsenic calamity in Bangladesh, the World Bank’s local chief mentioned tens of million of people are at risk & 43,000 villages out of 68,000 are presently or could be at risk in future. A report by World Health Organisation (WHO) predicts (Pearce, 1998) that, within a few years, one in 10 adults death occurs much of southern Bangladesh could be from cancers triggered by arsenic.

1. MAGNITUDE OF THE CALAMITY Table 1 shows the physical parameters of arsenic affected districts of Bangladesh & West Bengal-India. Figure 1 shows the arsenic affected districts of Bangladesh & West Bengal. Although the population in the affected districts are 79 & 38 million respectively, Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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but that does not mean all are drinking contaminated water & suffering, but no doubt they are at risk. To make an estimation of population drinking arsenic contaminated water & suffering from arsenical skin lesions in West Bengal, we surveyed for 4 years North-24-Parganas, one of the nine affected districts of West Bengal & a few blocks from other districts in detailed. We made an extrapolation of our generated data for the affected 9 districts & expecting about 5.0 million people are drinking contaminated water

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above & nearly 300,000 people may have arsenical skin lesions. Further, our theoretical calculation was applied to our preliminary dermatological field survey report where we had examined about 30,000 people from 154 villages of 7 affected districts. The comparative study indicates we have not over estimated. For Bangladesh so far we have not yet made such a systematic detailed study but from our last 4 years survey in Bangladesh in the affected villages with water & biological sample analyses & dermatological study in the affected villages, we feel Bangladesh is more affected than West Bengal (Table 1, Fig. 1).

2. ANALYSIS OF HAND TUBEWELLS FOR ARSENIC We have so far analysed, from Bangladesh 12,084 hand tubewells from 64 districts & out of that 10,781 samples are from 42 districts where groundwater contains arsenic above & from West Bengal’s arsenic affected 9 districts 55,166 hand tubewells. Figure 2 shows a comparative study. From the comparative study it appears that in Bangladesh arsenic groundwater concentration is much higher compared to West Bengal. Out of total 10,781 water samples from Bangladesh we have got 219 samples above 1,000 whereas from West Bengal out of 55,166 samples only 35 are above Arsenic speciation of water samples indicate ground water contains only arsenite & arsenate. Normally with poor nutrition status people drinking arsenic contaminated water above in Bangladesh & West Bengal may show arsenical skin lesions. We have also identified a few having arsenical skin lesions drinking arsenic contaminated water. In these two countries an average adults drink 4 litres of water & children 2 litres of water per day. Even those who work in field drink more water & as high as 10 litres per day.

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3. ANALYSIS OF BIOLOGICAL SAMPLES FOR ARSENIC We had analysed hair, nail, urine (metabolites), skin scale from people living in arsenic affected villages, from both Bangladesh & West Bengal. On an average out of our total biological samples analysed 40% are from the population having arsenical skin lesions. Table 2 shows the comparative study. It appears from Table 2 that good percentage of villagers who have no skin lesions have high arsenic body burden. Thus a good percentage of population may be subclinically affected. From West Bengal we had also analysed some blood samples (n = 160) from people who were drinking contaminated water during our survey. We had also analysed breast milk (n = 250) from the affected mothers & mothers drinking contaminated water. It appears from our study that arsenic level in breast milk is not elevated but blood samples show elevated level of arsenic.

4. DERMATOLOGICAL FEATURES From 7 arsenic affected districts out of 9 in West Bengal, we had so far carried out very preliminary survey in 151 villages & had examined 29,035 people & out of them we could identify 4,420 (15.02%) people with arsenical skin lesions. In Bangladesh during last 4 years we had examined 11,180 people from 112 villages, & 24% have arsenical skin lesions. Thus in Bangladesh more people are affected from arsenical skin lesions than in West Bengal. Children in the affected villages of Bangladesh are more affected (6.5%) than in West Bengal (1.7%). This is to note, that the reason of such high percentage patients in the affected villages is, we carried out our survey only in those villages where we had information of patients & people drinking highly arsenic contaminated water. When our survey will be done in district level & covering both affected & non affected villages this percentage will drastically reduce. Figure 3 shows comparative study of dermatological symptoms of adults in West Bengal & Bangladesh.

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5. ARSENICAL NEUROPATHY Both the peripheral nervous system (PNS) & the central nervous system (CNS) can be damaged due to arsenic exposure. But CNS impairment has been less frequently observed compared to the PNS effect. Arsenical neuropathy is the commonest type of peripheral neuropathy due to metal or metalloid far exceeding neuropathies caused by lead, mercury or other metals. Incidence of arsenical neuropathy was studied during the last one year in 413 patients with arsenical skin lesions of varying severity in Nadia & Murshidabad districts of West Bengal. Overall clinical neuropathy was evident in 154 patients (37.2%). 124 patients (30%) were affected with predominantly sensory neuropathy & 30 patients (7.2%) had sensorimotor neuropathy. Electrophysiological studies are helpful for both clinical & subclinical (electrophysiologic) forms of arsenical neuropathy. Studies in population exposed to arsenic contaminated drinking water showed varying difference of abnormal sensory & motor nerve conduction in 10 out of 33 in one series & 13 out of 147 in another series. By nerve conduction & electromyographic study (EMG & NCV study) on 20 patients of arsenicosis we found sensory nerve affection in 9 (45%) & motor nerve affection in 4 (25%) of our arsenic affected patients.

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6. CONCLUSION Working on West Bengal’s arsenic calamity for last 10 years & in Bangladesh for last 4 years even now we feel we are at the tip of the iceberg. Thus we need to know as early as possible the real magnitude of the arsenic calamity. According to WHO, the possibility of getting skin lesions exists among those drinking of arsenic per day for several years. & our analytical report on water indicates that a large sum of population are consuming above of arsenic per day. Our thousands of hair, nail & urine analyses from the affected villages indicate that more than 80% of population have higher arsenic body burden. Thus many may not be showing arsenical skin lesions but may be sub-clinically affected. Further if it is true that arsenic toxicity appears after several years of exposure, then the picture may actually be far more grim than it appears at present, & children our future generations are at a greater risk.

REFERENCES Biswas, B.K., Dhar, R.K., Samanta, G., Mandal, B.K., Faruk, I., Islam, K.S., Chowdhury, M.M., Islam, A., Roy, S., and Chakraborti, D., 1998, Detailed study report of Samata one of the arsenic affected villages of Jessore district, Bangladesh, Current Science, 74(2): 134–145. Dhar, R.K., Biswas, B.K., Samanta, G., Mandal, B.K., Chakraborti, D., Roy, S., Jafar, A., Islam, A., Ara, G., Kabir, S., Khan, A.W., Ahmed, S.A., and Hadi, S.A., 1997, Groundwater arsenic calamity in Bangladesh, Current Science, 73(l):48–59. Mandal, B.K., Roy Chowdhury, T., Samanta, G., Mukherjee, D.P., Chanda, C.R., Saha, K.C., and Chakraborti, D., 1998, Science of the Total Environment, 218:185–201. Pearce, F., 1998, Arsenic in water, The Guardian (London), February 19, Online 1–4.

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HEAVY METALS AND PERSISTENT ORGANIC POLLUTANTS IN NEWBORN CORRELATED WITH MATERNAL SMOKING

Bente Deutch and Jens C. Hansen Institute of Environmental and Occupational Medicine Aarhus University Aarhus, Denmark

Persistent Organic Pollutants (12 pesticides and 14 PCB-congeners) and heavy metals (Cd, Cu, Hg, Pb, Se, and Zn) were determined in 175 pregnant women and 169 newborn infants (umbilical cord blood) from Diskobay, Greenland, 1994–96, as part of the “Arctic Monitoring and Assessment Programme”, AMAP. The women were also asked to fill out questionnaires on demographic, anthropometric and lifestyle parameters, e.g. about drinking, smoking and intake of traditional Inuit food. The mean values of the measured organic contaminants in plasma were found to be among the highest in Arctic populations and among the highest in the world (Stone et al., 1998). Practically all the substances studied passed from the maternal blood, across the placenta barrier into the blood of the newborn with close to 100% effeciency. This means that the pollution burdens of the infants were equivalent of those of their mothers.The organic pollutants were mutually correlated. They were also correlated with several of the heavy metals and intake of some traditional food items and marine food in general, Table 1. A total of 135 women answered the questionnaires and among those, unmarried mothers were underrepresented compared to the general population of birthgiving mothers in the area. Consequently the ranges of birthweights and reported daily cigarette comsumption were slightly narrower than in the general polpulation and with significantly lower mean values. Nevertheless multiple linear regression analyses showed highly significant positive associations between the mothers smoking status (never = 0, previous = 1, present = 2) and plasma concentrations of all the studied organic pollutants both in maternal blood (Table 2) and umbilical cord blood. Appropriate corrections were made for the age of the mother, (bioaccumulation), Body Mass Index (turnover rate) and the intake of traditional food (source/vector of the contaminants). In addition also some of the heavy metals, Hg, Cd, and Se in maternal blood and Hg in cord blood showed significant positive correlations with smoking, Table 3. Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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The associations between smoking and blood contaminants were as strong or stronger than the association with traditional food allthough the traditional food is known to be the predominant source of both the organic and inorganic pollutants concerned (Hansen, 1981; Weihe et al., 1996; Mulvad et al., 1996; Dewailly et al., 1993). Only a few of the studied substances are known to occur in tobacco, namely cadmium and possibly lead and DDT (Hansen et al., 1991; Jensen and Clausen, 1979). All the other substances and in particular Hg and PCB’s are not likely to be found in tobacco. It thus seems that the higher accumulation rates of contaminants found in smokers need other explanations, namely that they are due to effects on the uptake or turnover mechanisms of xenobiotic substances, e.g. by influencing the P-450 cytochrome oxydase system (Pasanen and Pelkonen, 1990). This explanation is supported by the fact that in this study the more persistant (more slowly metabolized) pollutants typically the PCB-congeners 138, 153, 180 and 187 showed stronger correlations with smoking than the less persistent substances. Associations between lifestyle parameters and pollutant burdens or health effects found in epidemiological surveys may only be predictive of others factors and not necessarily causal, no matter how strong they are. It is therefore necessary to investigate these findings further, hopefully under more controlled conditions. Unfortunately intervention trials seem to be ethically problematic or impractical because of the longterm character of the proposed effects of smoking. However, since the burden of pollutants is lower in

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previous smokers than present smokers it should be posible to study the effect of smoking cessation. To further illuminate the phenomenon, we are doing a case-control study on a uniform selection of male Inuit hunters, smokers versus non-smokers and previous smokers. In addition we are planning to perform a metaanalysis including all the possible previously available data from Greenland and to perform POP analyses on stored plasma from previous epidemilogical surveys in Denmark.

REFERENCES Dewailly E., Ayotte P., Bruneau et al., 1993, Inuit exposure to organochlorines through the Arctic food chain in Arctic Quebec. Environ Health Perspect 101(7):618–620. Hansen J.C., 1981, A survey of human exposure to mercury, cadmiun and lead in Greenland. Mdd. Grl Man & Society, 3.1981. Hansen J.C., Jensen T.G., and Tarp U., 1991, Changes in blood mercury and lead levels in Pregnant women in Greenland 1983–1988. Proceedings of the 8 th International congress on Circumpolar Health. Postl B.D., Gilbert P., et al., (eds), University of Manitoba Press, Whitehorse, Yukon, May 20–25, 1990. Jensen G.E. Clausen J., 1979, Organochlorincompounds in adipose tissue of Greenlanders and southern Danes. J Tox Env Health 5:617–629. Mulvad G., Pedersen H.S., et al., 1996, Exposure of Greenlander Inuit to organochlorines and heavy metals through the marine food chain, an International study. Sci Total Environ 186(1–2):137–139. Pasanen M., Pelkonen O., 1990, Xenobiotic and steroid metabolizing monooxygenases catalyzed by cytochrome P 450 and glutation s-transferase conjugations in human placenta and their relation to maternal cigarette smoking. Placenta jan-feb 11(1):75–85. Stone D., Liljelund L.E., and Reirsen L.O., eds., 1998, AMAP Assessment report: Arctic Pollution Issues. Arctic Monitoring and Assessment Programme (AMAP) Oslo Norway. Weihe P., Grandjean P., Debes F., and White R., 1996, Health implications for Faroe Islanders of heavy metals and PCB’s from pilot whales. Sci Total Environ 186(1–2):141–148.

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COPPER AND EARLY CHILDHOOD CIRRHOSIS (ECC) A Retrospective Study In Germany

H. H. Dieter, W. Schimmelpfennig, E. Meyer, and M. Tabert Institut für Wasser-, Boden- und Lufthygiene des Umweltbundesamtes POB 330022, D-14191 Berlin Germany

1. INTRODUCTION The extent of a causality between ECC and excessive copper intake is a hotly debated issue in Germany (Eife et al., 1987/88; Eife et al., 1991; Eife et al., 1997). Authors from different countries have published recently on the problem (v. Mühlendahl and Lange, 1994; Horslen et al., 1994; Müller et al., 1996; Scheinberg and Sternlieb, 1994; Tanner et al., 1998). As far as exogenously enhanced copper exposure was brought into question as causative factor of ECC, this clinical entity outside India has been called “Idiopathic (idiopathic = arising spontaneously without recognizable cause) Copper Toxicosis (ICT)” by Scheinberg and Sternlieb (1994), whereas in India it figures under “Indian Childhood Cirrhosis (ICC)” (Sethi et al., 1993). We conducted a large-scale multicentric retrospective clinical study on the prevalence and distribution of ECC of whatever cause in Germany and its eventually copper-associated forms (Schimmelpfennig et al., 1996).

2. METHODS Patient Sample Our target group were children with confirmed ECC (ICD-10/No.571) of any etiology. A retrospective period of approx. 12 years (1982–1994) was taken into consideration. The most recent cases came from 1994. 40 of 63 pediatric centres (63.5%) in Germany responded to our request for cooperation. Cases of ECC of any cause had been Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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observed in only 16, representing a total of 103 diagnostically confirmed cases of ECC. The patient records supplied to us by the pediatric centers and the data on drinking water quality (either s.w./single well or c.s./centrally supplied drinking water) were evaluated case by case (Schimmelpfennig et al., 1996; Dieter et al., 1999).

Drinking Water Data and Assessment of Its Corrosivity Information on the type of water supply (c.s. or s.w.), the presence of copper plumbing and the extent of tap water use for preparing formula milk had to be inquired from the childrens parents. The rate of reply was 53/103 cases (=51.5%), including 8/13 households with private wells. Data to qualify the corrosivity of the consumed water were obtained either from the water suppliers serving the communities in which the diseased children had been living right after birth, or directly from the users of single wells. From the combination of striking corrosive potential (high base capacity of the consumed water and copper installations in the household we draw the conclusion that there might have been a particular exposure potential for the child with regard to a risk for “ICT” (details: see Schimmelpfennig et al., 1996; Dieter et al., 1999).

3. RESULTS Clinical: Confirmed Etiologies of ECC 103 cases of ECC of any etiology, diagnostically confirmed and secured by clinical, biochemical, virological, immunological and morphological criteria were documented. In each case there was a liver histology and in individual cases, also the re-sults of a laparoscopy or laparotomy (Schimmelpfennig et al., 1996). The observed range of diagnoses corresponded to clinical experience and knowledge: The overwhelming majority of the cases (47.5%) could be excluded to have a copper associated etiology but were biliary liver cirrhoses as a result of congenital biliary abnormalities. Metabolic liver diseases and liver cirrhoses of unclear or unclarified etiology were ranking second (18 cases or 17.5%, respectively). In one case was it possible to subsequently identify a hepatitis virus B infection. 8 cases were assigned to copper; 9 cases had subacute liver dystrophia (4) or congenital fibrosis (5).

Unclear Etiologies of ECC For 10 of the 18 unclear cases we were unable to identify any clear cause, despite adequate or clearly sufficient diagnostics. In the other 8 of these 18 cases, important diagnostic statements were lacking, e.g. on virus etiology or on the metabolic situation. In a closer look to the subgroup of the 10 “unequivocally unclear” cases, we considered the following criteria: Age of manifestation, breast-feeding practice, corrosive potential of the water, reported copper installations, clinical and hygienic features. In 9 out of these 10 cases certain well defined clinical and exposure indicators were present contradicting excessive copper exposure and hence copper intoxication. Only in 1 case would renewed examination of the exposure data have been worthwile, especially as a brother was likewise suffering from apparently congenital cirrhosis of the liver with a fatal course.

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Suspicion of Copper Etiology It could not been traced back from the hospital records whether the diagnoses were performed by medical doctors being blinded with respect to possible copper exposure. With this reservation, chronic copper intoxication should be taken into consideration as the main or sole cause of ECC for 8 out of our 103 cases of diagnostically confirmed ECC-cases, all 8 being supplied with (very) highly corrosive and/or presumed or confirmed acid water from single wells through copper pipes (Schimmelpfennig et al., 1996; Dieter et al., 1999). In 5 of these 8 cases, the copper diagnosis was regarded as probable since liver copper was proven to be very high and high copper exposure was confirmed or other etiologies could be excluded. They include 3 of those 5 cases having been already mentioned earlier among others by Eife et al. (1991) and exhibited the typical clinical and morphological picture, including extremely elevated hepatic copper concentrations. Their differential diagnostics showed that they could not have been caused or triggered by another etiology. In all 5 cases, acid single well water supplied via copper pipes was proven to have been used regularly during several weeks early after birth to prepare the baby’s formula, and all these babies had not been breast-fed at all or only for up to 4 weeks right after birth. One case exhibited (in addition to probably copper?) a cytomegalovirus infection and was characterized by two other particularities: the child’s Vena portae was transformed, and both parents showed elevated serum copper concentrations whose cause could not be cleared. 1 case was anti-HBs positive. For the 3 other cases copper intoxication could only be suspected as the cause of disease, since liver copper values were unknown and high copper exposure was not quantitatively confirmed or other etiologies were not reliably excluded (Schimmelpfennig et al., 1996; Dieter et al., 1999).

Drinking Water Data and Confirmed Exposure Situations For 59 of the 103 accepted cases we could gather details on the type of water supply: 46 were c.s. whilst 13 of them were s.w. Installations made primarily from copper were positively reported from 19 houses (11 c.s., 8 s.w.), mixed installations from 15 houses (14 c.s., 1 s.w.) and the absence of copper from 23 houses (21 c.s., 2 s.w.). From 2 s.w. we couldn’t get any information on the piping material. Analytic data of very different quality were reported from central suppliers for 78 out of 90 households, but of these 78, only 10 had positively reported to be equipped (primarily) with installations. Only 2 of these 10 had babies which were exclusively not breast-fed, whereas one other was partially breast-fed. The calculated base capacity or corrosivity of the drinking water in these 3 houses was either medium (1 case) or low (2 cases) and all three diagnoses were unequivocal: Inborn bile duct atresia or subacute liver dystrophia, but no excessive copper exposure. In 5 other c.s.-houses with primarily copper installations and babies fed exclusively with formula milk/drinking water, the water data were not complete enough to calculate the base capacity. However, also in these 5 cases the diagnoses unequivocally excluded a copper etiology for ECC. A total of only 2 complete water data sets could be gathered from the 13 s.w. waters. Both were assessed to have very high corrosion potential. For both we could also simulate exposure conditions and the corresponding copper concentrations (9–26.4 mg/l), whereas for a 3rd we could get only an exposure-relevant copper value (16 mg/l), but no

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water data. These three copper values were much higher than predicted by our experience with corrosive waters (see 2.2). The simulated exposure conditions indicated at least in 2 cases a 8–12 h stagnation time before the waters were used to prepare the baby’s milk (Fischer, 1994; Bent und Böhm, 1995; Schimmelpfennig et al., 1996; Dieter et al., 1999). For a 4th and a 5th case, although rating as probable, we merely obtained no value and copper values (1.3 mg/l and 13 mg/l) from later measurements only. From the remaining 10 s.w. households, 6 had reported no water data at all and 4 only some (mostly a pH-value below 6.5). 5 of these 10 children had an unequivocal, albeit not copper-related diagnosis and had not been living in houses with (primarily) copper installations. For the other 5 children, the probability/possibility of a copper caused or associated ECC and an etiology of excessive copper exposure via well water was concluded indirectly from the original hospital records (see above).

4. DISCUSSION Clinical Results The most comprehensive material on the results of (almost exclusively blind!) liver biopsies up to the age of 14 (n = 524) derives from Roschlau (1976). This sample apparently contained only one single case (= 0.2%) of liver cirrhosis. Our results confirm this observation on ECC being a relatively rare disease with different possible etiologies besides the possibility of excessive copper exposure. The rarity of the disease is also borne out by the health statistics in Germany as compared for former East and West Germany (Schimmelpfennig et al., 1996). Moreover, according to older statistics, relevant clinical experience and textbooks, no etiological factors can be traced in approximately 20% of cases (so-called cryptogenic cirrhoses), a percentage which goes well with our 17.5% of uncleared etiologies. ECC with a high hepatic copper content have occurred in association with (Adamson et al., 1992; Gormally et al., 1994; Müller et al., 1996; Müller-Höcker et al., 1987; Müller-Höcker et al., 1988; Price et al., 1996; Tanner, 1998; Trollmann und Behrens, 1998; Walker-Smith and Blomfield, 1973) and without (Aljajeh et al., 1994; Baker et al., 1995; Bartok et al., 1995; Lefkowitch et al., 1982; Lim and Choo, 1979; Tanner, 1998; Tauxe et al., 1994) excessive exposure to copper. In a rural area of Western Austria (Müller et al., 1996), pedigree analysis points to an autosomal recessive transmission of a non-Wilsonian gene (Wijmenga et al., 1998). Replacement of untinned copper by modern industrial vessels seems to have eradicated ecogenetic “ICT” from this region of Austria (Müller et al., 1996), but also ICC from India (Tanner, 1998). Therefore, copper associated ECC should not automatically be regarded to be “Idiopathic” Copper Toxicosis. A main argument against a postulated causative link between excessive copper intake and the occurrence of “ICT” was given by Scheinberg and Sternlieb (1994). They found no fatalities due to liver disease below 6 years of age among a total of over 60.000 child years in 3 Massachusetts cities with presumed high copper values (and low pH) in tap water. However, due to the presumed low absolute incidence of “ICT” and also of ECC, but also to limitations in sample size, that study might in principle not have been able to positively detect at least one or a few cases of either ECC or “ICT”. In contrast, our study was able to detect 103 cases of ECC, presumably because we examined a much larger population (at least child years).

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Summing up, we could demonstrate 1) the possibility to detect cases of ECC or even “ICT” if the sample size is chosen large enough. a 2) prevalence of at most 5 out of 103 for excessive copper intake being one or the sole causative environmental factor to trigger ECC. Therefore, even if including our 3 suspected and published by Müller-Höcker’s et al. (1987; 1988) earlier cases, the prevalence of “ICT” in Germany can be estimated to be distinctly less than that of Wilson’s Disease (IPCS/WHO, 1996). 3) that at least in some cases, as was postulated by an expert group of IPCS/WHO (1996), excessive exogeneous copper intake for the time being must be discussed as a cirrhogenic (co-) factor in early childhood.

Nevertheless, in the light of the international literature and case reports, it is not just a German problem to consider rare cases of ECC as possibly/probably caused by water born excessive copper exposure, although mean exposures to less than 2 mg/l have recently been reported to be safe at least for babies over 3 months of age (Olivares et al.,1998).

Drinking Water Regulatory Aspects From the regulatory point the situation remains unsatisfactory. Copper concentrations in households with drinking water from central suppliers on the one side, and in households with confirmed cases of copper-associated ECC on the other side, could differ at the tap sometimes by factors of only 3 to 5 if the water has run through copper pipes, be it acid or neutral with low or (very) high (Edwards et al., 1996). Tinned copper pipes and armatures being available since a few years in Europe could be suitable to overcome this uncertainty with regard to new copper pipes. Meanwhile, the question of whether “ICT” is associated merely by epidemiological bias with either “single wells” on the one side or “excessive chronic copper intake” on the other side must be kept open. The first possibility would implicate a high dark number of copper caused ECC (including early reversible and/or later benign states according to Müller-Höcker, 1998)— even in households with central drinking water supply and copper pipes. The latter being true would give a release to excessive copper as an exogenous trigger for “ICT”.

REFERENCES Adamson, M., Reiner, B., Olson, J.L., Goodman, Z., Plotnick, L., Bernardini, I., and Gahl, W.A., 1992, Indian childhood cirrhosis in an American child, Gastroenterol 102:1771–1777. Aljajeh, I.A., Mughal, S., Al-Tahou, B., Ajrawi, T., Ismail, E.A., and Nayak, N.C., 1994, Indian childhood cirrhosis-like liver disease in an Arab child, A brief report. Virch Arch 424:225–227. Baker, A., Gormally, S., Saxena, R., Baldwin, D., Drumm, B., Bonham, J., Portmann, B., and Mowat, A.P., 1995, Copper-associated liver disease in childhood, J Hepatol. 23:538–543. Bartok, I., Szabo, L., Horvath, E., and Ormos, J., 1971, Juvenile Zirrhose mit hochgradiger Kupferspeicherung in der Leber, Acta hepato-splenol 18:119–128. Bent, S. and Böhm, K., 1995, Kupferinduzierte Leberzirrhose bei einem 13 Monate alten Jungen. Gesundh-Wes 57:667–69. Dieter, H.H., Schimmelpfennig, W., Meyer, E., and Tabert, M., 1999, Early childhood cirrhosis (ECC) in Germany between 1984 and 1994 with special consideration of copper etiology, Europ. J. Med. Res. (Accepted for publication).

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Edwards, M., Schock, M.R., and Meyer, T.E., 1996, Alkalinity, pH and copper corrosion by-product release, J Am Wat Works Ass 88:81–94. Eife, R., Müller-Höcker, J., Kellner, M., Arleth, S., Schmölz, A., Weiß, M., and Bender-Götze, C., 1987/88, Kupferwasserleitungen als Ursache für Immundefizienz und frühkindliche letale Leberzirrhose (vom Typ der Indian Childhood Cirrhosis), pädiat prax 36:69–76. Eife, R., Reiter, K., Sigmund, B., Schramel, P., Dieter, H.H., and Müller-Höcker, J., 1991, Die frühkindliche Leberzirrhose als Folge der Kupferintoxikation, Bundesgesundhbl 32:327–29. Eife, R., Weiss, M., and Müller-Höcker, J., 1997, Vergiftungen durch Kupfer im Trinkwasser—Aktuelle Zusammenfassung der Beobachtungen über die Vergiftungen in Deutschland in den vergangenen zehn Jahren. Vortrag auf der 46. Jahrestagung der Süddeutschen Gesellschaft für Kinderheilkunde und Jugendmedizin, Pforzheim, 6. bis 8. Juni 1997. Zusammenfassender Bericht: Alete Wissenschaftlicher Dienst (München), ISBN 3-931310-03-5. Fischer, R., 1994, Probleme der morphologischen Diagnostik frühkindlicher Lebererkrankungen. in: Dieter, H.H. and Seffner, W., (eds.), Kupfer und frühkindliche Leberzirrhose, Umweltbundesamt Berlin, Institut für Wasser-, Boden und Lufthygiene, WaBoLu-Heft 9/1994:25–28. Gormally, S.M., Baker, A., Portmann, B., Mowat, A., and Drumm, B., 1994, High water copper content associated with Indian childhood cirrhosis in European children. Digestive Disease Week and the 95th Annual Meeting of the American Gastroenterological Association (AGA), May 15–18, 1994, New Orleans, LO, USA. Horslen, S.P., Tanner, M.S., Lyon, T.D.B., Fell, G.S., and Lowry, M.F., 1994, Copper associated childhood cirrhosis, Gut 35:1497–1500. IPCS/WHO (International Programme on Chemical Safety/World Health Organization), 1996, Environmental Health Criteria for copper: Summary, evaluation, conclusions and recommendations of IPCS Task Group—28 june 1996. United Nations Environment Programme/World Health Organization/International Labour Organization, PCS/EHC 96.28 (Unedited). Lefkowitch, J.H., Honig, Ch.L., King, M.E., and Hagstrom, J.W.C., 1982, Hepatic copper overload and features of Indian childhood cirrhosis in an American sibship, New Engl J Med 307:271–277. Lim, C.T. and Choo, K.E., 1979, Wilson’s disease—in a 2 year old child, J Singapore Paed Soc 21:99–102. Müller, T., Feichtinger, H., Berger, H., and Müller, W., 1996, Endemic Tyrolean infantile cirrhosis: an ecogenetic disorder, Lancet 347:877–880. Müller-Höcker, J., 1998, Different Pathomorphological patterns in exogenic Infantile Copper Intoxication in the liver, Pathol. Res. Pract. 194:377–384. Müller-Höcker, J., Meyer, U., Wiebecke, B., Hübner, G., Eife, R., Kellner, M., and Schramel, P., 1988, Copper storage disease of the liver and chronic dietary copper intoxication in two further German infants mimicking Indian childhood cirrhosis, Path Res Pract 183:39–15. Müller-Höcker, J., Weiß, J.M., Meyer, U, Schramel, P., Wiebecke, B., Belohradsky, B.H., and Hübner, G., 1987, Fatal copper storage disease of the liver in a German infant resembling Indian childhood cirrhosis, Virch Arch A 411:379–385. Olivares, M., Pizarro, F., Speisky, H., Lönnerdal, B., and Uauy, R., 1998, Copper in infant nutrition: Safety of World Health organization provisional guideline value for copper content of drinking water, J. Ped. Gastroenterol Nutr. 26:251–257. Price, L.A., Walker, I., Clague, A.E., Pullen, I.D., Smits, S.J., Ong, T.H., and Pastrick, M., 1996, Chronic copper toxicosis presenting as liver failure in an Australian child, Pathol 28:316–20. Roschlau, G., 1978, Leberbiopsie im Kindesalter. Gustav Fischer Verlag, Jena. Scheinberg, I.H. and Sternlieb, I., 1994, Is non-Indian childhood cirrhosis caused by excess dietary copper?, Lancet 344:1002–1004. Schimmelpfennig, W., Dieter, H.H., and Tabert, M., 1996, Cirrhosis of the liver in early childhood and copper content of drinking and well water, respectively. Multi-centric retrospective clinical study on frequency, distribution and etiology in Germany (supported by the University of Leeds/UK—The Environment Center). Federal Environmental Agency, Institute for Water, Soil and Air Hygiene, D-14191 Berlin, april 1996. Sethi, S., Grover, S., and Khodaskar, M.B., 1993, Role of copper in Indian childhood cirrhosis, Ann Trap Paediatr 13:3–6. Tanner, M.S., 1998, Role of copper in Indian childhood cirrhosis, Am. J. Clin. Nutr. 67:1074S–1081S. Tauxe, W.N., Reyes, J., Jaffe, R., and Knisely, A.S., 1994, “Indian childhood cirrhosis” in 2 female siblings of european descent, with abnormal radiocopper handling in father and brother, Lab Investig 70:9P. Trollmann, R. and Behrens, R., 1998, Späte Manifestation einer Indian Childhood Cirrhosis aufgrund einer chronischen Kupferintoxikation über das Trinkwasser, Gesundh.-Wes. 60:376–377.

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v. Mühlendahl, K.E. and Lange, H., 1994, Copper and childhood cirrhosis, Lancet 344:1515–1516. Walker-Smith, J. and Blomfield, J., 1973, Wilson’s disease or chronic copper poisoning?, Arch. Dis Childh 48:476–479. Wijmenga, C., Müller, T., Murli, I.S., Brunt, T., Feichtinger, H., Schonitzer, D., Houwen, R.H.J., Müller, W., Sandkuijl, L.A., and Pearson, P.L., 1998, Endemic Tyrolean infantile cirrhosis (ETIC) is not an allelic variant of Wilson’s Disease, Eur. J. Hum. Genet. 6:624–628.

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DIFFERENCE IN PENETRATION OF METALS INTO THE BRAIN

Takeshi Minami, Yuko Sakita, Yuko Okazaki, and Ryouhei Amano Department of Living Sciences & Program Processing Kinki University Toyo-Oka Junior College 160 Tobera, Toyo-Oka 668-8580 Japan Faculty of Pharmaceutical Sciences Kinki University 3-4-1 Kowakae, Higashi-Osaka 577-8521 Japan School Health Sciences Faculty of Medicine Kanazawa University Kanazawa 920-0942, Japan

1. INTRODUCTION Hydrophilic substances are difficult to penetrate into the brain as the blood-brain barrier (BBB) allows only certain substances to penetrate into the brain. However, its permeability is easily changed under various physiological conditions. We previously found that cisplatin, an antitumor platinum (Pt) compound, was penetrated into the mouse brain either by exposure under a short-term hypoxia or injection of lipopolysaccharide (LPS) (Minami et al., 1996a, 1998a). Free radicals, nitric oxide and prostaglandins may be related to induce those phenomena (Minami et al., 1996b, 1998a,b). Although various metals need in the brain, it was reported that a severe mental damage occurred in patients administered cisplatin (Higa et al., 1995). And aluminum (Al) is known as a substance inducing Alzheimer disease (Forbes et al., 1998). The aim of this study is to observe the different penetrations of metals into the brain under injection of LPS. Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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2. MATERIALS AND METHODS Male ddY mice (6w-old) were purchased from Japan SLC (Shizuoka, Japan), housed for 1 w before the experiments. They were housed in an air-conditioned room with 12/12 h light/dark cycle and fed commercial mouse chow and water ad libitum. LPS (Escherichia coli 026:B6) was purchased from Difco Laboratories (Detroit, MI, USA) and cisplatin was from Nihon Kayaku (Tokyo, Japan), Randa (50 mg of cisplatin/ 100 ml of injection solution). The other reagents were from Wako Pure Chemicals (Osaka, Japan). For observing on time dependency of the cisplatin injection after LPS treatment, conscious mice were intraperitoneally injected LPS at 3 mg/kg of body weight. After zero, 1, 3, or 5 h, 2.5 mg/kg of cisplatin were intravenously injected into the tail vein of mice, and the brains were resected 24 h after the injection of cisplatin. The control mice were injected with saline (10 ml/kg) instead of LPS, and then cisplatin was injected 3 h later. For observing on the concentration of Pt, zinc (Zn), or Al in the cerebral cortex-rich region (CCR), cisplatin (2.5 mg/kg, i.v.), zinc sulfate (2.5 mg/kg, s.c.) or aluminum chloride (10 mg/kg, i.p.) was injected 3 h after intraperitoneal injection of LPS. The brain was resected time-dependently, and the CCR was obtained. The control mice were injected with saline instead of LPS, and each metal solution was injected 3 h later. Twenty-four hours after the solution was injected, the brain was removed. For measurement of Pt concentration in CCR, the CCR was dried at 100 C for 20 h and then weighed. For measurement of Zn concentration in CCR, the CCR was weighed and dried at 100 C for 20 h. After a wet combustion with nitric acid (1.0 ml) and perchloric acid (0.5 ml), all samples were diluted to 10 ml with extra-pure water. The Pt or Zn content in the CCR was measured by ICP-MS (PIMS-3000, Shimadzu, Kyoto, Japan) as described by Minami et al. (1995) or ICP-AES method, respectively. For measurement of Al concentration in CCR, the freeze-dried CCR was heat-sealed in polyethylene tubes and irradiated for 5 min for the measurements of short Al-28 radioactivity produced by Al27 (n, r) reaction. The irradiation was carried out by the use of the pneumatic transport system connected to the thermal column TC-pn site of Kyoto University Research Reactor (KUR). At appropriate time intervals after irradiation, the gamma-ray spectra of the irradiated samples were measured without any chemical separation by Ortec high-purity Ge detector, having energy resolutions from 1.7 to 2.3 keV at 1333 keV photopeak, each coupled with a 4K-channel pulse height analyzer. Photopeak assignment and peak intensity evaluations were calculated and compared with the spectra of pure Al and reference materials. The half-life of Al-28 was also checked.

3. RESULTS AND DISCUSSION Pt was detected in the CCR of cisplatin-injected mice 3 or 5 hours after LPS injection, although Pt could not be detected in the CCR of cisplatin-injected mice either simultaneously or 1 hour after LPS injection. In addition, Pt was not detected in the CCR of cisplatin-injected mice without LPS injection. On the contrary, Pt was detected in the CCR after penetration into the CCR, and the level of Pt did not decrease till 7 days after cisplatin was injected. Therefore, the present results show that the permeability of BBB is changed several hours after LPS injection, and the Pt is penetrated into the brain. Furthermore, Pt is difficult to be excreted from the CCR after penetrated into the CCR.

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Figure 1 shows Al concentration in the CCR of Al-injected mice with or without LPS injection. Al level in the CCR increased at 2 hours after Al injection, but the level was not different between the saline- and LPS-injection groups. So, Al is easily penetrated into the CCR after Al injection, and quickly decrease from it. And LPS treatment does not affect the penetration of Al into the CCR. In contrast, Both the Zn injection and LPS treatment had no effect on the Zn content in the CCR as shown in Fig. 2. Zn may have a homeostatic balance in the brain and the Zn level in the CCR may be stable.

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From these results, the penetrations of metals into the CCR have different each other. And it is thought that metals are difficult to be excreted from the brain, when the metals unable to detect in the brain under a normal condition are penetrated into the brain.

REFERENCES Forbes, W.F. and Hill, G.B., 1998, Is exposure to aluminum a risk factor for the development of Alzheimer disease?—Yes, Arch. Neural. 55:740–741. Higa, G.M., Wise, T.C., and Crowell, E.B., 1995, Severe, disabling neurologic toxicity following cisplatin retreatment, Ann. Pharmacother. 29:134–137. Minami, T., Ichii, M., and Okazaki, Y, 1996a, Detection of platinum in the brain of mice treated with cisplatin and subjected to short-term hypoxia, J. Pharm. Pharmacol. 48:505–509. Minami, T., Ichii, M., Okazaki, J., Kawaki, H., and Okazaki, Y., 1996b, Free radical scavengers suppress the accumulation of platinum in the cerebral cortex, Biol. Trace Elem. Res. 55:1–7. Minami, T., Okazaki, J., Kawabata, A., Kuroda, R., and Okazaki, Y., 1998a, Penetration of cisplatin into mouse brain by lipopolysaccharide, Toxicology 130:107–113. Minami, T., Okazaki, J., Kawabata, A., Kawaki, H., Okazaki, Y., and Tohno, Y., 1998b, Roles of nitric oxide and prostaglandins in the increased permeability of the blood-brain barrier caused by lipopolysaccharide, Environ. Toxicol. Pharmacol. 5:35–41.

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EXPOSURE TO PLATINUM-GROUP METALS RELEASED BY AUTOMOTIVE CATALYTIC CONVERTERS The Case of Urban Youngsters

S. Caroli1, F. Petrucci1, B. Bocca1, M. Krachler2, F. Forastiere3, and A. Alimonti1 1

Istituto Superiore di Sanità Viale Regina Elena 299, 00161 Rome Italy 2 Karl-Franzens Universität Graz Institut für Analytische Chemie Universitätsplatz 1, A-8010 Graz Austria 3 Osservatorio Epidemiologico Regione Lazio Via S. Costanza 53, 00198 Rome Italy

1. INTRODUCTION Beginning in the 1970s and especially since the US Federal Clean Air Acts of 1985 and 1991 adopted increasingly strict environmental provisions, car manufactures have modified their exhaust systems to incorporate catalytic converters. At present, in the European countries all new cars are attached with catalytic converters capable of reducing the emission of aromatic hydrocarbons, CO and Although several designs are available, the dominant technology consists of a monolithic honeycomb support made of cordierite (a phase of on which the so-called washcoat is supported. The active metals Pd, Pt and Rh (Platinum Group Metals, PGMs), in various combinations or even alone, are contained in this washcoat along with stabilizers such as oxides of rare-earth elements. Although the great advantage of car catalysts are obvious, there are however disadvantages, typical for an “end-of-the-pipe” technique. This is the emission of the PGMs as a result of surface abrasion of the catalytic body during car operation. The Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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release of these elements into the environment can eventually affect human health (Hodge and Hollard, 1986; König et al., 1992; Lustig, 1997). The total extent of the emission, its composition in term of relative concentrations of the three elements and the average size of the emitted particles, strongly depend on the catalyst type and traffic conditions. The estimated release reported for Pt ranges from a few to several when the car has a speed of (König et al., 1992). The PGMs are well-known to be sensitizers in the etiology of allergenic pathologies such as asthma, conjunctivitis, dermatitis, rhinitis and urticaria (Rosner and Merget, 1990). In turn, Pt is a cy to toxic agent, widely used in several anticancer therapies as e.g. Cisplatin and Carboplatin, for the treatment of a number of malignancies (Dominici et al., 1989). Tumoral cells are more prone to the attack of the Pt-compounds than normal cells as a consequence of the higher permeability of the former to low-molecular weight Pt species formed in vivo as well as in conditions of acute exposure, i.e., high dose and short term of administration. The continuous presence of much lower concentrations of Pt in the environment inverts the scenario to the chronic exposure, the impact of which on living organisms is partly unknown. Pollution phenomena can more adversely affect particular sectors of the population such as elderlies and children. In this context a study was planned to quantify the PGMs concentration in the urine of schoolboys from the area of Rome.

2. EXPERIMENTAL In consideration of the above, it was decided to focus on 316 young subjects (age 6–10 years). Seven schools were selected to represent both heavy-traffic (HT) and lowtraffic (LT) zones of the metropolitan area of the town. The eligibility criteria adopted allowed to enroll in the study only those youngsters who had no specific pathologies. Samples of 24-h urine could not be collected for obvious and unsurmountable logistic problems. As a valid alternative to this, it was decided to sample only morning urine under the responsibility of school sanitary personnel specifically to this purpose. To account for the different urine density the concentration of PGMs was normalized by creatinine value. The samples were collected in pre-cleaned polyethylene tubes and frozen at –28 °C until analysis. As previously reported (Krachler et al., 1998), an aliquot of 5 ml urine was subsampled and added with 2.0 ml of 30% high-purity and 1.0 ml of 65% high-purity and subsequently UV-irradiated. After digestion, final 1 : 20 dilution of these solutions was carried out by means of high-purity water. Calibrants were prepared daily by dilution of stock solutions of of Pd of of Pt and of of Rh. Determination of PGMs were performed by means of the High Resolution—Inductively Coupled Plasma—Mass Spectrometry (HR-ICP-MS) combined with ultrasonic nebulization. An exhaustive description of the analytical approach adopted as well as of the possible mass interferences and their treatment has been already published (Krachler et al., 1998).

3. RESULTS AND DISCUSSION The average concentration values along with other figures of merit obtained for PGMs in urine (normalized with creatinine excretion) for the seven schools under test

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are given in Table 1. No statistically significant differences were found for each element among the seven sites of collection. This evidence is also corroborated considering the data grouped by traffic level (LT and HT columns of Table 2). These facts strongly support the view that overall exposure to PGMs is pretty the same in different zones of a vastly urbanized area, in spite of the particular density of traffic characterizing a given spot. Hence, all experimental data were merged and treated as if they pertained to one and the same population (Tot. columns in Table 2). Not surprisingly, this can be assumed to be the consequence of the long-range transportation of the particles of car exhaust fumes once these are released to the atmosphere. The proposed methodological approach appears to be fully adequate for the reliable quantification of Pd, Pt and Rh in urine. The actual concentrations of these metals in the tested group, although still rather low, raise some concern on the overall trend.

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More extensive monitoring campaigns are mandatory to fully assess the extent of this phenomenon in Europe.

REFERENCES Dominici, C., Petrucci, F., Caroli, S., Alimonti, A., Clerico, A., and Castello, M.A., 1989, J. Clin. Oncol., 7:100. Hodge, V.F. and Stallard, M.O., 1986, Environ. Sci. Technol., 20:1058. König, H.P., Hertel, R.F., Koch, W., and Rosner, G., 1992, Atmosf. Environ., 26A:741. Krachler, M., Alimonti, A., Petrucci, F., Irgolic, K.J., Forestiere, F., and Caroli, S., 1998, Anal. Chim. Acta, 363:1. Rosner, G., and Merget, R., in A.D. Dayan (Ed.), 1990, Immunotoxicity of Metals and Immunotoxicology, Plenum Press, New York, p. 93.

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URINARY COPPER AND MORTALITY AMONG INHABITANTS LIVING IN A CADMIUM POLLUTED AREA IN JAPAN M. Nishijo1, H. Nakagawa1, Y. Morikawa1, M. Tabata1, K. Miura1, K. Higashiguchi1, T. Seto1, T. Kido2, K. Nogawa3, K. Mizukoshi4, and M. Nishi4 1

Department of Public Health Kanazawa Medical University Uchinada, Ishikawa, Japan 2 School of Health Sciences Kanazawa University Kanazawa, Japan 3 Department of Hygiene Faculty of Medicine Chiba University Chiba, Japan 4 Division of Health and Welfare Ishikawa Prefecture, Japan

OBJECTIVES The Kakehashi River basin in Ishikawa Prefecture is one of the environmental cadmium (Cd) polluted areas in Japan due to mining activity of an upstream copper (Cu) mine. A high prevalence of renal tubular dysfunction induced by Cd was found in this area (Ishizaki, 1989). Also, high urinary excretion of Cu as well as Cd has been reported in inhabitants of Cd polluted areas and Itai-itai disease patients (Nogawa, 1984; Tohyama, 1988). We have previously investigated that Cd exposure and renal tubular dysfunction in this area increase the mortality of inhabitants living in this area (Nakagawa, 1993). This time, the relationship between urinary copper (Cu) and mortality was investigated to clarify the influence of Cu metabolic disturbance induced by Cd exposure on mortality. Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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SUBJECTS AND METHODS For 9 years, we have followed 3,005 inhabitants (1,344 men and 1,661 women) living in the Cd polluted Kakehashi River basin who participated in the health impact survey conducted by Ishikawa Prefecture in 1981–2. In this survey, urinary Cu, Cd were analyzed by atomic absorption spectrometry, and metallothionein (MT) which is induced by Cd exposure was measured by radio-immunoassay. These measurements were corrected by urinary creatinine measured by the Jaffe method. The survival status (alive or dead) was determined with the cooperation of the City Municipal Office and Prefecture Public Health Office, and the date was determined from death certificates. The relationship between urinary Cu and mortality was analyzed using Cox’s proportional hazard model to eliminate the influence of age and Cd or MT.

RESULTS Urinary Cu positive for men, for women: geometrical mean × 2S.D. of control subjects) rates were 6.5% in the men and 8.2% in the women, and significantly higher than those of the subjects living in a non-polluted area. During the observation period, 36 deaths (40.9% crude mortality rate) for men and 51 deaths (51% crude mortality rate) for women in Cu positive subjects, and 231 deaths (18.4% crude mortality rate) for men and 207 deaths (13.6% crude mortality rate) for women in Cu negative subjects were observed (Table 1). Crude mortality rates of urinary Cu positive subjects were higher than those of negative subjects in each age group, with significant differences noted between them in the 60–69 years old and all aged groups (Table 1). After adjustment for age using Cox’s proportional hazard model, the mortality risk ratio of the urinary Cu positive subjects was 1.41 and suggested to be increased in the men as compared with urinary Cu negative subjects. In the women, mortality risk ratio of urinary Cu positive subjects was 1.51 and it was significantly increased as well as the increase of mortality risk ratio of Cd positive subjects (Table 2).

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Among these subjects, positive rates of urinary Cu of Cd positive subjects or MT positive subjects were significantly higher than those of negative subjects in both sexes. These results suggest that urinary Cd or MT might be confounding the relation between urinary Cu and mortality. However, a significant increase of mortality risk ratio of urinary Cu positive subjects as compared to negative subjects was observed in the women, even after adjustment for urinary Cd or MT and age (Table 3). In the Cd positive group, a significant association was found between urinary Cu and mortality in the women, with the risk ratio of 1.53 in the urinary Cu positive subjects compared to the negative subjects.

DISCUSSION The Kakehashi River basin is an area of heavy metal pollution resulting from runoff water from an upstream copper mine, involving not only Cd pollution, but also to a lesser degree Cu pollution. However, no differences in Cu intake and blood Cu concentration were found between the inhabitants living in this area and control subjects (Nogawa, 1984). These facts suggest that Cu exposure has no major impact on health. However, increased excretion of Cu into urine was recognized in Itai-itai disease patients or inhabitants living in the Kakehashi River basin, and is speculated to be due to high excretion caused by renal tubular dysfunction of MT which is induced by Cd exposure and binding with Cu (Tohyama, 1988). In the present study, this disturbance of Cu metabolism induced by Cd exposure was suggested to influence mortality. Even after adjustment for Cd or MT as well as age, a significant increase of mortality risk ratio of urinary Cu positive subjects was recognized in the women. Moreover, high urinary excretion of Cu was related to increase of

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mortality in urinary Cd positive women who are supposed to have a high body burden of Cd. These facts suggested that urinary Cu is one of the prognostic factors in Cd exposed subjects.

CONCLUSION Cu metabolic disturbance induced by environmental Cd exposure influences the life prognosis of inhabitants living in a Cd polluted area.

REFERENCES Ishizaki, M., Kido, T., Honda, R., Tsuritani, I., Yamada, Y., Nakagawa, H., and Nogawa, K., 1989, Doseresponce relationship between urinary cadmium and 2-microglobulin in a Japanese environmentally cadmium exposed population, Toxicology, 58:121–131. Nakagawa, H., Nishijo, M., Morikawa, Y., Tabata, M., Senma, M., Kitagawa, Y., Kawano, S., Sugita, N., Nishi, M., Kido, T., and Nogawa, K., 1993, Urinary ß2-microglobulin concentration and mortality in a cadmium-polluted area, Arch. Environ. Health, 48:428–435. Nogawa, K., Yamada, Y., Honda, R., Tsuritani, I., Kobayashi, E., and Ishizaki, M., 1984, Copper and Zinc levels in serum and urine of cadmium-exposed people with special reference to renal tubular damage, Environ. Res., 33:29–38. Tohyama, C., Mitane, Y., Kobayashi, E., Sugihara, N., Nakano, A., and Saito, H., 1988, The relationships of urinary metallothionein with other indicators of renal dysfunction in people living in a cadmiumpolluted area in Japan, J. Apple. Toxicol., 8:15–21.

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SERUM SELENIUM IN RELATION TO BIOMARKERS OF LEAD IN MEN

J. Jurasovic, A. Pizent, and S. Telišman Institute for Medical Research and Occupational Health Ksaverska cesta 2, 10000 Zagreb Croatia

1. INTRODUCTION There is evidence from numerous experimental animal studies that selenium (Se) interacts with several metals, including lead (Pb). A protective effect of Se against Pb toxicity as well as an antagonistic effect of Pb on Se toxicity have been reported. Animal studies concerning acute simultaneous exposure to Se with a toxic metal generally showed increased tissue deposition of a toxic metal, as opposed to chronic exposure situation when Se decreased, rather than increased, toxic metal retention and tissue deposition. Because the effects of acute exposure may widely differ from those of chronic exposure to a particular metal, the majority of information derived from animal experiments at high doses and/or relatively short-term exposures may not be applicable to human exposure situations (Telišman, 1995). There are very few published data on possible interaction of Pb and Se in humans. A significant inverse association between blood Pb (B-Pb) and serum Se (S-Se) levels was reported in moderately Pb-exposed male workers (Gustafson et al., 1985) and environmentally Pb-exposed children (Osman et al., 1998), as opposed to no significant alteration of S-Se reported in male workers with relatively high occupational exposure to Pb (Giray et al., 1996). The present study considers the influence of low to moderate occupational levels of exposure to Pb on S-Se levels in adult male subjects. Activity of deltaaminolevulinic acid dehydratase (ALAD) in blood, the well-known highly sensitive biomarker of Pb exposure and/or effect in humans, was used in addition to B-Pb for better evaluation of long-term cumulative exposure to Pb of an individual (Telišman et al., 1982). Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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2. SUBJECTS AND METHODS The study was carried out in 178 healthy male subjects living in the urban area of Zagreb. There were 93 subjects occupationally exposed to Pb in a storage batteries factory, and 85 control subjects not occupationally exposed to Pb. The latter subjects were randomly selected from those reported for venipuncture in the course of routine medical examinations, such as pre-employment check-ups for health workers, periodical control for workers in the food industry, those applying for a licence to run a beauty parlour, restaurant, coffee-bar etc. None of the selected subjects had ever been occupationally exposed to other metals, or had hobbies involving risk of metal exposure, or used any medication that could influence metal metabolism. With the assistance of a physician, specialist in occupational health, a detailed questionnaire was completed for each of the subjects, including data on age, dietary habits, smoking habits, alcohol consumption, professional and medical history. All subjects gave their informed consent prior to inclusion in the study. Venous blood (total 10 mL) was sampled between 8 a.m. and 10 a.m. for each subject, all of whom were required to fast in the preceding 10 h and to abstain from alcohol in the preceding 24 h. The S-Se and B-Pb measurements were performed by electrothermal-AAS (Perkin-Elmer 5100/Zeeman). Analytical quality control was performed daily by analyzing three reference blood samples with certified B-Pb values (BCR No. 194–196, European Community Bureau of Reference) and two reference serum samples with certified S-Se values (Seronorm, Nycomed Pharma, Oslo, Norway, and Second Generation, J. Versieck, Gent, Belgium). The accuracy of measurements was also controlled by regular participation in the Trace Elements External Quality Assessment Scheme (Guildford, U.K.) for S-Se, and the National External Quality Assessment Scheme (Birmingham, UK) for B-Pb. The variance score of our results was consistently lower than the average variance for all participants. Activity of ALAD was measured less than 5 h after blood sampling (blood was stored at 4°C) using the European standardized method (Berlin and Schaller, 1974).

3. RESULTS AND DISCUSSION Table 1 shows relevant data in 93 Pb-workers and 85 control subjects. By using the Mann-Whitney U-test, significantly higher Pb-exposure was found in Pb-workers compared to the control subjects, as indicated by increased B-Pb and lowered ALAD (P < 0.0001). No significant difference between the groups was found for age, smoking habits, and alcohol consumption. The S-Se levels in both groups are similar to those reported

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in an earlier study of a population from the city of Zagreb (Beker et al., 1992), and considerably lower than those reported from many other areas of the world. The results showed significantly lower S-Se levels in workers occupationally exposed to Pb compared to control subjects (P < 0.005). Spearman rank correlation in all 178 subjects was highly significant between ALAD and B-Pb (r = –0.867, P < 0.0001). The results showed a significant inverse correlation between S-Se and B-Pb (r = –0.174, P < 0.05), an even more significant positive correlation between S-Se and ALAD (r = 0.202, P < 0.01) and inverse correlation between S-Se and occupational Pb exposure duration (r = –0.204, P < 0.01). Results of multiple regression showed no significant influence of age, smoking habits and alcohol consumption on the relationship between S-Se and B-Pb, or S-Se and ALAD.

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Figure 1 shows the relationship between the S-Se and B-Pb levels, and S-Se and ALAD levels, in the 178 subjects. The relatively better correlation of S-Se with ALAD compared to B-Pb may be attributed to the fact that B-Pb mainly reflects current or recent Pb exposure level, while ALAD better reflects long-term cumulative Pb exposure level of an individual (Telišman et al., 1982). Therefore it appears that chronic exposure to Pb can produce relative Se-deficiency in the body, and that duration of Pb exposure has important role.

REFERENCES Berlin, A. and Schaller, K.H., 1974, European standardized method for the determination of aminolevulinic acid dehydratase activity in blood, Z. Klin. Chem. Klin. Biochem. 12:389–390. Beker, D., Romi, Z., Kršnjavi, H., and Zima, Z., 1992, A contribution to the world selenium map, Biol. Trace Elem. Res. 33:43–9. Giray, B., Gürbay, A., Basaran, N., and Hincal, F., 1996, Selenium levels in lead exposed workers, in: Natural Antioxidants and Food Quality in Atherosclerosis and Cancer Prevention, (J.T. Kumpulainen and J.T. Salonen, eds.), pp. 208–210, The Royal Society of Chemistry, Cambridge. Gustafson, A., Schütz, A., Andersson, P., and Skerfving, S., 1987, Small effect on plasma selenium level by occupational lead exposure, Sci. Total Environ. 66:39–43. Osman, K., Schutz, A., Akesson, B., Maciag, A., and Vahter, M., 1998, Interactions between essential and toxic elements in lead exposed children in Katowice, Poland, Clin. Biochem. 31:657–665. Telišman, S., 1995, Interactions of essential and/or toxic metals and metalloids regarding interindividual differences in susceptibility to various toxicants and chronic diseases in man, Arh. Hig. Rada Toksikol. 46:459–476. Telišman, S., Keršanc, A., and Prpi-Maji, D., 1982, The relevance of arguments for excluding ALAD from the recommended biological limit values in occupational exposure to inorganic lead (WHO 1980), Int. Arch. Occup. Environ. Health 50:397–412.

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HEPATOTOXIC EFFECTS ASSOCIATED TO SHORT AND LONG TERM EXPOSURE TO EXCESS DIETARY COPPER IN RATS E. Aburto*, A. Cribb**, and C. Fuentealba* Department of *Pathology and Microbiology Department of **Anatomy and Physiology Atlantic Veterinary College University of Prince Edward Island Charlottetown, Canada C1A 4P3

1. INTRODUCTION Copper (Cu) toxicosis associated with increased hepatic Cu content occurs as a genetic disorder in humans (Scheinberg and Sternlieb, 1996) and animals (Ludwig et al., 1980; Wu et al., 1994). Toxicity due to consumption of excess dietary Cu is a wellrecognized problem in sheep (Kerr and McGavin, 1991). Excess dietary Cu appears to have a role in Indian childhood cirrhosis (Pandit and Bhave, 1996) and other copperassociated childhood cirrhosis (Müller et al., 1996; Scheinberg and Sternlieb, 1994). The mechanisms by which Cu exerts its toxic effect on the cell are still unclear. It has been suggested that Cu overload causes oxidative damage including lipid peroxidation with resulting lysosomal injury (Myers et al., 1993). The aim of this study was to investigate the liver response to short and long term exposure to excess dietary Cu, as well as the role of lipid peroxidation of copper-induced hepatocellular damage in rats.

2. MATERIAL AND METHODS Thirty 10-weeks old male Fischer 344 rats were randomly divided into two groups of 15 rats each. One group was fed a pelleted diet (Teklad Diets, Madison, Wisconsin) containing 500 ppm Cu (added as ), and the other group was fed a normal rodent diet (Purina, St. Louis Missouri) which contained 18 ppm Cu. Rats were housed five/cage and given free access to food and tap water. Food consumption and body weight were recorded once and 3 times per week, respectively. The animals were killed by exposure to CO2 after 3, 6, and 12 months on the experimental diet. Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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Livers were sampled and processed for routine histology, histochemical demonstration of Cu using the rhodanine method, and Cu analysis by atomic absorption spectrophotometry as described elsewhere (Fuentealba and Bratton, 1994). Assessment of lipid peroxidation was done using the spectrophotometric quantification of malondialdehyde (MDA) by the thiobarbituric acid (TBA) reaction (Sunderman et al., 1985). Analysis of variance and Duncan’s multiple range test were used for statistical analysis and the level of significance was set at

3. RESULTS There were no differences in body weight between copper-loaded and control rats at any sampling point. Copper-loaded rats had significantly (p < 0.01) increased levels of hepatic Cu after 3, 6 and 12 months of dietary copper overload compared to control rats. Copper levels after 12 months of copper overload were significantly higher (p < 0.01) than those detected after 3 and 6 months (Fig. 1). Liver from all copper-loaded rats exhibited histologic changes characterized by multifocal aggregates of macrophages with lesser numbers of neutrophils and lymphocytes surrounding small necrotic foci of parenchymal cells. Some individual necrotic hepatocytes, as evidenced by hypereosinophilic cytoplasm and pycnotic nuclei, were also seen. Histologic changes were not seen in control rat livers. Rhodanine stain demonstrated presence of Cu within the cytoplasm of some hepatocytes after 12 months of copper-overload. Copper was not detected in the livers of control rats or in livers from rats copper-loaded for 3 and 6 months.

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MDA concentration was significantly (p < 0.01) increased after 6 months of copper overload. After 12 months in the experimental diet a significant increase in MDA content was detected in both copper loaded (p < 0.05) and control rats (p < 0.01) (Fig. 2).

4. DISCUSSION Long term exposure to 500 ppm Cu did not cause severe hepatic lesions in Fischer 344 rats. Diets containing Cu levels similar to those used in the present study have been reported to cause liver injury in other species. Cow’s milk containing 63.3 ppm Cu was associated to cases of Endemic Tyrolean childhood cirrhosis (Müller et al., 1996). Chronic copper poisoning in sheep occurred in animals fed a mineral supplement containing 86.7 ppm Cu (Kamphues et al., 1991) and 110 ppm Cu (Hoff et al., 1998). Copper toxicity has been described in dairy cattle fed a mineral supplement with 37.5 ppm Cu (Bradley, 1993), and in rabbits fed a diet containing 100 ppm Cu (Cooper et al., 1996). In the present study, although the increase in liver copper was statistically significant in copper-loaded rats, these animals did not develop severe liver lesions. The highest concentration of hepatic copper in this study was 18.29ppm wet weight after 12 months on the experimental diet. It has been suggested that in the Wistar rat hepatic concentrations of over 400 ppm are needed to cause significant histomorphologic changes (Haywood, 1979). The low accumulation of hepatic copper and limited hepatic changes observed in this study may be associated to development of copper tolerance, as described in the Wistar rat (Haywood, 1985). The basis for the development of copper tolerance is

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unknown, but it could be due to efficient transport and unloading through increased synthesis of ceruloplasmin and metallothionein (Fuentealba and Bratton, 1994). High levels of MDA in copper-loaded and unloaded rats after 12 months is likely an age-related change. In aged rats the rate of reactive oxygen species generation exceeds the induced hepatocellular antioxidant ability, favoring oxidative stress and peroxidation (Sanz et al., 1997). Increase in MDA content indicative of lipid peroxidation was not detected in this study. The highest level of hepatic MDA detected in copper-loaded rats was 0.7 nmols/mg of protein. In contrast, hepatic MDA levels may reach 2.3 nmols/mg of protein in Long-Evans cinnamon rats before the onset of hemolytic crisis (Rui and Suzuki, 1997), and up to 3.5 nmols/mg of protein in rats exposed to aflatoxin B1 (Shen et al., 1994). Although it may be argued that 500 ppm Cu is a low dose to cause hepatic damage in Fischer 344 rats, a deleterious effect on liver function has been reported in Fischer rats fed diets containing 150–600 ppm Cu (Sugawara et al., 1995). Alternatively, excess dietary copper may not be solely responsible for the development of copperassociated cirrhosis. It has been suggested that a second xenobiotic agent is required with excess copper to produce Indian Childhood Cirrhosis (Aston et al., 1996). Further studies are needed to determine whether the low levels of hepatic copper accumulation and limited hepatic changes are due to copper tolerance or if a synergistic hepatotoxicity is needed in the rat to experimentally induce liver damage similar to that observed in spontaneous cases of copper-associated diseases.

REFERENCES Aston, N., Morris, P., and Tanner, S. 1996, Retrorsine in breast milk influences copper handling in sucking rat pups, J. Hepatol. 25:748–755. Bradley, C.H. 1993, Copper poisoning in a dairy herd fed a mineral supplement, Can. Vet. J. 34:287–292. Cooper, G.L., Bickford, A.A., Charlton, B.R., Galey, F.D., Willoughby, D.H., and Grobner, M.A. 1996, Copper poisoning in rabbits associated with acute intravascular hemolysis, J. Vet. Diagn. Invest. 8:394–396. Fuentealba, I.C. and Bratton, G.R. 1994, The role of the liver, kidney and duodenum in tolerance in the copperloaded rat, Anal. Cell. Pathol. 6:345–358. Haywood, S. 1979, The effect of the sex of weaned rats on the accumulation of dietary copper in their livers, J. Comp. Pathol. 89:481–486. Haywood, S. 1985, Copper toxicosis and tolerance in the rat. I. Changes in copper content of the liver and kidney, J. Pathol. 145:149–158. Hoff, B., Boermans, H.J., and Baird, J.D. 1998, Retrospective study of toxic metal analyses requested at a veterinary diagnostic toxicology laboratory in Ontario (1990–1995), Can. Vet. J. 39:39–43. Kamphues, J., Matschullat, G., and Muller, E. 1991, Copper poisoning of sheep after ad libitum supplementation of rations by a mineral feed, DTW. Dtsch. Tierarztl. Wochenschr. 98:109–111. Kerr, L.A. and McGavin, H.D. 1991, Chronic copper poisoning in sheep grazing pastures fertilized with swine manure, J. Am. Vet. Med. Assoc. 198:99–101. Ludwig, J., Owen, C.A., Barham, S.S., McCall, J.T., and Hardy, R.M. 1980, The liver in the inherited copper disease of Bedlington terriers, Lab. Invest. 43:82–87. Müller, T., Feichtinger, H., Berger, H., and Müller, W. 1996, Endemic Tyrolean infantile cirrhosis: an ecogenetic disorder, Lancet 347:877–880. Myers, B.M., Prendergast, F.G., Holman, R., Kunts, S.M., and Larusso, N.F. 1993, Alterations in hepatocyte lysosomes in experimental hepatic copper overload in rats, Gastroenterology 105:1814–1823. Pandit, A. and Bhave, S. 1996, Present interpretation of the role of copper in the Indian childhood cirrhosis, Am. J. Clin. Nutr. 63:830S–835S. Rui, M. and Suzuki, K.T. 1997, Copper in plasma reflects its status and subsequent toxicity in the liver of LEC rats, Res. Commun. Mol. Pathol. Pharmacol. 98:335–345. Sanz, N., Diez-Fernandez, C., Alvarez, A., and Cascales, M. 1997, Age-dependent modifications in rat hepatocyte antioxidant defense systems, J. Hepatol. 27:525–534.

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Scheinberg, I.H. and Sternlieb, I. 1994, Is non-Indian childhood cirrhosis caused by excess dietary copper?, Lancet 344:1002–1004. Scheinberg, I.H. and Sternlieb, I. 1996, Wilson disease and idiopathic copper toxicosis, Am. J. Clin. Nutr. 63:842S–845S. Shen, H.M., Shi, C.Y., Lee, H.P., and Ong, C.N. 1994, Aflatoxin B1-induced lipid peroxidation in rat liver, Toxicol. Appl. Pharmacol. 127:145–150. Sunderman, F.W., Marzouk, A., Hopfer, S., Zahaira, O., and Reid, M. 1985, Increased lipid peroxidation in tissues of nickel chloride-treated rats, Ann. Clin. Lab. Sci. 15:229–236. Sugawara, N., Li, D., Sugawara, C., and Miyake, H. 1995, Response of hepatic function to hepatic copper deposition in rats fed a diet containing copper. Biol. Trace Elem. Res. 49:161–169. Wu, J., Forbes, J.R., Chen, H.S., and Cox, D.W. 1994, The LEC rat has a deletion in the copper transporting ATPase gene homologous to the Wilson disease gene, Nat. Genet. 7:541–545.

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217

THE BIOLOGICAL AND MEDICAL IMPORTANCE OF THE INTERACTIONS BETWEEN NICKEL AND ZINC, MAGNESIUM AND MANGANESE IN VIVO

M. Anke, A. Trüpschuch, and G. Gunstheimer Institute of Nutrition and Environment Friedrich Schiller University Dornburger Str. 24, D-07743 Jena Germany

According to the present level of knowledge, the Ni offer meets the Ni requirements of the flora, the fauna and humans. Therefore, Ni deficiency symptoms need not be reckoned with in real life. On the other hand, nickel allergies often occur in Ni-sensitive women (8 to 14%). The effect of Ni exposure with 0, 250, 500 and 1,000 mg Ni/kg ration dry matter was systematically investigated in young hens at the beginning of the laying period. Ni supplementations of 250 mg/kg ration increased the feed intake by 6%, higher amounts (1,000 mg/kg ration dry matter) decreased it by 27%. The egg production of young hens followed the feed consumption significantly. Nickel offers of 50 to 1,000 mg Ni/kg ration decreased the reproduction performance of hens significantly, that of cocks only individually and not generally. The composition of the eggs without shells laid by young hens with 0 to 1,000 mg Ni/kg ration dry matter showed that the egg is used for the excretion of the surplus Ni. This is particularly true for the beginning of the laying period. The Ni exposure did not take a significant effect on the Fe, Cu, Co and Ca content whereas the Zn, Mg and Mn content in the eggs without shells laid by Ni-exposed hens decreased significantly. The individual organs and tissues reacted to the Ni exposure with a redistribution of the inorganic components. Apart from egg shells, all investigated parts of the body of the Ni-exposed hens increased their Ni content significantly. Muscle stomach and down feathers are excellent indicators of an Ni exposure. The Ni application generally increased the Zn, Mg and Mn content in the liver and reduced that in skeleton (femur), ovary and egg significantly. The Fe, Cu, Co and Ca concentrations in the tissues did not show any significant reaction to the Ni offer. 685

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The Ni application tended to increase the percentage of unfertilized eggs and reduced the hatching rate from 90% in hens without Ni supplementation to 75, 61 and 33% in Ni-exposed hens (250; 500; 1,000 mg Ni/kg ration dry matter). Ni offers of 500 and 1,000 mg/kg ration increased the number of dead, stuck and malformed chicks significantly. The pre- and post-natal Ni exposure and the Zn, Mg and Mn deficiency resulting therefrom are very probably the cause of the bad growth and the high mortality of chicks hatched from eggs of Ni-exposed hens. The clinical picture of an Ni exposure, which was developed in 3 species (pig, cattle, hen, chick) is similar to that of parakeratosis, a Zn deficiency syndrome, which occurs most intensively in domestic pigs (Anke et al. 1984a, b) and it is also in accordance with the skin injuries caused by the Ni allergy (Anke et al. 1995) which occurs in Ni-sensitive women after the filling of their body depots. The protective effect of Zn and the negative one of Ni on the development of gastric ulcer after administrating phenylbutasone was impressively confirmed in rats by Nechifor et al. (1998). More attention must be paid to the interactions of Ni with Zn (and Mg, Mn) in the case of Ni allergy in women (Trüpschuch 1997).

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EFFECT OF ALUMINUM ON MANGANESE SUPEROXIDE DISMUTASE (MnSOD) EXPRESSION

C. Garrel, C. Carron, and A. Favier Laboratoire de Biologie du Stress Oxydant (LBSO) UFR Pharmacie Medecine Grenoble, France

Aluminum is a neurotoxic metal which has been implicated in various neurological diseases as Alzheimer’s disease. However, despite numerous studies on aluminum-treated animals and cells in culture, the sites at which aluminum is bound within cells still remains unclear. We have recently shown, by electrophoretic mobility shifft assay on nuclear extraits of HeLa cells, that various concentrations of aluminum sulfate led to a decrease of SP1 DNA binding activity. Yet, the MnSOD gene expression is under control of an SP1containing and TATA-less promotor. So, we hypothesis that aluminum could act on MnSOD expression at the initiation of transcription level, by a mechanism which involves SP1 sites. To verify this hypothesis, we used a plasmid containing human MnSOD promoter –210/+37 (pMnSOD Luc) link to luciferase gene as a reporter. HeLa cells are transfected with the reporter plamid, using calcium-phosphate methods, and stimulated during 3 and 24 hours by various concentrations of aluminum sulfate. The reporter gene activity is measured by a luciferase base assay system (Promega). We reported that aluminum sulfate decrease luciferase activity from the reporter construct as a dose dependant manner. These date support the hypothesis that one of the delerious effect of aluminum may be to decrease the level of the antioxidant enzymes MnSOD throught an interaction with DNA SP1 sites and thereby alter the ability of cells to be effectively protect from an oxidative stress. Moreover this effect of aluminum could be link to abnormalities in the cellular regulation and expression of antioxidant enzymes described in Alzheimer disease.

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INDICATORS FOR SENSITISATION TO NI, CO, AND CR, A EU-PROJECT

Olof Vesterberg Department of Occupational Medicine National Institute for Working Life S17184 Solna Sweden

Project partners and participants: 1. O. Vesterberg, F. Acevedo, F. Lottspeich; 2. J. Molin Christensen, L. Borg, J. Kristiansen; 3. L. Clerici, M. Serra Beltrán, M. Ermolli, G. Bresia; 4. T. Menné, NHN Nielsen; 5. D. Taramelli; General objectives: studies of effects of exposure to Ni, Cr and Co concerning: • Clinical and biochemical studies for establishment of threshold concentrations of the metals for reaction in metal sensitised individuals. Development of in vitro-assays for early detection of sensitisation and for risk assessment. • Studies of responses at cellular level to metal exposure, including uptake of metals, cytotoxicity and intracellular distribution as well as immune system hyperreactivity. Results. To accomplish the objectives methods have been developed and many studies have been made. Methods for determination of nickel in skin tape strippings and finger nails were established as well as an assay for lymphocyte proliferation, LP. Procedures avoiding metal contamination at sampling and analysis were elaborated. ELISA assays were validated for the cytokines IL 4, IL 5, and The preliminary study gave interesting results. Clinical examinations of exposed skin areas demonstrated that the exposure levels are satisfying. The selected clinical, chemical and biochemical parameters were relevant. Increased concentrations of cytokines were observed in sensitised persons. Nickel induced LP indicating that lymphocytes from nickel allergic individuals react to nickel ions. We have developed two procedures for quantification of the extent of complement activation and found that nickel at low concentrations stimulated the activation in plasma samples. This stimulation was significantly higher in nickel-allergy patients compared to non-allergic controls. The complement system is involved in important immunologic 688

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reactions in the body and thus of significance also in metal allergy. Studies on the potential use of measurement of complement activation to screen nickel-allergy is in progress. We also found changes in haemoglobin related to nickel-allergy. Two independent principles to detect changes in blood proteins related to nickel-allergy have thus been developed. With a special reagent (NiNTA-AP) nickel binding proteins (NBP) are detected. The sensitive methods developed means that the demand for plasma sample volume is minimal and that less abundant NBP can be studied. A keratinocyte cell line is a model to evaluate human keratinocyte activation and has given interesting information on Ni subtoxic doses. Nickel mainly accumulated in the cytosol and induced concentrations of IL 1, IL6 and Studies for identification of metal binding proteins and protein expression using electrophoretic methods give interesting results. Findings suggest that IL-12 may be an early marker of nickel effect on keratinocytes and that keratinocytes in vivo play a role in allergic contact dermatitis. Conclusions: the project is successful and results are prepared for publication. Economic supported from the EU Commission is acknowledged.

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FISH CONSUMPTION AND MERCURY SPECIATION IN HAIR OF INDIGENOUS POPULATION OF THE AMAZON

Antonio C. Barbosa, José G. Dórea, Jurandir R. Souza, and Glauce L. Oliveira Departament of Chemistry Universidade de Brasilia 70919-970 Brasilia Brazil

The riverine inhabitants of the Amazon Basin depend heavily on fish to meet their nutritional needs. Per capita fish intake has been estimated as 200 g/day. Mercury concentration in fish depends on feeding habits and age/size of fish. As a result contamination of fish eating population will depend not only on the quantity of fish consumed but also on the species of choice. We determined mercury concentration on samples of different species of fish most consumed by the local population of the Rio Negro. The species of fish sampled included piscivorous, herbivorous, omnivorous, and detritivorous. A qualitative survey of number of fish meals consumed per day was made with a sample of the riverine population of Rio Negro. In these people we studied the levels of mercury contamination and the distribution of organic (methyl mercury) and inorganic mercury in hair. Total mercury concentrations in fish ranged from 32.1ng/g to 2,441.1ng/g with mean concentrations higher in piscivorous species. Total mercury in hair of indigenous inhabitants ranged from to Mercury speciation in this fish eating population showed that inorganic mercury ranged from 0 to of hair while methyl mercury ranged from 0 to of hair. The relative mean distribution between inorganic and organic mercury was respectively 28.7% and 71.3%.

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METABOLISM OF PHOSPHOLIPIDS IN PITUITARY GLAND Effect of Ion Cadmium

Calderoni Ana María and Giménez María Sofía Department of Biochemistry and Molecular Biology Faculty of Chemistry Biochemistry and Pharmacy National University of San Luis San Luis Argentina

Exposure of mammalian cells to cadmium triggers a complex reprogramming of the expression of a variety of genes. Cadmium administration is known to be followed by deleterious effects on the endocrine system although its action mechanism is not well understood. It is known that animals subchronically treated with body weight gain was lower than in the control rat and provoke diminution in the prolactin levels in serum and increased plasma level of growth hormone, thyroid stimulating hormone and follicle stimulating hormone, showing that Cd modify pituitary hormone secretion differentially and specifically. Previous results obtained in our laboratory showed that 15ppm cadmium (CD) in the drinking water administrated during seven weeks increase the amount of phospholipid in pituitary gland. In the present work we explored the effect of Cd on phospholipid metabolism in “in vitro” experiments. Slices of pituitary of adult male rat weighing around 300 g were incubated in Dulbeco Modified Eagle Medium, 5% calf fetal serum and antibiotic with or without Cd en presence of Choline for 3 h at 37 °C in 5% air. After this time the slices were washed three times with ice-cold Hank’s buffer saline solution, and fenol red free. The lipids of pituitary gland slices were extracted with isopropanol: hexane 3:2. The different species of phospholipids were separated by Thin Layer Chromatography

Email [email protected]

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(TLC) using Chloroform: methanol: water 65:25:4 v/v/v as solvent. The band of phosphatidyl choline (PC) and Sphingomielyn (Spm) identified with iodine vapor were scrapped. On the other hand, the total phospholipids were separaed by TLC using hexane: ether: acetic acid 80:20:1 as solvent. The radioactivity incorporated in total phospholipids, PC and Spm was measured in a Beckman Liquid Scintillation Counter. The proteins were determined by Lowry et al method. The statistic analyses was made using students test. The results showed that the incorporation of expressed as cpm/mg protein was higher in phospholipids, PC and Spm of pituitary incubated with Cd compared with those incubated without Cd. The amount of protein was not modified by Cd. In other experiment we used slices of pituitary from rat that received 15ppm of in the drinking water for seven weeks. The slices were incubated as was described above except that Cd was omitted in the culture medium. The result showed that the incorporation of in total phospholipids and PC while decreased in Spm. Cd by a mechanism no determined modifies the metabolism of phospholipids in pituitary gland.

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INVESTIGATIONS ON THE ELEMENTS STATUS MARKERS IN HUMANS. LEAD. SIGNIFICANT CORRELATION BETWEEN PB CONTENT IN MILK TEETH AND BLOOD OF 6-YEAR-OLD CHILDREN

H. Barton, Z. Zachwieja, and M. Folta Department of Food Chemistry and Nutrition Jagiellonian University School of Medicine Medyczna 9, 30-688 Cracow

Poland

In order to develop non-invasive diagnostic methods of evaluation of elements status, the analysis of lead in scalp hair, milk teeth and finger blood of ca.100 6-year-old randomly selected children was undertaken. Concentrations of toxic and bio-elements in drinking water was also estimated. The samples were taken in spring 1998 in Cracow region. The hair samples were washed according to the IAEA recommendation for environmental studies (1986), then digested in using the microwave system MDS 2000. Blood was analysed with phosphate modifier without other pre-treatment and teeth were dissolved in prior to analyses. The analysis were carried out by flameless AAS methods using a Perkin-Elmer ZL-5100 spectrometer. The methods were validated by certified standards, an internal and external control system. Results: – strong positive correlation was found between Pb content in teeth and blood: rS (Spearman) = 0.57, N = 66, p = <0.001 regressions: log PbBlood = (0.54 ± 0.04) + (0.52 ± 0.11) log PbTeeth, r = 0.50 ±0.11 log PbTeeth = (0.40 ± 0.03) log PbBlood, r = 0.87 ± 0.06; (units: teeth ugPb/g], blood [ugPb/dL]) – geometric mean of Pb content was—GM (geometric SD): 4.68 (1.47) ug/dL in blood, 693

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– Pb concentration in blood did not correlate with Pb in hair and the ratio of Pb content in teeth/hair. – concentration of lead in drinking water correlated weekly with lead in teeth and hair Conclusion: Strong positive correlation of lead content in milk teeth and blood of 6-year old children suggests a potential usefulness of analysis of milk teeth in population studies, even for the populations not highly exposed to lead.

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MORPHOLOGIC AND BIOCHEMICAL ASSESSMENT OF THE ROLE OF LIPID PEROXIDATION IN THE PATHOGENESIS OF COPPER TOXICITY IN COPPER-LOADED RATS

E. Aburto*, A. Cribb**, and I. C. Fuentealba* Department of *Pathology and Microbiology and Department of **Anatomy and Physiology Atlantic Veterinary College University of Prince Edward Island 550 University Avenue Charlottetown, PEI, C1A 4P3 Canada

The aim of this study was to determine the amount of excess dietary copper necessary to induce hepatocellular injury in rats and to evaluate the role of lipid peroxidation in copper-induced liver damage. Male Fisher 344 rats of uniform age (10 weeks) and weight (140 grams) were divided in 6 groups of five rats each, and fed a rodent diet containing 18 (control), 750, 1,000, 1,250, 1,500, and 2,000 ppm copper. Rats were killed by exposure to after 3 months in the experimental diets and their livers were sampled and processed for histology, histochemistry (rhodanine stain), and copper analysis by AA spectrophotometer. Assessment of lipid peroxidation was performed using the spectrophotometric quantification of malondialdehyde (MDA) by the thiobarbituric acid (TBA) reaction. The data were analyzed using multivariate analyses of variance within general linear models. With the exception of rats receiving 750 ppm copper, copper levels (m/g tissue (wet weight) in the liver were significantly higher (p < 0.01) in rats receiving excess dietary copper compared to the controls (MEANS: control diet = 4.8m/g, 750 ppm Cu diet = 39.6 m/g, 1,000 ppm Cu diet = 111.2 m/g, 1,250 ppm Cu diet = 389 m/g, 1,500PPM Cu diet = 509.4m/g, and 2,000ppm Cu diet = 766m/g). Histological sections from copper-loaded rats exhibited multifocal accumulations of macrophages and lymphocytes surrounding small necrotic foci. Numerous necrotic hepatocytes were seen scattered throughout the parenchyma. Abundant hyaline remnants and a mild to moderate inflammatory response, consisting predominantly of lymphocytes with lesser numbers of macrophages, were detected in the centroacinar zones (portal areas). Copper 695

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was histochemically demonstrable in the cytoplasm of centroacinar hepatocytes in rats receiving over l,250ppm Cu. The severity of the histological lesions and the number of cells or groups of cells positive histochemically for copper increased gradually according to the level of copper in the food. Similarly, a strongly positive correlation (r = 0.98) between dietary copper and hepatic copper content was noted. In general, no significant differences were found for MDA levels between groups, and there was no correlation between dietary copper, MDA levels and histological changes. The amount of excess dietary copper required to induce lesions typical of copper-associated hepatitis has not been clearly established in rats. The present study demonstrates that in Fisher 344 copperloaded rats the levels of excess dietary copper required to induce substantial hepatocellular damage is very high (1,500–2,000 ppm Cu) compared to other species. This is probably due to copper tolerance, as described in the Wistar rat. Increase in MDA content, indicative of lipid peroxidation, was not detected in this study. It is known that MDA can avidly react with proteins through the formation of a Schiff base. In such a case, MDA would not be detectable by the TBA reaction since this method can only identify free MDA. It is also possible that lipid peroxidation may not play a major role in this model of copper toxicity.

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EFFECT OF DOLOMITE AND THIAMINE SUPPLEMENTATION ON TISSUE TRACE ELEMENTS CONCENTRATIONS IN LEADINTOXICATED RATS

Z. Krejpcio, D. Olejnik, R. W. Wojciak, and J. Gawêcki Department of Food Hygiene and Human Nutrition Agricultural University Wojska Polskiego str. 31 60-624 Poznan, Poland

Despite efforts to limit environmental lead exposure, lead toxicity remains the most common preventable pediatric disease in some countries. The toxic effect of this metal may be mediated or enhanced by interactions or deficiencies of nutritionally essential metals. The best defined and clinically important relationships between lead and essential elements include calcium, iron, zinc and copper. There is both experimental and clinical evidence that nutritional factors have an impact on health outcoms from lead exposure. The objective of these studies was to investigate the effect of Ca, Mg and thiamine supplementation on tissue Fe, Zn, Cu and Pb concentrations in rats exposed to lead acetate. The studies were carried out on 6-weeks old male Wistar rats fed for 8-weeks semipurified diets containing the 4-combinations of experimental factors: dolomite (powdered as a source of Ca and Mg; 20 and 40g/kg diet) and thiamine (25 and 50mg/kg diet). Additionally, rats were exposed to lead acetate (500mgPb/kg diet). Besides, two reference groups (K0- standard diet, K1-standard diet + Pb) were established. At the end of the experiment rats were sacrificed (Thiopental injection) to draw blood and remove inner organs. The Fe, Zn, Cu and Pb concentrations in animal tissues, after wet digestion and appropriate dilution of mineral solutions, were determined by the FAAS method. For statistical evaluation of the results Fisher test and linear regression analysis at p < 0.05 have been applied. It was found that: dolomite and thiamine supplementation significantly decreased liver and kidney Pb in comparison to the control group (K1), 697

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higher level of thiamine supplementation significantly increased liver and kidney Cu and decreased femur Zn in the rats exposed to lead, higher level of Ca and Mg supplementation decreased kidney Fe and increased liver Cu and femur Zn in the rats, there were significant correlations between some pairs of metals in animal tissues: liver: Pb/Fe (r = 0.457, p < 0.01) and kidneys: Pb/Fe (r = –0.341, p < 0.05), Pb/Zn (r < –0.538, p < 0.01), Pb/Cu (r = –0.494, p < 0.001).

CONCLUSIONS Dolomite and thiamine supplementation affects trace element content in the body thus showing protective effect against lead poisoning.

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ACUTE HUMAN MOLYBDENUM TOXICITY FROM A DIETARY MOLYBDENUM SUPPLEMENT—A NEW MEMBER OF THE “LUCOR METALLICUM” FAMILY

Berislav Momcilovic

Institute for Medical Research and Occupational Health Ksaverska cesta, Zagreb Croatia

“Jay”, a young Californian health practitioner in a full health of his late 30s wanted to be even healthier. His fellow profesional suggested he try a Mo supplement (Molybdenum chelated, Mo per tbl., Nutriwest Co., Duglas, WY) to cure his “allergy” to the perfume from the skin of his patients. Until then “Jay” had a remarkable empty medical record and was outstaning in his educational curriculum. “Jay” consumed a HCl supplement for “indigestion” before he started to take Mo on July 1, 1997. After 7 days he showed the first signs of anxiety and agitation. Fourteen days later he became mildly psychotic and experienced visual and auditory hallucinations. On day 18 he could “smell the Mo all around him” and discontinued Mo supplements. On day 19 he had severe psychosis with strong audio and visual hallucinations, insomnia, intense craving for salt, diarrhea, and painful and cold extremities. On day 22 his hallucinations were accompanied with a petit mal seizures and on day 24 he tried to take his life with a knife and, “chased by the devil”, ran through a plate glass window and jumped headlong off a 20 feet wall. The emergency room physicians first considered him drugged, then believed that he had an attack of maniac-depressive psychosis. There were no signs of skull fracture or hemorrhage on NMR. He was placed on heavy neuroleptics but continued to have fits, seizures, and hallucinations. “Jay’s” devoted wife insisted unsuccessfully that he should be treated for Mo poisoning. “Jay’s” blood Mo was 7.7ng/ml (normal 0.9– 1.8ng/ml; Mayo Med. Lab., Rochester, MI). Only after “Jay” was chelated with EDTA on August 22, 1997 did his fits, seizures, and psychosis stop within two hours. Subsequently, he underwent chelation therapy 10 more times. After the psychotic signs, fits, and seizures were under control, the first sequel to Mo poisoning was an immediate, severe depression totally non responsive to Zoloft and Nortriptilyne. His sex drive was nil and six month later it was found that his blood testosterone was low and his depres699

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sion appeared responsive to testosterone patch treatment. A year later evident frontal cortical damage was confirmed by an extensive battery of neuropsychological tests and by the Emission computer tomographic images (SPECT). Milder impairment was registered in the descending degree in the parietal lobe, temporal lobe, and even basal ganglia. The degree of the brain involvement is such that it is questionable when and if at all “Jay” would be able to reassume his previous successful professional carrier. Heavy metal damage to the brain is well documented for mercury, manganese, lead, and cadmium and even the term “Lucor Manganicum” (Manganese Madness) was coined. This report is the first case of acute human Mo poisoning to my knowledge and a new, unwelcome addition, to the “Lucor Metallicum” family. It was to be expected that, sooner or later, the appearance of a wide range of mineral supplements, some of them with neurotoxic potential, on the market would lead to tragic accidents like this one. Indeed, it is absolutely unpredictable what possible “cocktail” mixtures the prospective customers would be able to create. Considering the fact that the complex dose-dependent interactions between the mineral supplements and other “nutriceuticals” are largely unknown and poorly understood it would be advisable to state in the “red ink” on a product label that their safety is guaranteed only if not mixed with other supplements. Poison Control Centers should be warned of the Mo as a new member of the “Lucor Metallicum” family and the doctors should be advised about the necessity of an urgent chelation treatment.

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EFFECTS OF LEAD ON THE MATURATION OF THE FEMALE GAMETE AND FERTILITY B. Lefèvre1, J. Poupon2, A. Pesty1, V. Machelon1, and J. Testart1 1

INSERM U355 & IFR 14 32 rue des Carnets, 92140-Clamart France 2 Laboratoire de Biochimie-Toxicologie Hôpital Fernand Widal Paris

France

INTRODUCTION Toxic effects of lead on the reproduction and growth have been described. Most of the studies were conducted on the male and only a few dealt with the female reproductive organs. The aim of this work is to study the deleterious effects of lead at different levels of the female reproductive function: in vivo and in vitro on meiotic maturation, ovulation, fertilization, implantation, first steps of the mouse embryo development.

MATERIAL AND METHODS 1) In vivo: mice were exposed to lead either in acute or chronic conditions. Lead levels were measured by Electrothermal Atomic Absorption Spectrometry in several tissues including ovary. Contralateral ovaries were fixed for histological examination. Mice were mated and sacrificed at different times after mating. 2) In vitro: Effects of lead were studied on two steps of the oocyte maturation (rupture of the nuclear membrane or GVBD and extrusion of the first polar body and formation of the metaphase II.

RESULTS In vivo, acute intoxication induces an accumulation of lead in the ovaries and a total sterility associated with the atrophy of uterine horns. Chronic intoxication does not impair the ovulation since corpus lutea are observed on the ovaries, but decreases the fertility rate. In vitro, culture of immature oocytes in presence of lead acetate 1 or 701

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induces a slowing up of the first step of meiosis (GVBD) and a partial inhibition of the second step (MII). Moreover, half of the MII oocytes exhibit an abnormal position of the polar body with regard to the metaphase plate.

CONCLUSION It has been demonstrated in many cellular models that ions can replace ions thus impairing the -dependent functions of the cell. As calcium metabolism plays an important role in the ooctyte meiosis, deleterious effects of lead on the female mouse fertility could be linked to the disturbance of this metabolism.

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RHAMNOGALACTURONAN II (RG-II), A PECTIC POLYSACCHARIDE PRESENT IN THE VEGETABLE-DERIVED PRODUCTS, STRONGLY DECREASES LEAD TISSUE ACCUMULATION Potential Interest in Chronic Lead Exposure

M. Tahiri1, P. Pellerin2, J. C. Tressol1, T. Doco2, Y. Rayssiguier1, and C. Coudray1 1

Centre de Recherches en Nutrition Humaine Unité Maladies Métaboliques et Micronutriments INRA de Theix, 63122 St. Genès Champanelle France 2 Unité de recherches biopolymeres et arômes Institut des Produits de la Vigne Centre INRA de Montpellier 2, Place Viala, 34060 Montpellier Cedex France

The Rhamnogalacturonan II (RG-II), a pectic polysaccharide, present in the vegetable-dervived products, forms complexes in vitro with lead (Pb) and other divalent and trivalent cations. The purpose of this study was to examine the effect of RG-II on the bioavailability and the tissue distribution of Pb, as well as of magnesium, zinc and iron in rats after chronic Pb exposition. 40 male Wistar rats were divided into 4 groups A, B, C, and D. Group A received a semi-purified conventional diet. Groups B, C, and D received the same diet supplemented with 3mgPb/Kg, 3mgPb/Kg + 0.5gRG-II/kg, or 3mgPb/kg + 5gRG-II/kg respectively for 3 weeks. During the last 5 days, a chemical balance study was conducted during which individual fecal and urinary excretions of each animal were collected. Whole blood Pb concentration was increased by about 18, 13, and 9 fold for groups B, C and D respectively compared to control group. Bone and kidney Pb levels were significantly increased in Pb-treated rats comparing to controls (B vs A). Interestingly, addition of RG-II decreased bone and kidney Pb levels, and this effect was dose dependent. Pb urinary excretion was significantly increased in animals exposed to Pb, and animals receiving RG-II showed a significant decrease in their Pb urinary 703

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excretion when compared to the other Pb-treated animals. Means apparent absorption of Pb were: 79.4%, 15.2%, 12.3% and –9.6% for groups A, B, C, and D respectively. Balance of Pb was 4.36, 9.56, 5.97, and –4.76µg/day for groups A, B, C, and D respectively. The level of magnesium, zinc and iron in the tibia, kidney and liver was unchanged either with Pb or RG-II treatments. Faecal and urinary excretions, apparent absorption and balance of these essential minerals also were unaffected by RG-II treatment. These results suggest that RG-II may decrease the intestinal absorption and the tissue retention of Pb, reducing then its toxicity without affecting essential cation status. The ingestion of this product may thus contribute to decrease the toxic effect associated with chronic lead exposure.

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DIETARY EXPOSURES TO THE PLATINUM GROUP ELEMENTS

G. E. Ysart, P. F. Miller, H. Crews*, P. Robb*, M. Baxter*, C. De L’Argy*, S. Lofthouse*, C. Sargent*, and N. Harrison Joint Food Safety and Standards Group Ministry of Agriculture Fisheries and Food, Ergon House c/o Nobel House Smith Square, London SW1P 3JR UK *CSL Food Science Laboratory Norwich Research Park, Colney Norwich, NR4 7UQ, UK

There is little information on the concentrations of the platinum group elements in food or dietary exposures. To obtain information on dietary exposures of the UK population, analyses for the platinum group elements (iridium, palladium, platinum, rhodium and ruthenium) were carried out on samples from the 1994 UK Total Diet Study using Inductively Coupled Plasma-Mass Spectrometry (ICP-MS). The results of these analyses together with consumption data for the average UK household were used to estimate population dietary exposures. This is the first time that analyses for the platinum group elements have been included in an UK Total Diet Study and that dietary exposures have been assessed. Concentrations of these five elements in the 20 food groups comprising the Total Diet Study were very low and generally at or below the limits of detection (LOD) in the fresh weight foods. Iridium was only detected at concentrations above the LOD of 0.001 mg/kg in the miscellaneous cereals group (0.002mg/kg). Palladium was present in most food groups above the LOD of 0.0003 mg/kg with the highest concentrations found in nuts (0.003mg/kg), bread, offal, and fish groups (all 0.002mg/kg). Platinum was only present above the LOD of 0.0001 mg/kg in the oils and fats group (0.0002mg/kg). Rhodium was detected at concentrations above the LOD of 0.0001 mg/kg in bread (0.002mg/kg), offal (0.0005 mg/kg), fish (0.0002mg/kg), milk (0.0003 mg/kg) and nuts (0.004mg/kg). Ruthenium was not detected above the LOD of 0.002mg/kg. To estimate dietary exposures of the general UK population it was assumed that for those elements below the LODs that they were present at concentrations equal to the 705

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LODs. The resulting values can therefore be regarded as worst-case estimates of exposure. Average dietary exposures for the UK population were estimated to be: iridium— 0.002 mg/person/day; palladium—0.001 mg/person/day; platinum—0.0002 mg/person/ day; rhodium—0.0003 mg/person/day; and ruthenium—0.004 mg/person/day.

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ACUTE AND CHRONIC EFFECTS OF ACID PHENOL COMPOUNDS ON ALUMINUM RETENTION IN RATS Z. Deng1, L. Gouzoux2, C. Coudray3, A. Mazur3, Y. Rayssiguier3, and D. Pépin2 1

Permanent address: Food Nutrition Laboratory Food Science Department Nanchang University Nanchang 330047, China 2 Laboratoire de Controle des Eaux Institut Louise Blanquet Faculté de Médecine et Pharmacie 28 place H. DUNANT 63001 Clermont-Ferrand, France 3 Centre de Recherches en Nutrition Humaine Unité Maladies Métaboliques et Micronutriments INRA de Theix, 63122 St. Genès Champanelle France

Aluminum (Al) uptake seems to be very low, but many factors can modify its absorption and distribution in animals and humans. In the present study, we investigated the acute and chronic effects of citrate and of different acid phenol compounds on Al metabolism in rats by monitoring Al levels in blood and several tissues. In each study, the rats were divided into 7 groups: control, Al, Al+ sodium citrate, gallic acid, chlorogenic acid, caffeic acid and protocatechuic acid. In the acute study, after 14 hours fasting period, animals were dosed orally with, respectively, 2ml of desionized water, or 2mmol Al chloride, or 2mmol Al chloride plus 2mmol sodium citrate or 2mmol Al chloride plus 2mmol of studied acid phenol compound. Blood samples were taken before and 2 and 6 hours after the gavage. Animals were sacrificed 6 hours after the gavage and tissues were then removed. In the chronic study, the rats were first adapted for a semi-purified diet for one week and then received during 4 weeks 16mmol of AlCl3 per kg of diet, except the group 1, plus 16 mmol of the studied molecules per kg of diet, except the group 1 and 2. At the end of forth week of diet, animal were sarified and blood and tissues were sampled. Al concentration in blood, liver, tibia, kidney and brain were determined 707

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by ICP-OES. As expected, citrate highly increased Al blood concentration and its tissue retention, in the acute study. The most important result is the particular behavior of caffeic and chlorogenic acids. In the chronic study, Al levels in urines were significantly increased in all the Al groups in comparison to control group, indicating that Al was absorbed. Significantly higher Al levels in tibia, kidney and brain were observed in the citrate group. A significant increase in brain Al level was also noted in the chlorogenic acid group. This may suggest a possible relation structure-activity of acid phenol compounds. Further studies are still necessary to better understanding of the influence of acid phenols on Al metabolism, in particular that of chlorogenic acid.

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DETERMINATION OF TOTAL AND SOLUBLE TITANIUM IN SOFT TISSUES COVERING TITANIUM MICROPLATES IN STOMATOLOGY An Analytical Study

J. Poupon1, J. P. Méningaud2, C. Chenevier1, M. Galliot-Guilley1, and J. Ch. Bertrand2 1

Laboratoire de Biochimie-Toxicologie Hôpital Fernand Widal, Paris France 2 Service de Chirurgie Maxillo-Faciale Hôpital de la Pitié-Salpêtrière Paris, France

AIMS The following reports a study conducted to investigate a relationship between duration of plating and metal release from titanium (Ti) miniplates in craniofacial surgery.

METHODS 1) We used a prospective cohort study design. All patients who underwent removal of titanium miniplates from January to September 1998 in the Salpêtrière University Hospital (27 cases) have been evaluated in relation to the concentration of Ti in the soft tissues covering the plates. Total Ti and soluble Ti rates were compared to duration of plating (ranged from 15 days to 3 years, mean = 9 months). Correlation coefficients were computed. 2) Ti determination: In a first set of patients (n = 15), tissues were divided in two fragments. One was rinced with NaCl (1-A) while the other was not (1-B). The two fragments were weighed before and after drying. Tissues were mineralized in teflon bombs with nitric acid in a microwave digestion system. When present, insoluble residues were solubilized with sulfuric acid. Total Ti in mineralisates was determined by Inductively-Coupled Optical Emission Spectrometry 709

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(ICP-OES). In a second set (n = 12), tissues were sonicated before enzymic digestion with protease type VIII plus collagenase. After centrifugation, the pellet was dissolved in sulfuric acid and Ti was measured by ICP in both fractions. Reference material was used to check the accuracy of Ti determination.

RESULTS In the first set, total Ti levels showed very large variations with no incidence of rincing the tissues before treatment: mean 1-A was dry, sd = 2,729, range: 3–11,000 and mean 1-B was dry, sd = 2,203, range: 3–8,625. Macroscopic examination after nitric acid mineralization evidenced metallic Ti particles and their visual estimation correlated with Ti content measured by ICP. After enzymic digestion, measurement of Ti in the supernatant (soluble Ti) and in the pellet indicated that only less than 5% of Ti was in a soluble form. No correlation was found between soluble Ti or total Ti and the duration of plating.

CONCLUSION According to our own experience, ICP-OES is a more convenient method than Atomic Absorption Spectrometry for Ti measurement in tissues. As others, this study demonstrates that titanium is found in the soft tissues in contact with the titanium plates but it is the first time that soluble and non soluble fractions are measured. Our results indicate that almost of Ti is insoluble likely corresponding to metallic particles liberated during the implantation of the plates. Time-dependent leakage of Ti, if exists, seems to be negligible.

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TISSUE SELENIUM LEVELS IN LAYING HENS ARE INFLUENCED BY DIETARY OILS AND FATTY ACIDS INCLUDING TRANS FATTY ACIDS

Klaus Schäfer Institut für Tierernährung Freie Universität Berlin 14195 Berlin, Germany

1. INTRODUCTION Feeding fat-added diets to farm animals is becoming a common practice (Herber and van Elswyk, 1996; Lopez-Bote et al., 1997), and also in human diets the use of fat supplements, particularly the consumption of polyunsaturated fatty acids, has been increased in recent years to reduce the risk of cardiovascular diseases and other afflictions (Simopoulos, 1991). Experimental data indicate that dietary fat may be an important factor in trace mineral metabolism (Lukaski and Johnson, 1992). However, the interactions between dietary fat and selenium as well as the influence of dietary fat on tissues selenium concentrations and also the influence of dietary fat on the metabolic fate of selenium in the body and the influence of dietary fat on selenium needs are largely unknown. The present study with laying hens was done to investigate whether dietary fat influences tissue selenium concentrations and the selenium metabolism.

2. MATERIALS AND METHODS The influence of omega 6 fatty acids as well as monounsaturated trans fatty acids on tissue concentrations of selenium in laying hens were examined. Soybean oil (SBO), or free linoleic acid (LA), or partially hydrogenated soybean oil (PHS), were fed to laying hens as supplements. The soybean oil diet and the linoleic acid diet contained a particularly high percentage of n-6 fatty acids, while the PHS diet contained more monounsaturated fatty acids, particularly monounsaturated trans fatty acids (Schäfer and Schwabe, 1997). The birds of six dietary treatments were fed 115 grams feed/day during a period of 8 weeks. Each dietary group of 10 hens were fed either the unsupplemented control diet, or the control diet supplemented with 30 grams soybean oil per kg diet, or linoleic Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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acid, or partially hydrogenated soybean oil. The control diet and the fat supplemented diets contained 0.09mg selenium per kg diet. Additionally two dietary groups were fed a fat supplemented diet with sodium selenite added. The daily selenium uptake was 10.4 microgram respectively 67.9 microgram in the two selenium supplemented groups. All diets were supplemented with 60 milligram tocopheryl-acetate per kg diet.

3. RESULTS AND DISCUSSION Supplementation of dietary oil or fatty acids has a pronounced effect on selenium profiles in all tissues examined with the exception of breast muscle. The tissue concentration of selenium in the supplements were depressed to levels which could be characterized as marginal or deficient. Figure 1 shows the effects of dietary oils and fatty

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acids on the selenium status in some tissues of laying hens examined. The mean value of plasma selenium in hens fed the unsupplemented diet was The selenium concentration in the blood plasma of supplemented hens dropped from 1.47 to 0.98 to Activities of the selenium enzyme GSH-Px in plasma were also evaluated as an indicator of the selenium status. Like the plasma selenium levels, the enzyme activities in the plasma of control birds were significantly higher than the enzyme activity of animals feeding on fat added diets. The decrease depended on the dietary selenium levels. Birds feeding a selenium-supplemented diet showed similar activities like the control group. In egg yolk selenium trends, as expected, like the hepatic selenium results. Supplementation with SBO, PHS or LA decreased the selenium concentration in egg yolk significantly. Dietary fat can provide eggs with high polyunsaturated fatty acid contents but at the same time dietary fat may deplete selenium in egg yolk if the dietary selenium is not adapted to changed needs of selenium. Tissue selenium levels and the loss of selenium in tissues are not affected by fat supplementation in the same way, results appear to be tissue specific and fatty acid specific

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as well (Table 1). However, the decrease of selenium concentrations in tissues did not depend primarily and noticeably on the degree of unsaturation of the dietary fatty acids. Partially hydrogenated soybean oil, depressed selenium contents in heart muscle more than LA, and more than SBO, whereas in red blood cells fat supplements showed no different effects. The depressive effect of trans fatty acids in heart is remarkable. In recent years attempts have been made to elucidate the risks of consuming trans fatty acids (Koletzko, 1994). It may be that another issue on the question of adverse health effects of trans fatty acids should be considered. The mechanism by which fat supplementation alters the selenium status is not known. One explanation could be that the observed decrease of selenium in different tissues was the result of decreased selenium absorption caused by an additional amount of fat in the diet. Selenium compounds are absorbed efficiently from all segments of the small intestine. If the presence of fat in the small intestine made selenium less available for absorption, then fat supplementation would be expected to raise selenium contents in the intestinal chyme. However, the remaining selenium in the intestinal chyme in the fat supplements was not increased (Fig. 1) indicating that it is not likely that dietary fat influences the selenium status in tissues via altered intestinal absorption of selenium. The postabsorptive utilisation of selenium includes the redistribution and exchange of selenium among tissues, for example liver selenium is diverted to target tissues. It has been reported by Behne and Höfer-Bosse (1984) and Behne et al., (1988) that there exists a hierarchy of need within the body selenium pool. It is hypothesized that in birds fed supplemented fat diets selenium was preferentially diverted to critical tissues, probably to endocrine tissues and small intestine tissue, leaving less selenium in heart, liver and egg yolk. Feeding fat added diets could also affect the endogenous excretion of selenium in the intestine tissue. The selenium concentration in the colon contents showed a decreased excretion of selenium when PHS or LA were fed as supplements (Fig. 1), but the difference between fat-added birds and control birds was not significant. Additional data points would be needed to elucidate the influence of fat supplements on the rate of total endogenous excretion of selenium, including urinary excretion. In summary, this study provides further evidence that the selenium metabolism in laying hens as well as in animals and humans is sensitive to the amount and type of fat added to the diet. The dietary requirement for selenium may depend partly on the dietary fat intake.

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REFERENCES Behne, D. and Höfer-Bosse, T., 1984, Effects of low selenium status on the distribution and retention of selenium in the rat, J. Nutr, 114:1289–1296. Behne, D., Gessner, H., and Scheid, S., 1988, Selenium and selenoproteins in tissues with endocrine functions. In: Trace elements in Man and Animals, (Hurley. L.S., Kfeen, C.L., Lonnerdal, B., and Rucker, R.B. eds.) pp. 55–57, Plenum Press, New York. Herber, S.M. and van Elswyk, M.E., 1996, Dietary marine algae promotes efficient deposition of enriched shell eggs, Poultry Sci. 75:1501–1507. Koletzko, B., 1994, Trans fatty acids and human infant. In: Galli, C., Sinopoulos, A.P., Tremoli, E. (eds.): Fatty acids and Lipids: Biological Aspects, World Rev. Nutr. Diet, Basel, Karger. Vol. 75:82–85. Lopez-Bote, C.J., Rey, A.I., Sanz, M., Gray, J.I., and Buckley, D.J., 1997, Dietary vegetable oils and alphatocopherol reduce lipid oxidation in rabbit muscle, J. Nutr. 127:1167–1182. Lukaski, H.C. and Johnson, P.E., 1992, Dietary fatty acids and minerals. In: Fatty acids in foods and their health implications (Chow, C.K. ed.). Marcel Dekker, Inc. New York, pp. 501–516. Schäfer, K. and Schwabe, M., 1997, Beeinflussung essentieller Fettsäuren in verschiedenen Geweben der Legehenne bei unterschiedlicher Versorgung mit Fettsäuren und Selen. In: Fett in Nahrung und Ernährung (Wenk, C., Amado, R., and Dupuis, M. eds.) Wissenschaftliche Verlagsgesellschaft mbH Stuttgart, Germany, pp. 116–123. Simopoulos, A.P., 1991, Omega-3 fatty acids in health and disease and in growth and development, Am. J. Clin. Nutr. 54:438–463.

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DIETARY Mo AS AN ANTAGONIST TO Cu ABSORPTION Stable Isotope (65Cu) Measurements in Grazing Sheep

S. O. Knowles, N. D. Grace, J. R. Rounce, A. Litherland, D. M. West, and J. Lee AgResearch, Grasslands Research Centre PB 11008, Palmerston North New Zealand

1. INTRODUCTION We have modified the method of Buckley (1991) using stable isotopes of Cu to estimate Cu absorption in sheep and cattle at pasture. With this technique, the impact of dietary Mo and S on Cu status of grazing livestock can be quantified. Increasing Mo intakes in the presence of S reduces the absorption, storage and utilisation of Cu via the synthesis of thiomolybdates in the reticulorumen and formation of insoluble Cu thiomolybdates in the digesta and tissues (Suttle and Field, 1983). Mo-induced Cu deficiency can lead to nerve dysfunction (enzootic ataxia) (Fell et al., 1965), and bone and connective tissue disorders (Hogan et al., 1971). In the current study we have used a protocol of isotope tracer infused i.v. over 4 hours and serial collection over 180 days of blood and liver biopsy samples to examine the impact of low and high Mo intakes on the absorption and metabolism of Cu in grazing Romney sheep.

2. METHODS Seventeen 1 hectare pastures, 2 to 6 replications per treatment, were topdressed with Mo-amended fertiliser at the rate of 0, 500, 1,000 or 1,500 g Mo/ha on day 1 of the study. Representative samples from each pasture were collected every 3 weeks thereafter for elemental analysis of herbage. On day 44, groups of wether lambs of mean liveweight 27 kg and matched for similar initial Cu stores, were eartagged and randomly distributed among the experimental pastures, where they grazed until the end of the study on day 224. Liver samples of 50 to l00mg were obtained by biopsy procedure under local Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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anaesthesia on days 44, 111, 128, 148, 174, 198 and 224. Blood was collected from the jugular vein on days 44, 148, 174, 198 and 224. The total elemental concentrations in acid digests of freeze-dried liver, blood plasma and pasture samples were measured by ICP-OES. On day 125, 4 to 8 sheep per treatment were brought indoors overnight to receive l0mg of stable isotope tracer in 100ml 0.9% saline delivered by constant i.v. infusion over 4 hours. Isotope ratio measurements in liver biopsies were made by ICP-MS. Isotope enrichment of above natural abundance was calculated according to Coudray et al. (1998), from which single-compartment exponential tracer kinetics were determined. Turnover time, total liver Cu pool size, and Cu absorption were calculated (see footnotes to Table 1).

3. RESULTS Concentration of Mo in the herbage of treated pastures increased steadily for the duration of the trial. Mean pasture Mo concentrations over the 100-day post-infusion period were 2.2, 4.2, 7.0 and 11.7mgMo/kg dry matter (DM) for the four treatments, respectively. Pasture Cu and S content was not affected by time or treatments, averaging 7.8 ± 0.land 3,210 ± 58mg/kg DM, respectively. Sheep DM intake was determined to be 1.4kg pasture DM/day (Geenty and Rattray, 1987), which provided dietary Mo intakes of 3.1, 5.9, 9.8 and 16.4 mg Mo/day. Liver Mo concentration was significantly increased only in the highest treatment (3.8 ± 0.2 vs. 3.1 ± 0.1 mg Mo/kg tissue DM). The effects of increasing pasture Mo concentration on liver and plasma Cu concentrations are shown in Fig. 1. Liver Cu concentration decreased with time and with increasing Mo exposure, such that by day 224 several animals were marginally Cu deficient. Plasma Cu also decreased significantly with time but was not affected by treatments. Plasma caeruloplasmin activity was not changed. Liver Cu pool size decreased substantially over time in all treatment groups (Table

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1). This was an important consideration when calculating fractional loss rate, turnover, and Cu absorption, because a changing pool size violates the assumption of steady state. Nevertheless, sheep tracer kinetics could be adequately approximated by a singlecompartment (liver) model. Fractional loss from the liver Cu pool showed a dose response to Mo treatment; rates in sheep grazing the higher Mo pastures were 50% to 70% faster than controls. Increasing mean pasture Mo concentration from 2.2 to 4.2mg Mo/kg DM reduced by half the amount of Cu absorbed by sheep, from 0.50 down to

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0.28mg Cu/day. Those dose responses agree with the results of a preliminary Cu isotope trial performed using sheep grazing untreated very low Mo pastures (<0.5 mg Mo/kg DM); fractional loss was 25% slower than controls, and the Cu absorbed was 0.57mg Cu/day.

4. DISCUSSION The range of pasture Mo concentrations produced by the Mo treatments was representative of New Zealand farms. Increasing Mo intakes significantly decreased sheep liver Cu concentration, liver Cu pool size, and the quantity of Cu absorbed. Although liver Cu stores were being depleted, no clinical signs of Cu deficiency were observed. Grace (1994) has shown by a factorial approach that growing sheep gaining 0.1 kg/day have a net Cu requirement of 0.23 mg/day. Using the stable isotope method, we estimated the amount of Cu absorbed by sheep on the lowest and highest Mo treatments to be 0.50 and 0.28 mg Cu/day, respectively. Had the sheep continued to graze the highest Mo pasture for longer than 180 days, signs of Cu deficiency would likely have been observed. Impaired growth and connective tissue lesions have been observed in young sheep grazing pastures containing 5 to 7mg Cu and 5 to 8 mg Mo/kg herbage DM for at least 6 months (Hogan et al., 1971). Blincoe (1992) and Symonds and Forbes (1993) have described multi-compartment models of Cu metabolism. In sheep the liver Cu pool contains 50–60% of total body Cu, and was assumed in this experiment to behave as a single compartment. Thus the loss of Cu from liver is equivalent to gain from dietary Cu intake times fraction absorbed. The coefficients of Cu absorption (% of Cu intake) determined for sheep in this study ranged from 4.6% to 2.2%, and are similar to those reported by Suttle (1983). The use of stable isotopes to study in vivo Cu kinetics offers distinct advantages over short half-life radioactive tracers (Blincoe, 1992). The technique has previously been applied to ruminants in only a few trials of cattle fed and housed indoors (Buckley, 1991). By extending the method to sheep, we enhance our understanding of Cu metabolism and can improve strategies to diagnose and prevent Cu deficiency. Additionally, sheep might serve as an animal model for research on human Cu disorders, as sheep are able to accumulate large quantities of Cu in liver, a condition similar to the symptoms of Wilsons disease.

REFERENCES Blincoe, C., 1992, Simulation of copper metabolism by mammals, Comput. Biol. Med. 22:113–122. Buckley, W.T., 1991, A kinetic model of copper metabolism in lactating dairy cows, Can. J. Anim. Sci. 71:155–166. Coudray, C., Bousset, C., Tressol, J.C., Pépin, D., and Rayssiguier, Y., 1998, Short-term ingestion of chlorogenic or caffeic acids decreases zinc but not copper absorption in rats, utilization of stable isotopes and inductively-coupled plasma mass spectrometry technique, Br. J. Nutr. 80:575–584. Fell, B.F., Mills, G.F., and Boyne, R., 1965, Cytochrome oxidase deficiency in motor neurones of copperdeficient lambs; a histochemical study, Res. Vet. Sci. 41:417–419. Geenty, K.G. and Rattray, P.V., 1987, The energy requirements of grazing sheep and cattle, in: Livestock Feeding on Pasture, (A.M. Nicol, ed.), pp. 39–52, New Zealand Society of Animal Production Occasional Publication No. 10. Grace, N.D., 1983, Amounts and distribution of mineral elements associated with fleece-free empty body weight gains in the grazing sheep, N.Z. J. Ag. Res. 26:59–70.

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Grace, N.D., 1994, Copper, in: Managing Trace Element Deficiencies, pp. 35–51, N.Z. Pastoral Ag. Res. Inst. Ltd., Palmerston North, New Zealand. Hogan, K.G., Money, D.F.L., White, D.A., and Walker, R., 1971, Weight responses of young sheep to copper and connective tissue lesions associated wit the grazing of pastures of high molybdenum content, N.Z. J. Ag. Res. 14:687–701. Suttle, N.F., 1983, Effects of molybdenum concentration in fresh herbage, hay and semi-purified diets on the copper metabolism of sheep, J. Ag. Sci. 100:651–656. Suttle, N.F. and Field, A.C., 1983, Effects of dietary supplements of thiomolybdates on copper and molybdenum metabolism in sheep, J. Comp. Path. 93:379–389. Symonds, H.W. and Forbes, J.M., 1993, Mineral metabolism, in: Quantitative Aspects of Ruminant Digestion and Metabolism (J.M. Forbes and J. France, eds.), pp. 363–379, CAB International, Wallingford.

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COPPER-ASSOCIATED CIRRHOSIS IN NORTH RONALDSAY SHEEP A Possible Model for Idiopathic Copper Toxicosis

S. Haywood, T. Müller*, W. Müller*, and Z. Dincer Department of Veterinary Pathology University of Liverpool Liverpool, U.K *Department of Paediatrics Community Hospital Reutte 6600 Reutte, Austria

1. INTRODUCTION Idiopathic copper toxicosis (ICT) is a rapidly fatal liver disease of young children endemic in the Tyrol from 1900–1974 (Müller et al., 1996). ICT, as exogenic infantile copper toxicosis, has also been identified in Germany (Müller-Höcker et al., 1998) and sporadically throughout the world. ICT is clinically and pathologically indistinguishable from Indian childhood cirrhosis (ICC), Whilst excess copper intake has been implicated in the majority of reports of ICT, a hereditary predisposition has been suspected in others and confirmed in the Tyrolean subset. Indeed, ICT is regarded as an ecogenetic disorder requiring synergy of both genetic predisposition and environmental factors for manifestation of the disease (Müller et al., 1998). ICT and ICC differ from Wilson disease in their earlier age of onset, normal serum caeruloplasmin and distinct histopathology. The liver changes are characterised by a marked pericellular and panlobular fibrosis, associated with a mixed inflammatory infiltrate and culminating in fibrosis/cirrhosis with minimal regeneration. Usually but not invariably there is ballooning degeneration of hepatocytes with Mallory body inclusions (Müller et al., 1998), although atypical pathomorphological changes have been reported in some children (Müller-Höcker et al., 1998). North Ronaldsay sheep have adapted to a copper-impoverished (<5 ppm Cu) environment on the island foreshore, although the breed are susceptible to copper poisoning elsewhere (Maclachan and Johnston, 1982). A flock of North Ronaldsay sheep has been maintained at Liverpool University field station on a copper-monitored diet (7–11 ppm Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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Cu). Despite this liver fibrosis has been observed in culled Ronaldsay sheep and some sheep have died with very high liver copper (>2,000g/g) and coarsely-nodular fibrosed livers. This is remarkable since neither fibrosis nor cirrhosis has been reported previously in ovine copper poisoning. Copper toxicosis in the North Ronaldsay breed appears to be an ecogenetic disease similar in this respect to ICT. It was decided to evaluate the liver pathology of these sheep from archival material at our disposal to see if Ronaldsay sheep copper toxicosis (RCT) could be a possible model of the human disease.

2. MATERIALS AND METHODS Formalin fixed, paraffin-embedded livers were utilised from North Ronaldsay sheep accessioned at the University of Liverpool. These included 13 randomly culled sheep of different ages and sex, six sheep which had been used as controls in an experimental study (Dincer, 1994) and two sheep that had been put down in liver failure; all sheep having been reared on the mainland. In addition three liver samples from culled island-bred sheep were used as controls. Liver sections were stained with H&E, reticulin van Gieson for fibrous connective tissue and rhodanine for copper. The liver samples had been previously analysed for copper by atomic absorption spectrophotometry.

3. RESULTS The mainland sheep had accumulated copper compared with the island sheep and were divided into categories 1–3 (Table 1). In the first category, 9 sheep had accumulated liver copper to without evidence of liver damage. In the second, 11 sheep with a liver copper elevation upto exhibited piecemeal periportal necrosis, cholangioplasia, mixed inflammatory infiltrate, portal bridging fibrosis with a marked periportal and pericellular reticulofibrosis. The two sheep in liver failure with copper in excess of had more extensive hepatocellular degeneration and necrosis with early cirrhosis. Histochemical copper was variable, most prominent periportally but often panlobular or negative. Macrophages and Kupffer cells frequently stained Cu + ve. Portal hyaline cells (Mallory-like bodies) were identified of macrophage, or possibly hepatocellular, origin, and often contained copper or flocculent material. Giant, multinucleate sinusoidal cells, likewise containing copper and/or flocculent material were prominent especially around the central veins. Hepatocytes exhibited a variable degree of polyploidy or karyomegaly. Mitoses were rare, except in one case of hepatic failure, which was associated with nodular regeneration.

4. DISCUSSION AND CONCLUSIONS The pathological consequences of liver copper accumulation in North Ronaldsay sheep in which piece-meal necrosis, or cell drop out, accompanied by an early florid fibrosis culminating in cirrhosis is very different from copper poisoning in other breeds of sheep. The pathomorphological patterns of periportal and pericellular reticulofibrosis, copper retention, cholangioplasia and mixed inflammatory infiltrate are similar in RCT and ICT. Hyaline portal “cells” identified in the portal tracts were similar to the “Mallory-like bodies” in copper poisoned rats (Haywood, 1985) but were not necessar-

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ily the same structures as the Mallory bodies which are so characteristic a feature of ICT and ICC. More work will be needed to clarify their derivation. Finally, RCT and ICT appear to share a similar pathogenesis with respect to the dual roles of exogenous copper and genetic susceptibility substantiated by the very similar pathologies. RCT should be a useful model for investigating ICT.

ACKNOWLEDGMENTS We wish to acknowledge the support of KME Metal AG, Osnabruck, Germany.

REFERENCES Dincer, Z., 1994, Copper toxicity in sheep: studies on copper chelation by ammonium tetrathiomolybdate (TTM) and metallothionein, Ph.D. thesis, Liverpool University. Haywood, S., 1985, Copper toxicosis and tolerance in the rat I–Changes in copper content of the liver and kidney, J. Pathol. 145:149–158. Maclachan, G.K. and Johnston, W.S., 1982, Copper poisoning in sheep from North Ronaldsay maintained on a diet of terrestrial herbage, Vet Record III:299–302. Müller, T., Feichtinger, H., Berger, H., and Müller, W., 1996, Endemic Tyrolean cirrhosis: an ecogenetic disorder, The Lancet, 347(No. 9005):877–880.

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Müller, T., Müller, W., and Feichtinger, H., 1998, Idiopathic copper toxicosis, Am. J. Clin. Nutr. 67(suppl):1082S–1086S. Müller-Höcker, J., Summer, K.H., Schramel, P., and Rodeck, B., 1998, Different pathomorphologic patterns in exogenic infantile copper intoxication of the liver, Pathol. Res. Pract. 194:377–384.

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DIETARY COPPER AFFECTS LIPID AND CHOLESTEROL METABOLISM IN FINISHING STEERS

T. E. Engle, J. W. Spears, C. L. Wright, and T. A. Armstrong Department of Animal Science and Interdepartmental Nutrition Program North Carolina State University Raleigh, NC, 27695-7621 USA

1. INTRODUCTION Supplementation of copper (Cu) to nonruminants above their recommended requirement has been shown to decrease plasma and breast muscle cholesterol concentrations in poultry (Bakalli and Pesti, 1994) and decrease the saturated to unsaturated fatty acid ratio in swine adipose tissue (Amer and Elliot, 1973). In ruminants, Cu supplementation to diets marginal in Cu led to an increase in adipose cell volume and lipolytic rate in sheep (Sinnett-Smith and Woolliams, 1987) and decreased backfat depth and increased rib eye area in cattle (Ward and Spears, 1997). Therefore, the present studies were conducted to determine the effects of dietary Cu source and level on lipid and cholesterol metabolism in beef cattle.

2. MATERIALS AND METHODS In both experiments, steers were stratified by weight and randomly assigned to treatments. Steers were housed in pens equipped with individual electronic Calan gate feeders. Body weights and blood samples were obtained every 28 days. Carcass data and a final liver sample were obtained from each animal postmortem. In experiment 1, sixty Angus (n = 36) and Angus x Hereford (n = 24) steers (average initial BW 250kg ± 4.9) received one of six treatments. Treatments consisted of: 1) control (no supplemental Cu), 2) 20mg Cu/kg diet from Cu sulfate 40mg Cu/kg diet from 4) 20 mg Cu/kg diet from Cu citrate, 5) 20 mg Cu/kg diet from Cu proteinate, and 6) 20 mg Cu/kg diet from tribasic Cu chloride. Steers were fed a corn silage-soybean Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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meal-based growing diet (89% corn silage and 11% of a soybean meal-mineral-vitamin supplement; basal diet contained l0mg of Cu/kg DM) for 56 days. After the 56 day growing phase, steers were switched to a high concentrate diet (basal diet contained 5 mg of Cu/kg diet) but remained on the same dietary treatments that they received in the growing phase until they reached a finished weight of approximately 560kg. Equal numbers of steers per treatment were slaughtered after receiving the finishing diets for either 101 or 121 days. In experiment 2, sixty Angus steers (average initial BW 391.1 kg ± 6.1) received one of three treatments. Treatments consisted of 0 (control), 10, and 20 mg supplemental Cu/kg diet from Steers were fed a high concentrate diet (basal diet contained 5.1 mg of Cu/kg diet) until they reached a finished weight of approximately 580kg. Equal numbers of steers per treatment were slaughtered after receiving the finishing diets for either 96 or 112 days.

3. RESULTS AND DISCUSSION Experiment 1 Serum cholesterol was lower (P < 0.05) in steers supplemented with Cu by day 84 of the finishing period (Table 1) and remained decreased at each subsequent sampling period thereafter. Longissimus muscle cholesterol concentration and percentage saturated fatty acids tended to be reduced (P < 0.11; Table 1) while percentage polyunsaturated fatty acids were increased (P < 0.10) by Cu supplementation. Backfat was lower (P < 0.05) in animals receiving supplemental Cu, but intramuscular fat was similar across treatments (Table 1). Throughout this experiment, there were few differences between Cu sources and additive effects of 40 mg of Cu/kg DM when compared to 20 mg of Cu/kg DM.

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Experiment 2 Serum total cholesterol concentrations were reduced (P < 0.05) by day 56 (Table 2) and at subsequent sampling dates thereafter. Longissimus muscle cholesterol concentrations were lower (P < 0.10) in steers receiving supplemental Cu (Table 2). Backfat depth was lower (P < 0.05) in animals receiving supplemental Cu, but intramuscular fat was similar across treatments. Percent unsaturated fatty acid in longissimus muscle was increased (P < 0.05) and percent saturated fatty acid tended to be reduced (P < 0.12) in steers supplemented with Cu (Table 2). Polyunsaturated fatty acid (18:2 and 18:3) concentrations were higher (P < 0.05) in steers supplemented with Cu (Table 2). These results indicate that Cu addition to high concentrate finishing diets alters lipid and cholesterol metabolism in steers.

REFERENCES Amer, A.M. and J.I. Elliot, 1973, Effects of level of copper supplement and removal of supplemental copper from the diet on the physical and chemical characteristics of porcine depot fat, J. Anim. Sci. 53:1039–1145. Bakalli, R.I. and G.M. Pesti, 1994, Dietary copper in excess of nutritional requirements reduces plasma and breast muscle cholesterol of chickens, Poultry Sci. 74:360–365. Sinnett-Smith, P.A. and J.A. Woolliams, 1987, Adipose tissue metabolism and cell size: Variation between subcutaneous sites and the effects of copper supplementation, Anim. Prod. 45:75–80. Ward, J.D. and J.W. Spears, 1997, Long-term effects of consumption of low-copper diets with or without supplemental molybdenum on copper status, performance, and carcass characteristics of cattle, J. Anim. Sci. 71:2748–2755.

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A COMPARISON OF TWO METHODS OF COPPER SUPPLEMENTATION FOR GRAZING RED DEER (CERVUS ELAPHUS)

D. V. Illingworth, D. W. Jackson, N. R. Kendall, and S. B. Telfer Centre for Animal Sciences Leeds Institute for Biotechnology and Agriculture School of Biology University of Leeds Leeds LS2 9JT United Kingdom

1. INTRODUCTION The deleterious effects of clinical copper deficiency reduce stock rearing efficiency. The problems of copper deficiency conditions, such as swayback, on British red deer farms require effective supplementation. Previous reports have supplemented copper in the form of injections and glass boluses (Telfer, 1994), or oxidised copper wire (Booth et al., 1989). The advantage of long term slow release copper supplementation is minimal animal disturbance. Two such commercially available preparations, extensively used in cattle and sheep production, were compared in a field trial of a deer herd which had reported previous cases of swayback in the young stock.

2. MATERIALS AND METHODS Yearling stags (120) were randomly assigned to two treatment groups at turnout at the beginning of April 1998 following a diagnostic blood sample (Week 0). One group was treated with a single deer sized (75 ± 5 g ) copper, cobalt and selenium soluble glass bolus (Cosecure©, Telsol Ltd. ). The bolus used was the same glass formulation as the sheep bolus containing 10.1 g copper, 0.4g cobalt and 0.1 g selenium as and was expected to provide supplementation for 6 months. The second group was treated with two ewe/calf gelatin capsules each containing 2.5g copper oxide rods (= 4.2g copper, Copacaps©, Merial Animal Health Ltd). The deer were raised to slaughter weight (>80kg ). Equal numbers of animals were slaughtered at a commercial deer abattoir Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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during July (15–16 weeks post treatment) and August (18–20 weeks). Blood samples were obtained during exsanguination. Liver samples were obtained within 20 mins of slaughter. Blood samples were analysed for total plasma copper concentration (Pl-Cu) and trichloro-acetic acid soluble plasma copper concentration (TCA-Cu) by atomic absorption spectrometry (Mackenzie et al., 1997). Serum caeruloplasmin activity (CP) was measured using the method of Henry (1974), erythrocyte superoxide dismutase activity (SOD) using the method of Misra and Fridovich (1974), amine oxidase activity (Amox) by the method of Mulryan and Mason (1985) and glutathione peroxidase acivity by the method of Paglia and Valentine (1967) as adapted for the Cobas Mira (Roche). Serum concentrations were measured by assay (ICN Pharmaceuticals Ltd). Liver samples were analysed for total copper concentration after microwave wet digestion in nitric acid (Kendall et al., 1997). Statistical analysis was carried out by ANOVA using GLM on MINITAB 11 with Week 0 as covariate.

3. RESULTS AND DISCUSSION For the July slaughtered deer, copper supplementation raised blood copper parameters in both groups (Fig. 1). The bolus treated animals were significantly higher than needle treated animals with regard to Pl-Cu, TCA-Cu, CP and CP/Pl-Cu ratio (all p < 0.05). SOD and Amox showed no significant differences, due to large inter-animal variations in both groups, but the activity levels indicated copper adequacy. The CP/Pl-Cu ratio has been shown in cattle to be an indicator of thiomolybdate toxicity (Mackenzie et al., 1997). The ratios obtained in this experiment (<1.5) would indicate a significant thiomolybdate problem. However, at present, there is insufficient trial information in red deer to show that the same ratio values that apply in cattle also apply in this species. GSH-Px (Fig. 2) activities were significantly greater in the bolus group (p < 0.01) indicating effective selenium supplementation. levels were not significantly

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greater in the bolused group in either July or August (214 ± 18pM, total mean ± SE) but were within the previously reported range for red deer using the radioreceptor assay (Alexander, 1986). The similarity to cattle values and the lack of any supplementation effect suggest that, as in cattle, values obtained by radioreceptor assay are not an unequivocal indicator of cobalt status (Carlos et al., 1987). The low number of Cosecure boluses recovered (approximately 12%) and their pitted condition indicated that bolus dissolution was almost complete in four months and not the expected six months. Bad weather immediately after turnout caused the animals to be rehoused and fed a non-grass diet until May. An increase in rumen acidity due to the feeding regime would have contributed to a faster bolus dissolution, a situation seen regularly in dairy cows (Telsol technical information). Signs of healed ulceration were observed in stomachs of some of the needle treated deer, possibly following the lodging of the copper oxide needles in the abomasal wall. For the August slaughtered animals no boluses were recovered, and there were no significant differences between the two groups for any of the copper parameters. GSHPx was still significantly higher for the bolus group (p < 0.05) due to re-utilisation of selenium and the 4–6 week erythrocyte half-life. All week 0 and July deer had plasma copper values as had all but two of the August slaughtered animals. The liver copper concentrations showed no significant differences between the groups of stags at either slaughter period (overall, 107 ± 14mg/Kg DM). However, the July Cosecure treated group showed the greatest range (34–230 mg/kg DM), indicating a varied dissolution rate of the Cosecure boluses and hence variable storage of the copper in the liver. No symptoms of copper toxicity were observed in any animal with the higher values for copper parameters. There was also no statistical correlation between plasma copper levels and liver copper concentrations. The final slaughtering also included a small group of hinds (n = 5) from the same farm. These animals had not received any copper supplementation during the grazing season and all the measured copper parameters at slaughter showed copper deficiency e.g. CP <2mg/dl, P1 Cu liver copper <30mg/kg DM. The copper deficient status

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of the hinds suggests that the supplementation of the trial stags was necessary to maintain an adequate copper status.

CONCLUSIONS The soluble glass bolus was more effective at 16 weeks after administration than the copper oxide needles. However, the treatments were similar at 20 weeks in supplying extra copper to red deer at pasture. The bolus also provided effective selenium supplementation. The equivocal results indicate caution in using this parameter to assess cobalt status in this species until further evaluated.

ACKNOWLDGMENTS We are grateful for the assistance of the staff at the Whitlebury Park and Round Green Deer Farms.

REFERENCES Alexander, T.L., 1986, Management and diseases of deer: a handbook for the veterinary surgeon p 195 Edited by T.L. Alexander. Veterinary Deer Society, London. Booth, D.H., Wilson, P.R., and Alexander, A.M., 1989, The effect of oral oxidised copper wire on liver copper in farmed red deer. NZ Vet. J. 37(3):98–101. Carlos, G.M., Telfer, S.B., Johnson, C.L., Givens. D.I., Wilkins, R.J., and Newberry, R.D., 1987, Microbiological assay of blood serum for the vitamin B12 status of dairy cows. J. Dairy Res. 54:463–470. Henry, R.J., Cannon, D.C., and Winkelman, J.W., 1974, Clinical Chemistry, Principles and Technics p 860. Harper & Row, Publishers, London. Kendall, N.R., Mackenzie, A.M., and Telfer, S.B., 1997, Effect of a soluble cobalt, selenium and zinc glass bolus on humoral immune response and trace element status in lambs. In Trace Elements in Man and Animals -9: Proceedings of the Ninth International Symposium on Trace Elements in Man and Animals. Edited by P.W.F. Fischer, M.R. L’Abbé, K.A. Cockell, and R.S. Gibson. NRC Research Press, Ottowa, Canada, pp. 442–443. Mackenzie, A.M., Illingworth, D.V., Jackson, D.W., and Telfer, S.B., 1997, A comparison of methods of assessing copper status in cattle. In Trace Elements in Man and Animals -9: Proceedings of the Ninth International Symposium on Trace Elements in Man and Animals. Edited by P.W.F Fischer, M.R. L’Abbé, K.A. Cockell, and R.S. Gibson. NRC Research Press, Ottowa, Canada, pp. 301–302. Misra, H.P, and Fridovich, I., 1977, Superoxide dismutase: a photochemical augmentation assay. Arch. Biochem. Biophys. 181:308–312. Mulryan, G. and Mason, J., 1992, Assessment of liver copper status in cattle from plasma copper and plasma copper enzymes. Ann. Rech. Vet. 23:233–238. Paglia, D.E. and Valentine, W.N., 1967, Studies on quantitative and qualitative characterisation of erythrocyte glutathione peroxidase. J. Lab. Clin. Med. 70:158–169. Telfer, S.B., 1994, Trace element supplementation of deer. In Proceedings of the European Association of Animal Production p 117 Edinburgh, August 1994.

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TRACE ELEMENTS AND VITAMINS IN BLOOD OF ALPACAS (LAMA PACOS) AND SHEEP GRAZING THE SAME PASTURE 1

2

G. J. Judson , B. A. McGregor , and A. M. Howse

2

1

SA Research & Development Institute 33 Flemington Street, Glenside SA 5065, Australia 2 Agriculture Victoria Victorian Institute of Animal Science 475 Mickleham Road, Attwood Victoria 3049, Australia

1. INTRODUCTION Many of the 20,000 alpacas in Australia are raised in areas where ruminants are at risk of trace element and vitamin deficiencies. A survey of alpacas at pasture in southern Australia showed that plasma concentrations of zinc and copper were lower than the concentration of these elements in healthy ruminants and that plasma concentrations were intermediate of those concentrations found in healthy sheep and cattle (Judson et al., 1996). We are not aware of any reports of a direct comparison of trace constituents in the blood of alpacas and ruminants although a number of inter-species differences have been observed with sheep and llamas (Espinoza et al., 1982) and sheep and guanacos (Illingworth et al., 1998) grazing the same pasture. The present study was undertaken to compare the concentration of trace constituents in blood of sheep and huacaya alpacas grazing the same pasture.

2. MATERIALS AND METHODS The animals were held at Attwood in a paddock (about 3 ha) supporting improved pasture composed mainly of annual ryegrass with smaller amounts of subterranean clover, annual grasses and capeweed (McGregor, 1998). The climate is temperate with an average annual rainfall of about 560mm. Five alpaca wethers (2–11 years old) and five alpaca males (2–6 years old) were introduced to the paddock in 1994 followed by ten Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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Merino wethers (3 years old) in October 1995. Grazing pressure was increased from October 1996 until August 1997 by introducing extra sheep and excluding animals from about 25% of the paddock. No supplementary feed was offered to the animals. Blood samples were collected from the jugular vein of the ten alpacas and ten sheep on 12 occasions at 1–3 monthly intervals between November 1995 and August 1997. Pasture samples, collected at time of blood sampling, were assayed for mineral elements using an inductively coupled plasma mass spectrometer. Pasture, whole blood and plasma samples were assayed for selenium by a fluorometric procedure and plasma samples were assayed for using a radioactive assay kit (Solid Phase No Boil, Diagnostic Products Corporation, USA) and for trichloroacetic acid soluble copper and zinc using an atomic absorption spectrophotometer. High pressure liquid chromatography with fluorescence detection was used for the assay of plasma and feed vitamin E (alpha tocopherol) and plasma vitamin A (retinol). The alpaca results were subjected to an analysis of variance to test for differences between males and wethers. If differences were found then alpaca males and wethers were compared with sheep. An analysis of variance of a randomised design was used to compare the two species over time. Difference between means was compared using the least significant difference procedure.

3. RESULTS AND DISCUSSION Alpaca wethers were heavier (P < 0.05) than alpaca males and both were heavier (P < 0.001) than sheep at all stages of the sampling period. The animals gained weight from November 1995 until October 1996 and thereafter lost weight. The loss in weight coincided with increased grazing pressure and reduced pasture growth due to poor rainfall in May 1997 to August 1997 (McGregor, 1998). The overall mean weights are given in Table 1. Mean concentrations of trace constituents in the blood of alpaca males and alpaca wethers were similar (P > 0.05) apart from blood selenium and plasma and A concentrations: males had lower concentrations (P < 0.05) of blood selenium and plasma vitamin A but greater concentrations (P < 0.05) of than alpaca wethers. The overall mean values are given in Table 1. Statistical analysis indicated a species effect for all blood constituents (P < 0.05) and a species by sampling time interaction (P < 0.05) for all constituents except plasma selenium concentrations. Compared to sheep, alpacas had lower mean concentrations of

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blood selenium and plasma copper, zinc and and E and greater mean concentrations of plasma selenium and vitamin A. The overall mean concentrations are given in Table 1. In general, the differences between species were evident at all sampling times for all constituents except vitamin E. It was only in the latter stages of the experiment that mean vitamin E concentrations were greater in sheep than in alpacas. The marked species differences in plasma and blood selenium concentrations (Table 1) suggest that the selenium concentration in blood cells is markedly lower in alpacas than in sheep. Mean trace element and vitamin concentrations in blood of sheep were in the adequate range for this species (Paynter, 1996; Puls, 1994) at all sampling times. Similarly, the mean concentration of these nutrients in the blood of alpacas was typical of those concentrations observed in apparently healthy alpacas in southern Australia (Judson, 1996; Judson et al., 1996) indicating that dietary intake and/or tissue reserves were meeting the needs of both species. Trace element concentrations in the pasture were usually above the minimum desirable concentration for sheep (SCA, 1990) apart from cobalt and copper. Cobalt concentrations were low in summer—autumn of 1996 and in 1997 cobalt and copper concentrations were low in autumn and vitamin E concentrations were low in summer—autumn. Mean concentrations (and range) for pasture constituents (mg/kg dry matter) were: 0.13 (0.01–0.40) for cobalt; 6.8 (4.3–8.7) for copper; 621 (134–1,396) for iron; 0.8 (0.1–1.3) for molybdenum; 2,300 (1,200–3,800) for sulfur; 0.14 (0.07–0.23) for selenium; 45 (33–66) for zinc, and 57 (4–125) for vitamin E. Studies with sheep and alpacas on improved pasture indicate species differences in grazing behaviour; sheep were found to have a greater preference for ryegrass (Sharp et al., 1995) and legumes (San Martin and Bryant, 1989) than alpacas. In the present study it appears unlikely that the marked species differences in blood constituents, observed at most sampling times, were due to differences in grazing behaviour. Both species exhibited a marked preference for short grazed pasture despite the abundance of pasture in 1996 (McGregor, 1998) and in 1997 the opportunity for selective grazing was reduced due to increased grazing pressure and reduced pasture growth. The present findings show that marked differences in the concentration of all blood trace constituents examined, apart from plasma vitamin E, exist between sheep and alpacas when retained on the same pasture. For these trace constituents, reference ranges indicating adequacy in sheep may not be appropriate for alpacas.

ACKNOWLEDGMENTS The skilled assistance of Peter Bansemer, Petra Hajduk, Katrina Melnyczyn, Athena Mitsioulis and Peter Zviedrans in the laboratory and Ralph Behrendt, Mark Ferguson, Renee King and Meagan Nimbs in the field is gratefully acknowledged. We are grateful to Debra Partington, Biometrics SA for the statistical analyses. This study was supported by a grant from the Rural Industries Research and Development Corporation.

REFERENCES Espinoza, J.E., McDowell, L.R., Rodriguez, J., Loosli, J.K., Conrad, J.H., and Martin, F.G., 1982, Mineral status of llamas and sheep in the Bolivian Altiplano, J. Nutr. 112:2286–2292. Illingworth, D.V., Jackson, D.W., Fraser, M.D., and Telfer, S.B., 1998, Plasma copper and cobalt metabolism of guanacos (Lama guanicoe) and sheep grazing the same upland pasture, Proc. Brit. Soc. Anim. Sci. p. 218.

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Judson, G.J., 1966, Survey of the mineral, trace element and vitamin status of alpacas, in: Camelid Medicine & Surgery, Proceedings 278, pp. 311–317, Post Graduate Foundation in Veterinary Science, University of Sydney. Judson, G.J., Tuckwell, C.D., Ponzoni, R.W., Kenyon, R.V., McGregor, B.A., and Carmichael, I.H., 1996, Blood trace element and vitamin levels of alpacas, Proc. Aust. Soc. Anim. Prod. 21:481. McGregor, B.A., 1998, The effects of excellent nutrition and drought on the live weight, fibre growth and quality and grazing behaviour of alpacas and Merino sheep, Proceedings of the International Alpaca Industry Conference, pp. 89–95, Australian Alpaca Association Inc., Melbourne. Paynter, D.I., 1996, Diagnosis of mineral deficiencies, in: Detection and Treatment of Mineral Nutrition Problems in Grazing Sheep, ACIAR Monograph No. 37, (D.G. Masters and C.L. White, eds.), pp. 45–56, ACIAR, Canberra. Puls, R., 1994, Vitamin Levels in Animal Health, Diagnostic Data and Bibliographics, Sherpa International, Clearbrook. San Martin, F. and Bryant, F.C., 1989, Nutrition of domesticated South American llamas and alpacas, Small Rum. Res. 2:191–216. Sharp, P., Knight, T.W., and Hodgson, J., 1995, Grazing behaviour of alpacas and sheep, Proc. N.Z. Soc. Anim. Prod. 55:183–186. Standing Committee on Agriculture (SCA), 1990, Feeding Standards for Australian Livestock. Ruminants, CSIRO, East Melbourne.

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A COMPARISON OF THE EFFICACY OF PROPRIETARY PRODUCTS IN THE TREATMENT OF MOLYBDENUM INDUCED COPPER DEFICIENCY

N. R. Kendall, C. Middlemas, H. Maxwell, F. Birch, D. V. Illingworth, D. W. Jackson, and S. B. Telfer Centre for Animal Sciences Leeds Institute of Biotechnology and Agriculture School of Biology University of Leeds Leeds LS2 9JT, United Kingdom

1. INTRODUCTION Clinical copper deficiency has been described in ruminant animals with various visual symptoms being observed. One such visual symptom is the alteration of hair/wool pigmentation. The cause of clinical copper deficiency has until recently been defined as a lack of copper that is available for absorption into the animal, with the antagonistic interactions of iron/sulphur and molybdenum/sulphur acting to reduce the amount of “available” copper. However, work by Phillipo, Humphries, Atkinson, and Henderson (1987) has shown that clinical copper deficiency is due to a toxic effect of molybdenum rather than a lack of copper for metabolic function. Copper in excess of that required for essential metabolic function (<1 mg/kg DM, Zervas, 1983) is used as an agent to react with the toxic molybdenum/sulphur compounds (thiomolybdates) to render them inactive and harmless to metabolic function. The efficacy of copper supplementation of ruminant animals is the ability to correct the symptoms of clinical copper deficiency and should not be judged by the supplement’s ability to raise the copper content of the body. Liver copper and total blood copper are still used alone as indicators of copper status, these do not take any account of the correction of the symptoms of clinical copper deficiency. The experiment described here uses a visual symptom (alteration in wool pigmentation) as one of an extensive set of copper status indicators to assess the efficacy of a variety of propriety copper supplements. Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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2. MATERIALS AND METHODS Twelve hebridean lambs (black wooled) were housed in two pens of six. They were fed a concentrate diet low in copper (3 mg/kg), high in iron (l,217 mg/kg) and molybdenum (3.6 mg/kg) with moderate sulphur (2 g/kg) with ad libitum access to barley straw (3 mg Cu/kg, 997 mg Fe/kg, 0.7 mg Mo/kg and 1g S/kg). Additional sulphur (0.4 g/sheep day) and molybdenum (1.2 mg/sheep day) were added to the feed. The sheep were sheared at the start of the experiment and a patch approximately 10 cm × 10 cm kept trimmed to enable easy comparison of emerging wool colour to existing wool. Once the sheep were “copper deficient” and exhibiting white wool they received the ewe dose of one of five proprietary treatments or were maintained untreated as controls. The treatments were 2 ml of a veterinary surgeon prepared drench (40 mg Cu), 20 ml of Liquithrive (Agri-Lloyd) a copper chelate drench (80 mg Cu), a 33 g Cosecure (Telsol Ltd) soluble glass bolus (4.4 g Cu), a 4 g capsule of Copporal (Beecham) copper oxide needles (3.2 g Cu) and 2 ml of Cuvine (C-vet) copper heptonate injection i.m. (25 mg Cu). Copper status was assessed by plasma copper concentration (PICu), serum caeruloplasmin activity (CP), erythrocyte superoxide dismutase activity (SOD), trichloroacetic acid soluble copper in the plasma (TCA), serum amine oxidase activity (AMOX) and the ratio between CP and PlCu (CP/PlCu) using the methods of Mackenzie, Illingworth, Jackson, and Telfer (1997) and Mulryan and Mason (1992).

3. DISCUSSION OF RESULTS The control sheep were the sheep in each pen with the best copper status (judged by wool pigmentation) at time of treatment, and this has dulled the response of the treatments when compared to the controls. Wool colour especially illustrates this as in one of the control sheep the wool did not turn white due to alterations in pigmentation but in fact ceased growth altogether, became loose and fell out. The treatments were allocated in reverse order of expected duration of efficacy with the two oral liquids (drench and chelate) being administered to the next two highest copper status sheep, followed by the injection, needles and bolus. The plasma copper concentrations (Fig. 1) were in the normal range for most of the sheep after treatment, although the control sheep were not as low as may have been expected (normal to marginal range) based on assessing their clinical symptoms. The other sheep were occasionally classified as marginal except for one of the drench treated sheep after week 7, which was deficient. Three sheep (injection, chelate and needles) had plasma copper concentrations at levels where toxicity would be a concern The TCA soluble copper (Fig. 2) paints a different picture with the control and drench treated sheep having deficient amounts of copper when the TCA insoluble copper tetrathiomolybdate was precipitated off. The chelate was only able to increase the TCA soluble copper from marginal on three samplings and for one sheep only. The injection gave a good initial boost but was drifting back towards marginal as the experiment progressed. The needles gave a better response than did the injection which was sustained throughout for one of the two sheep. The bolus maintained an adequate TCA soluble copper concentration throughout the experiment. The TCA insoluble fractions illustrate how the different treatments are working. A high TCA insoluble copper fraction indicates a large amount of copper tetrathiomolybdate (Cu-TTM) present in the blood therefore suggesting any effective copper treatment to be by providing copper to react

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with absorbed TTM in the blood thus preventing the stripping of metabolic copper. A low TCA insoluble fraction indicates that there is little or no Cu-TTM in the blood due to either there being no uptake of TTM into the blood (i.e. by chelating copper to form CU-TTM in the rumen which is not absorbed by the sheep) or that there is no copper available in the blood (plasma amino acid copper chelate) to react with the absorbed TTM and hence the TTM will react with sources of metabolic copper and depress enzyme

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activities (illustrated by the CP/PlCu ratio) (Mackenzie et al., 1997). Figure 3 shows the TCA insoluble fractions, and it can be seen that both bolus treated sheep were maintaining low TCA insoluble fractions indicating a prevention of the uptake of free TTM into the blood. This effect was also shown by one of the needle treated sheep. The caeruloplasmin activities (Fig. 4) were mainly normal (>15 mg/dl) apart from one of the control and both drench treated sheep. The injection and needles gave the largest increase in CP. The bolus and injection maintained CP in the normal range throughout whilst the chelate and needles were both deficient in one sheep on one sampling. The CP/PlCu ratio (Fig. 5) can be used to give an indication of free tetrathiomolybdate in the blood. The ratio was lowest for the drench treated sheep and the controls. The chelate was variable with one of the sheep having a very poor response whilst the other showed a ratio response for most of the experimental period. The injection had the greatest immediate effect, however this effect was lost by week 5. The needles and bolus were the best treatments for this parameter, although the needles seemed to be very effective in one sheep and poorer in the other, losing the effect after week 5. Serum amine oxidase activities (Fig. 6) were low for the control sheep and the second chelate treated sheep. The superoxide dismutase activities (Fig. 7) have a longer half-life than the other parameters and reflect the copper status over the previous six weeks. In terms of SOD the needles gave the quickest response which was maintained until the end. The bolus and injection were lower initially although the response was sustained throughout. The chelate had a three week transitory response, whilst the drench only had a week response. The wool colour (Fig. 8), an analysis of a clinical symptom (alteration of wool pigmentation), was not maintained black by either the chelate or the drench. The bolus had the quickest response in wool colour, which was maintained throughout the rest of the trial, as was the injection and needle response. The control sheep that maintained black wool throughout had in fact ceased wool growth and this wool was loose, with bare patches occurring.

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The drench, chelate and needles primarily rely on the absorption of the copper in order to exert a beneficial effect. The absorbed copper (and copper from injection) then acts probably as plasma amino acid bound copper, allowing the TTM to strip copper from the amino acids in preference to stripping the copper from its metabolically active roles (copper dependant enzymes etc.). The longevity of response is dependant on the ability of the treatment to resupply copper to the plasma amino acids and hence release

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enough amino acid bound copper to counteract the free TTM. The needles also maintain longevity by the nature of the slow dissolution of the oxidised copper. The bolus works in a different manner, with the release of copper into the rumen which binds to the free TTM to form Cu-TTM which is not absorbed and is egested in the faeces, thus giving the animal a lowered copper requirement as there is little free TTM requiring detoxification in the blood of the animal.

CONCLUSIONS Over the 10-week duration of the trial, the bolus gave the best maintenance of copper status and correction of clinical copper deficiency by preventing uptake of free tetrathiomolybdate into the sheep. The needles and injection also corrected the clinical copper deficiency, with the injection increasing the plasma copper and caeruloplasmin more, whilst the needles were better when the CP/PlCu ratio and TCA insoluble copper were assessed. The chelate was better than the drench for copper status although neither could sustain the correction of the clinical deficiency.

REFERENCES Mulryan, G. and Mason, J. 1992. Assessment of liver copper status in cattle from plasma copper and plasma copper enzymes. Ann Rech Vét 23:233–238. Mackenzie, A.M., Illingworth, D.V., Jackson, D.W., and Telfer, S.B. 1997. A comparison of methods of assessing copper status in cattle, in: Trace Elements in Man and Animals—9: Proceedings of the Ninth International Symposium on Trace Elements in Man and Animals (P.W.F. Fischer, M.R. Abbé, K.A. Cockell, and R.S. Gibson, eds.) pp. 301–302, NRC Research Press, Ottawa, Canada.

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Phillipo, M., Humphries, W.R., Atkinson, T., and Henderson, G.D. 1987. The effect of dietary molybdenum and iron on copper status, puberty, fertility and oestrus cycle in cattle. Journal of Agricultural Science 109:321–336. Zervas, G.P. 1983. The prevention of copper deficiency in ruminants by means of soluble glass rumen bullets. Ph.D. Thesis, University of Leeds, UK.

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THE EFFECT OF A COPPER, COBALT, AND SELENIUM BOLUS ON SHEEP FROM THREE UPLAND SCOTTISH FARMS A. M. Mackenzie1, N. R. Kendall2, D. V. Illingworth2, D. W. Jackson2, I. M. Gill3, and S. B. Telfer2 1

Animal Science Research Centre Harper Adams University College Newport, Shropshire, TF10 8NB UK 2 Centre for Animal Sciences Leeds Institute of Biotechnology and Agriculture School of Biology University of Leeds LS2 9JT, UK 3 Thrums Veterinary Group 1 Morrison Street, Kirriemuir Angus DD8 5DB, UK

1. INTRODUCTION Effects of clinical trace element deficiencies, particularly copper, cobalt and selenium in sheep include decreased growth rates and increased mortality. Clinical copper deficiency in sheep results from complex interactions between copper, molybdenum iron, and sulphur. As with the interaction between copper, molybdenum and sulphur the interaction between copper, iron and sulphur forms a complex that prevents the absorption of copper. However, high levels of dietary iron have been shown to decrease plasma and liver copper concentrations, but not to induce clinical signs of copper deficiency (Humphries et al., 1983). Levels of trace elements in the herbage are influenced by soil pH, soil drainage and grass species and methods to improve Scottish hill pastures have resulted in a decrease in copper levels and an increase in molybdenum levels (Burridge et al., 1983). The use of a soluble copper, cobalt and selenium glass bolus has previously been shown to prevent these trace element deficiencies occurring (Telfer et al., 1985; Zervas et al., 1988). However, since these early reports, the method of bolus production has changed to a sinter method producing a copper, cobalt polyphosphate Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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glass bolus containing sodium selenite. The aim of this trial was to investigate the effects of this sintered bolus on the trace element status of wintered store lambs in the Scottish Highlands, an area known to be deficient in these minerals.

2. MATERIALS AND METHODS On each of three hill farms in the Angus region of Scotland, 70 gimmer lambs (year old females) were allocated to the two treatment groups of 35 lambs. One group was administered a single Cosecure© bolus (Telsol Ltd) on day 0 (Copper) and the other group was not bolused (Control). The boluses were 33 ± 3g and contained (w/w) 15.4% copper, 0.15% selenium and. 0.5% cobalt. Blood samples were collected by jugular venepuncture at day 0 (pre-bolusing) and on days 100, 119 and 121 for farms 1, 2 and 3 respectively. Trace element status was determined by methods stated by Mackenzie et al. (1997b). The copper parameters measured were: 1. plasma copper (Pl-Cu) by atomic absorption spectrophotometry, 2. serum caeruloplasmin (CP) activities, 3. erythrocyte superoxide dismutase (SOD), and 4. amine oxidase were all measured on a Cobas-Mira (Roche). 5. Copper status was determined by the ratio of caeruloplasmin activity to plasma copper concentration (CP/Pl-Cu) to give an indication of effects of thiomolybdate on the copper enzymes (Mackenzie et al., 1997a). Selenium status was measured by the activity of the erythrocyte glutathione peroxidase (GSHPx) on the Cobas-Mira and serum vitamin concentrations were measured to assess cobalt status (Kendall et al., 1997). Statistical analysis was carried out on each farm by analysis of varience with day 0 as a covariate using GLM on MINITAB 11.

3. RESULTS The bolused lambs showed a significant increase in serum vitamin concentration and erythrocyte GSHPx activity compared with the unbolused controls (p < 0.001) on day 100, 119 and 121 on farm 1, 2 and 3 respectively (Table 1). The bolused lambs on all farms had significantly increased plasma copper concentrations compared with the controls (p < 0.01 for farm 1 and p < 0.001 for farm 2 and 3). Caeruloplasmin activity was significantly higher in the bolused animals (p > 0.001) (Table 2). The activities of superoxide dismutase and amine oxidase were also signifi-

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cantly higher in the bolus group. The bolused lambs also had a significantly higher caeruloplasmin to plasma copper ratio compared with the control lambs.

4. DISCUSSION The results of this trial show that the soluble glass bolus significantly increased the glutathione peroxidase and serum vitamin concentration of wintered lambs. Measurement of erythrocyte GSHPx activity is widely used to assess selenium status in animals. The mean GSHPx activities in the lambs on two of the farms on day 0 (Farms 2 and 3) would classify them as deficient in selenium. On the second sample day farm 1 would also be classified as deficient and farm 3 may be marginal. Similar to the previous reports using the former bolus formulation (Zervas et al., 1988), the present work also shows that the bolus ensures an adequate supply of selenium for the lambs. The mean vitamin concentrations of the lambs prior to treatment (day 0) and in the control groups on the second sample date were all in the normal range indicating an adequate dietary supply of cobalt from the herbage. However, the bolus significantly increased vitamin concentrations in the treated lambs compared with the controls. The copper status, as measured by plasma copper, caeruloplasmin activity and the caeruloplasmin to plasma copper ratio were also significantly increased by the bolus. At the start of the trial (day 0), the mean copper status of all lambs would classify them as normal. However, on the second sample date, the control lambs on farms 1 and 3 had plasma copper concentrations and caeruloplasmin activities that would classify them as deficient. The bolus significantly increased all copper parameters on days 100, 119 and 121 for farms 1, 2 and 3 respectively.

CONCLUSIONS In conclusion, the sintered cobalt and copper status of the lambs.

bolus significantly increased the selenium,

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REFERENCES Burridge, J.C., Reith, J.W.S., and Berrow, M.L. (1983). Soil factors and treatments affecting trace elements in crops and herbage. In: Trace Elements in Animal Production and veterinary Practice (Suttle N.F., Gunn R.G., Allen W.M., Linklater K.A. and Wiener G. eds), pp 77–85, BSAP, Occasional Publication No. 7. Humphries, W.R., Phillippo, M., Young, B.W., and Bremner, I. (1983). The influence of dietary iron and molybdenum on copper metabolism in calves British Journal of Nutrition: 49, 77–86. Kendall, N.R., Mackenzie, A.M., and Telfer, S.B. (1997a). The effect of a soluble cobalt, selenium and zinc glass bolus on humoral immune response in lambs, in: Trace Elements in Man and Animals—9: Proceedings of the Ninth International Symposium on Trace Elements in Man and Animals, (Fischer P.W.F., Abbé M.R., Cockell K.A., and Gibson R.S. eds), pp 442–444, NRC Research Press, Ottawa, Canada. Mackenzie, A.M., Illingworth, D.V., Jackson, D.W., and Telfer, S.B. (1997a). The use of caeruloplasmin activities and plasma copper concentrations as indicators of copper status in ruminants In: Trace Elements in Man and Animal -9: Proceedings of the Ninth International Symposium on Trace Elements in Man and Animals, (Fischer P.W.F., L’Abbé M.R., Cockell K.A., and Gibson R.S. eds), pp 137–138, NRC Research Press, Ottawa, Canada. Mackenzie, A.M., Illingworth, D.V., Jackson, D.W., and Telfer, S.B. (1997b). A comparison of methods of assessing copper status in cattle. In: Trace Elements in Man and Animal—9: Proceedings of the Ninth International Symposium on Trace Elements in Man and Animals, (Fischer P.W.F., L’Abbé M.R., Cockell K.A., and Gibson R.S. eds), pp 301–302, NRC Research Press, Ottawa, Canada. Telfer, S.B., Illingworth, D.V., Anderson, P.J., Zervas, G., and Carlos, G. (1985). Effect of soluble-glass on copper, cobalt and selenium status of sheep. Biochemical Society Transactions: 13, 529. Zervas, G., Telfer, S.B., Carlos, G., and Anderson, P.J. (1988). The effect of soluble-glass boluses containing copper, cobalt and selenium on the blood composition of ewes. Animal Feed Science Technology: 21, 23–29.

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SELENIUM AND FLUORIDE TOXICOLOGY Water Quality Guidelines for Dohne Merino Sheep in Southern Africa

J. A. Meyer and N. H. Casey Department of Animal and Wildlife Sciences University of Pretoria Pretoria 0002, South Africa

1. INTRODUCTION The validity of water quality guidelines (WQG) for livestock watering in southern Africa was researched due to discrepancies reported between predicted effects and clinical observations. Typically WQG’s are static tabulated water quality constituents (WQC) with a single value cut-off concentration suggesting recommended allowable limits or maximum levels (Adelaar, 1974; Kempster et al., 1985; Marincowitz and Conradie, 1985; Smith, 1988; Borehole Water Association of Southern Africa, 1990; Ensminger et al., 1990; DWA&F, 1993; 1996; Casey et al., 1998; Meyer et al., 1997). These WQG’s are inadequate as site-specific factors impacting on ingestion, dose, physiology, and thus toxicology, are not catered for sufficiently. This leads to a wide range of possible effects at the same [mg/L]. Using sheep, cattle and goats, biological trials were conducted to model WQG’s on a site-specific constituent ingestion rate risk assessment basis, as mg to develop solutions for water not fit for use, and to provide accurate risk assessments. Groundwater is often the sole source of water in the semi-arid and arid regions of South Africa with over 300 towns and small settlements largely dependent on it (DWA&F, 1991; Parsons and Tredoux, 1993). South Africa is characterised with seasonal dry periods, droughts and nutritional imbalances. Emphasis is not to provide no risk judgements or no adverse effect guarantees, but rather management supported WQG’s allowing for more efficient usage. Groundwater quality investigations in South Africa found Se and F occurring at concentrations exceeding local and international WQG’s for animals, namely 0.05 mg Se/L and 2–6mgF/L. Table 1 provides some of the concentrations recorded. A F trial tested a potential alleviator, boron (B), mainly tested in monogastric animals (Wheeler Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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and Fell, 1983), in a ruminant model. A Se trial tested the validity of WQG’s over a period of growth from weaning to slaughter weight.

2. PROCEDURES Fluoride and Boron Alleviatory Trial Treatments commenced at lambing with F+B (20 mgF/L + 25 mgB/L), F (20 mg F/L), and C (F+B < 0.09 mg/L), each with 10 Dohne Merino lambs, tested under pen and pasture systems, receiving a pelleted feedlot ration (DM% > 90%), and Fescue and Kikuyu pastures, respectively. At market weight (ca. 4 months exposure) thyroid, liver and kidney samples were collected and prepared for histopathological examination as described by Williams (1990). Mineral content of these tissues and metacarpal samples was determined by the wet-ash method (atomic absorption spectrophotometer, model Varian SpectrAA). Macroscopic evaluation for skeletal and dental fluorosis was conducted monthly (Buck and Osweiler, 1988). Traditional fluoro metric methods used have been shown to be unreliable (Van Staden and Janse van Rensburg, 1991).

Selenium Growth Trial At weaning 30 Dohne Merino wethers were assigned to 0 mg Se/L, 0.1 mg Se/L and 1 mg Se/L treatments, housed in pens and offered a feedlot ration (ME = 10.18 MJ/kg). Treatments were administered daily (exposure 13 weeks) and chosen due to WQC results

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and reports that groundwater can contain up to 1 mg Se/L (Galvin, 1996). Sodium selenite AR-grade was used (Pehrson, 1993; Oldfield et al., 1994). Whole blood samples were taken by jugular venipuncture according to Van Ryssen et al. (1998), and relevant tissue histopathology was conducted.

3. RESULTS AND DISCUSSION Fluoride and Boron Alleviatory Trial Pen system final live weights were significantly (P < 0.05) heavier in F+B treated animals, compared to the F treatment. The F treatment was significantly (P < 0.05) heavier than that for C and F+B in the pasture system. Low DM% (<20%) of the pasture system reduced F ingestion allowing for osteoblast and osteoclast benefits in the F group. Lower pen system moisture % ration increased F intakes, reduced growth of the F group, and led to an alleviatory effect in the F+B group. Significant (P < 0.05) liver Ca results suggested lower Ca absorption in F treatment groups compared to F+B and C groups for both systems, with F+B groups returning values higher than their comparative F groups. Mineral results (notably Ca in bone, liver and kidneys) accorded with findings in monogastric animals by Elsair et al. (1980). The significantly lower renal [B] (P < 0.05) obtained for F and C treatments compared to the F+B treatment indicated the B dose to be sufficient, and considering tubular transport maximums, probably more than required. No clear histopathological lesions were observed. The pen system F group presented with mild enamel hypoplasia.

Selenium Growth Trial No significant (P < 0.05) differences for live weight were observed between or within subgroups of the various treatments. The results accord with Van Ryssen (1998) regarding plateaus reached after an initial linear relationship between Se intake and whole blood [Se], as the high Se treatment group (did not show a proportional dose increase compared to the low Se treatment. All treatments yeilded significantly different (P < 0.05) whole blood [Se], values in ng/g being 236.2, 386.62 and 445.16 for 0, 0.1 and 1 mg Se/L treatments respectively. No histological abnormalities were found in the sections reviewed.

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4. CONCLUSION Successful livestock production is dependent on the provision of water with a specific chemical composition. The aim of a WQG is to recommend what the composition should be, not just to prevent adverse effects, but to achieve optimal water utilisation. It is concluded that under the trial conditions experienced in this study, adverse effects due to the provision of F in the drinking water to a concentration of 20 mg/L from birth to slaughter weight, may be mitigated by use of B as an alleviator at a concentration of ca. 25 mg/L. Where water [F] is marginal in terms of being potentially hazardous, it is concluded that 25 mgB/L will not in itself result in any adverse effects and can thus be recommended for use over the production phase used. Based on growth, histopathological and whole blood [Se] results and reduced synergistic factors in the trial design, it is concluded that drinking water containing 0–1 mgSe/L with a maximum total ingestion of 3.884 mg Se/sheep/d and [Cu] of <0.001 mg/L, used for mutton production over the period of weaning to slaughter weight, could be recommended for use.

ACKNOWLEDGMENTS The authors acknowledge the Water Research Commission of South Africa, Onderstepoort Veterinary Institute of the Agricultural Research Council, and SoftyCompCc for their valuable contributions.

REFERENCES Adelaar, T.F., 1974, Veeartsnykunde Onderstepoort (Reuter and Gouws, 1974). Borehole Water Association of Southern Africa, 1990, Groundwater—Guidelines for Boreholes. Compiled by Borehole Water Association of Southern Africa, CSIR (Ematek), Department of Agricultural Development, Department of Water Affairs and the Geological Society of South Africa. CSIR (Ematek), Pretoria. 3–27. Casey, N.H., Meyer, J.A., and Coetzee, C.B., 1998, An investigation into the quality of water for livestock production with the emphasis on subterranean water and the development of a water quality guideline index system. Water Research Commission. Project No K5/644/1/98. ISBN No: 1 86845 739 0. Vol. 1:1–37. DWA&F, 1991, Department of Water Affairs and Forestry, Water Quality Management policies and strategies in the RSA. Pretoria. DWA&F, 1993, Department of Water Affairs and Forestry, South African Water Quality Guidelines (1st edn.) Agricultural Use: Livestock Watering. 67–134. DWA&F, 1996, Department of Water Affairs and Forestry, South African Water Quality Guidelines (2nd edn.) Vol. 4: Agricultural Use: Livestock Watering.

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Elsair, J., Merad, R., Denine, R., Reggabi, M., Alamir, B., Benali, S., Azzouz, M., and Khelfat, K., 1980, Boron as an antidote in acute and subacute fluoride intoxication in rabbits: its action on fluoride and calciumphosphorus metabolism. Fluoride. 13:129–138. Ensminger, M.E., Olfield, J.E., and Heinemann, W.W., 1990, Feeds and Nutrition, (2nd edn.). The Ensminger Publishing Co., California. 168–169. Galvin, R.F., 1996, Occurrence of metals in waters: An overview. Water SA. Vol. 22(1):16. Hart, B.U., Angehern-Bettinnazzi, C., Campbell, I.C., and Jones, M.J., 1992, Australian Water Quality Guidelines. Australian and New Zealand Environmental and Conservation Council. Draft for public comment. Kempster, P.L., Hattingh, W.H.J., and van Vliet, H.R., 1985, Summarized Water Quality Criteria. Dept of Agriculture and Water Affairs, Hydrological Research Institute. Technical Report TR 108. Marincowitz, G. and Conradie, W.J., 1985, Boorgatwater vir Veesuipings op Soutpanproefplaas. Department of Agriculture and Water Affairs, Transvaal Region. Agrivaal 7(4): 1–5. Meyer, J.A., Casey, N.H., and Coetzee, C.B., 1997, Water quality guidelines for livestock watering in Southern Africa. Water SA. Vol. 23(1):7—12. Oldfield, J.E., Burau, R., Moller, G., Ohlendorf, H.M., and Ulrey, D., 1994, Risks and benefits of selenium in agriculture. Issue paper supplement No 3, Council for Agricultural Science and Technology, Ames IA, USA. Parsons, R. and Tredoux, G., 1993, The Development of a Strategy to Monitor Groundwater Quality on a National Scale. Water Research Commission. Report No. 428/1/93. 4–22. Pehrson, B., 1993, Diseases and diffuse disorders related to selenium deficiences in ruminants. Norwegian J.Agric.Sci., Suppl. 11. 79. Ramsay, K.A., Reed, D.S., Bothma, A.J., and Lepen, M.J., 1994, Profitable and environmentally effective farming with early domesticated livestock in Southern Africa. Conservation of Early Domesticated Animals of Southern Africa. Conference held at Willem Prinsloo Agricultural Museum, 3–4 March, 1994. 105–136. Smith, R., 1988, Water Quality Criteria for Livestock Watering and Human Consumption. CSIR: Division of Water Technology. Project No. 670 2170 9. Van Ryssen, J.B.J., de Villiers, J.F., and Coertze, R.J., 1998, Supplementation of selenium to sheep grazing kikuyu or ryegrass. I. Selenium status of unsupplemented sheep and animal performance upon supplementation. S.Afr.J.Anim.Sci. (In press) Van Staden, J. and Janse van Rensburg, S.D., 1991, Improvement on the microdiffusion technique for the determination of ionic and ionisable fluoride in cows milk. Analyst 116:807–810. Wheeler, S.M. and Fell, L.R., 1983, Fluoride in Cattle Nutrition. Nutrition Abstracts and Reviews—Series B. Commonwealth Bureau of Nutrition. 53(12):741–766. Williams, M.C., 1990, The pathology of experimental Lasiospermum Bipinnatum (thunb.) druce poisoning in sheep. I. Hepatic lesions. Onderstepoort J.Vet. Res. 57:249.

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EFFECT OF DIETARY COPPER LEVEL AND HIGH SULFATE WATER ON COPPER METABOLISM AND GROWTH IN CATTLE

C. L. Wright, J. W. Spears, T. E. Engle, and T. A. Armstrong Department of Animal Science and Interdepartmental Nutrition Program North Carolina State University Raleigh, NC, 27695-7621 USA

1. INTRODUCTION Presence of high sulfate water is quite prevalent in certain regions of the United States, and this is of particular importance when dealing with the copper (Cu) nutriture of ruminant species. Previous research has defined a number of mechanisms by which Cu and sulfur may interact alone or in complexes with other minerals to decrease Cu availability in the ruminant. Early researchers observed that liver Cu concentrations were reduced in ruminants when diets were supplemented with molybdenum (Mo) and sulfur (S). Rumen microflora rapidly reduce dietary sulfate to sulfide, which may interact with molybdenum (Mo) forming thiomolybdates, which bind Cu. Copper and S may also interact independently of Mo forming CuS which decreases the bioavailability of Cu to the animal. Wittenburg and Boila (1988) observed lower plasma and liver Cu concentrations and decreased ceruloplasmin activity in steers when dietary S was raised from 1.3 to 3.7 mg/kg DM. Addition of CuS to the diet of hypocupremic sheep failed to increase plasma Cu, furthermore replacement of CuSO4 with CuS resulted in a decrease in plasma Cu (Suttle, 1974). Supplemental concentrations of Cu may overcome these detrimental interactions, however feeding high concentrations of Cu may induce metallothionein, thus sequestering the Cu (Bremner, 1987). This experiment was designed to determine the effects of supplemental Cu and high water on Cu metabolism and growth of cattle. Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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2. MATERIALS AND METHODS The experiment was divided into an 84 d depletion phase and 70 d repletion phase. In the depletion phase, twenty-nine Angus and Angus x Hereford heifers were blocked by weight and randomly assigned to one of two water treatment groups. One treatment received ad libitum access to water containing l,500 mg/L added as the remaining treatment received control water. All heifers were individually fed using electronic gates and were housed in covered pens with slat flooring. The diet for the depletion phase was a corn silage-soybean meal based diet that contained 6 mg Cu/kgDM and was supplemented with 1 mg Mo/kg DM. Liver biopsies were collected on d 0, 42 and 84. Plasma samples were collected on d 0, 28, 56 and 84. Rumen fluid was obtained via stomach tube on d 84. In the repletion phase, heifers continued to receive their appropriate water treatment from the depletion phase and were assigned to one of three dietary treatments. Dietary treatments included: 1) basal diet (Con), 2) basal diet + 10 mg supplemental Cu/kg as (10 Cu), and 3) basal diet + 100 mg supplemental Cu/kg as (100 Cu). The basal diet from d 0 through d 36 was a corn silage-soybean meal based diet that contained 6 mg Cu/kg DM. On d 37 the basal diet was changed to a high corn based diet that contained 5 mg Cu/kg DM. Supplemental Mo was removed from all diets in the repletion phase. Rumen fluid, liver biopsies and plasma samples were collected on d 0, 37 and 70. On d 70 all heifers were transported 320 km, held overnight and slaughtered. Final tissue samples and bile were collected at slaughter. Carcass data were collected 2d post-mortem to allow for adequate cooling and bloom of the carcass.

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3. RESULTS In the depletion phase, rumen soluble Cu concentration was decreased (P < 0.01) in heifers receiving high water. High water reduced liver Cu concentration (Table 1) on d 42 (P < 0.01) and d 84 (P < 0.01). High water had no effect on plasma Cu concentration. These data agree with previous literature which demonstrates the negative effects of the Cu: Mo: S interaction in the rumen on Cu status. High water depressed (P < 0.01) liver Cu concentration during the repletion phase. Liver Cu was increased (P < 0.01) in the 100 Cu treatment relative to the 10 Cu and Con treatments, and tended to be greater (P < 0.07) in the 10 Cu treatment relative to the Con treatment (Table 2). Copper supplementation increased (P < 0.05) liver Cu in heifers receiving control water and to a lesser extent in heifers receiving high water (Table 2). Rumen soluble Cu was increased (P < 0.01) in the 100 Cu treatment relative to 10 Cu and Con treatments (Table 3). These data demonstrate that supplementation of diets with high levels of inorganic Cu is an effective means to overcome the negative impact of high sulfate water on Cu stores. Copper supplementation decreased (P < 0.03) average daily feed intake. Feed intake from d 37 to d 70 was depressed (P < 0.04) in the 100 Cu treatment relative to 10 Cu and Con treatments. Neither dietary copper supplementation nor high water affected plasma ceruloplasmin activity, biliary Cu level, feed efficiency and average daily gain.

CONCLUSIONS High levels of supplemental inorganic Cu depress intake and increase hepatic Cu stores in the presence of high water. High water decreases liver and rumen soluble Cu concentrations.

REFERENCES Bremner, I. 1987. Involvement of metallothionein in the hepatic metabolism of copper. J. Nutr. 117:19–29. Bremner, I. and B.W. Young. 1978. Effects of dietary molybdenum and sulfur on the distribution of copper in plasma and kidneys of sheep. Br. J. Nutr. 39:325–336.

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Suttle, N.F. 1974. Effects of organic and sulfur on the availability of dietary copper to sheep. Br. J. Nutr. 32:559–568. Wittenburg, K.M. and R.J. Boila. 1988. Supplementary copper for growing cattle consuming diets high in molybdenum or molybdenum plus sulfur. Can. J. Anim. Sci. 68:1143–1154.

241

APPARENT TRACE ELEMENT ABSORPTION IN GROWING PIGS FED RATIONS OF INCREASING CALCIUM CARBONATE CONTENT

Torben Larsen, José A. Fernandéz, and Ricarda M. Engberg Danish Institute of Agricultural Sciences Research Centre Foulum P.O. Box 50, Tjele, Denmark

INTRODUCTION Fast growing production animals do have a calcium requirement higher than intrinsic content of normal feed stuffs. Calcium is consequently supplied to the rations to fulfil the demand for solid bone construction. Several studies show that interactions between minerals ands trace elements may take place in the intestine before absorption, resulting in uptake deviating from expected amounts. The present study investigated the effect of increasing supplementation of calcium carbonate to pigs feed. Apparent absorption of manganese, iron, zinc and copper were calculated in the animals and related to relevant parameters.

MATERIALS AND METHODS 30 female pigs (crossbred L*Y), consisting of 10 litters with three litter mates with an initial mean body weight of 20 kg were involved in the study. Litter mates were allocated to one of the experimental groups. Over a period of 62 days the experimental animals received a conventional diet based on barley, wheat and soybean meal. The basal diet was formulated to contain increasing amounts of calcium carbonate, i.e. 2.0 g/kg diet (low), 7.3 g/kg diet (recommended, medium), and 12.7 g/kg diet (high), respectively. No other ingredients were varied in the experiment. The animals were housed individually and fed twice a day, semi ad libitum, to avoid feed residues. Two separate faeces balances were carried out using stainless steel metabolism cages, i.e. d 20–d 29 and d 40–d 49,

Email: [email protected] Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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respectively. Apparent absorption of elements were calculated as intake minus faecal excretion.

RESULTS Feed intake, growth, and feed conversion ratios were not affected by the experimental conditions, Table 1. Nor was nitrogen retention affected by dietary calcium level. Apparent manganese, iron, zinc, and copper absorption is shown in Table 2, both at approximately 35 kg live weight (d 20–29 in experiment) and at approximately 60 kg live weight (day 40–49 in experiment). Mean value of manganese absorption was 6.2 mg/d and was apparently not affected by either the dietary calcium level or live weight of the animal. Iron absorption was on average 41 mg/d throughout the period. Apparent absorption ‘peaked’ at the medium calcium level (p < 0.05), iron uptake at 60 kg live weight was furthermore significantly enhanced compared to uptake at 35 kg live weight, 47.3 mg/d vs. 36.8 mg/d. Zinc absorption was significantly reduced at the low calcium dietary level (p < 0.05); apparent absorption moreover increased from 35 kg live weight to 60 kg live weight, 31.1 mg/d vs. 42.0 mg/d. Copper absorption, on the other hand, appeared significantly enhanced at the high dietary calcium intake (p < 0.001), i.e. 12 mg/d vs. 6 mg/d for the high Ca diet and medium Ca diet, respectively. Absorption pattern was consistent at both 35 kg and 60 kg live weight.

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DISCUSSION Nitrogen retention and mineral/trace element absorption have previously been shown to be closely associated (Larsen and Poulsen, 1996). In accordance with this, the present study found an increased trace element absorption at 60 kg live weigh compared to 35 kg live weight, because daily N retention (= lean body accretion) is greater at this stage of growth. On the other hand, average animal growth was not influenced by dietary calcium; but iron, zinc, and copper absorption was affected by the calcium addition at the intestinal level. Interactions between calcium and trace elements at the intestinal level have formerly been described. We have recently observed similar—although not entirely identical—effects between dietary calcium and trace element absorption in the pig (Larsen and Sandström, 1993; Larsen et al., 1999).

REFERENCES Larsen, T. and Sandström, B. Effect of dietary calcium level on mineral and trace element utilization from a rapeseed (Brassica napus L.) diet fed to ileum-fistulated pigs. Br J Nutr, 69:211–224. Larsen, T. and Poulsen, H.D. 1996. The relationship between mineral and nitrogen balances in growing pigs fed diets supplemented with zinc oxide. Can J Anim Sci, 76:409–415. Larsen, T., Skoglund, E., Sandberg, A.-S., and Engberg, R.M. 1999. Effect of soaking and pelleting of the diet on apparent absorption and retention of minerals in pigs. Submittet.

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HEAT PROCESSING OF PROTEIN SOURCES ON THE FAECAL EXCRETION OF THEIR SELENIUM IN DAIRY COWS

J. B. J. van Ryssen* and G. E. Schroeder** *University of Pretoria 0002 Pretoria, South Africa **IAPI, Irene, RSA

Natural selenium (Se) in many animal feedstuffs is present in the protein, largely as selenomethionine. Using the in situ bag technique, a high correlation was measured between the rumen degradability of protein and the disappearance of Se from the bags in the rumen (Van Ryssen et al., 1996, TEMA-9). Heat processing decreases the rumen degradability of proteins, thus increasing the quantity of their amino acids reaching the lower digestive tract. The amino acids will be available to the animal, if the heat treatment did not denature the protein. It was postulated that heat processing should affect the availability in the lower digestive tract of Se in protein sources, similar to its effect on availability of amino acids. Different plant and animal protein sources were evaluated. The plant proteins were processed at different temperatures for different time intervals. The animal protein sources were collected from commercial factories where different processes were used to dry the product. After incubating the feeds for 16 hours in small dacron bags in the rumens of dairy cows, followed by a pepsin digestion, the bags were placed into the duodenums of cows (the mobile bag technique) and collected when excreted in the faeces. Selenium analyses were performed on the feeds and residues in the bags. The proportion of the Se that remained in the bags relative to the original Se concentration in the feed and that entering the duodenum was calculated. Between 12.3 and 45.6% of the Se in lupin seed entering the duodenum, were excreted in the faeces. After heating cottonseed oilcake at 150°C for 40 min. (150/40), 24.6% of the Se was excreted compared to 75.5% in the 150/60 treatment. For sunflower oilcake, 27.5% of its Se was excreted when treated at 130/90 and 45.5% when treated at 150/60. Between 15 and 77% of the Se in blood meal were present in the bags when excreted in the faeces. Although the Se that disappeared from the bags is not necessarily available to the animal, it can be assumed that the Se remaining in the bag would have been unavailable to the animal. This suggests that heat exposure of protein sources during processing could have a significant effect on the bio-availability of the Se in protein sources.

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DIETARY ANTAGONISTS TO COPPER ABSORPTION Stable Isotope Measurements in Grazing Ruminants

J. Lee, N. D. Grace, J. R. Rounce, and S. O. Knowles New Zealand Pastoral Agr. Research Inst. PB 11008, Palmerston North New Zealand

A method using stable isotopes of Cu has been developed which allows Cu absorption to be estimated in sheep and cattle at pasture. With this technique, the impact of dietary S and Mo on Cu absorption in pastoral livestock can be quantified. The data will underpin a decision-support model of Cu metabolism used for managing Cu deficiency. Copper absorption in six Romney wethers, matched for similar initial Cu stores was estimated by measuring changes in the 65 Cu to 63 Cu ratio in liver and blood after intravenous administration of a single bolus of purified 65 Cu. A full feed balance study was carried out, and liver Cu pool size and turnover time were calculated. Average Cu absorption was 0.43 ± 0.08 mg Cu/day, which was 6.53 ±1.5 percent of total Cu intake. Net copper requirement was estimated to be 0.29 ± 0.03 mg Cu/day. Analytical procedures were validated by adding know, amounts of 65 Cu to liver tissue taken from non-experimental animals. Digestion, recovery, and overall experimental methodology were optimised prior to analysis of experimental samples. Sheep served as a model to develop the method, but current work in progress applying the tracer technique to cattle grazing pastures treated with Mo will also be reported.

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THE EFFECT OF A ZINC, COBALT, AND SELENIUM BOLUS ON RAM SEMEN QUALITY AND TRACE ELEMENT STATUS

N. R. Kendall, A. Green, S. McMullen, and R. G. Rodway Centre for Animal Sciences Leeds Institute of Biotechnology and Agriculture School of Biology University of Leeds Leeds LS2 9JT, United Kingdom

Thirty-three ram lambs at pasture with access to quality grass silage and a barley/pea concentrate were split into two groups by restricted randomisation of liveweight. On day 0, one group (n = 17) was bolused with a zinc, cobalt and selenium soluble glass bolus (Zincosel©, Telsol Ltd) with the other group unbolused as controls (n = 16). The rams were blood sampled on days 0, 23, 44, 65 and 86. Plasma zinc concentrations (PlZn) and erythrocyte glutathione peroxidase activities (eGSHPx) were analysed (Kendall et al., 1999). Collection of semen samples by natural mount and diversion into an artificial vagina were attempted from all rams on days 44, 52, 58, 65, 73, 79 and 86. Semen samples were assessed for ejaculate volume (Vol.), spermatocrit (Sc.), total sperm count (Count), motility (Mot), proportion of live sperm (Live) (negrosin-eosin stain) and abnormalities (total, coiled tail, no tail, two tail, tail droplets and others). The functional integrity of the sperm’s plasma membrane was assessed using the hypoosmotic swelling test (HOS) (Correa et al., 1997). Seminal plasma zinc concentration (spZinc) and glutathione peroxidase activity (spGSHPx) were analysed using the same assays as for the blood. Statistical analysis was carried out by ANOVA using GLM on MINITAB 11 (blood parameters used day 0 as a covariate, proportionality data (Sc., Live, HOS, Mot) used the arcsine transformation and abnormality data used the square root transformation with zero value adjustment. Seven of the control and five of the bolused rams failed to produce a valid semen sample on any of the seven semen collections. The bolused rams had significantly higher erythrocyte glutathione peroxidase activities than the control rams throughout the post bolusing period (p < 0.001). The groups did not have significant differences in plasma zinc concentrations. MOT (d 44,73), HOS (d 73) and Live (d 73,79) were all significantly higher for the bolus group. Count (d 65) and spZinc (d 73) were significantly higher 769

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(p < 0.05) for the control group. There were no significant differences in Sc, Vol., spGSHPx, total abnormalities or any of the individual abnormalities. The selenium status was significantly increased in the bolused rams, however, all rams remained adequate (>40 U/m1 PCV) throughout. Apart from day 0, PlZn was maintained above for both groups. The increased Mot, Live and HOS were probably due to the increased spGSHPx, which although not significant was consistently higher for the bolused group (6 of 7 samplings), this is in agreement with findings of Vézina et al. (1996) in humans. In conclusion, the bolus significantly increased the selenium status of the rams resulting in increased sperm motility, % live sperm and sperm responding to the hypoosmotic swelling test.

REFERENCES Correa, J.R., Pace, M.M., and Zavos, P.M., 1997, Relationships among frozen-thawed sperm characteristics assessed via the routine semen analysis, sperm functional tests and fertility of bulls in an artificial insemination, Theriogenology 48:721–731. Kendall, N.R., Middlemas, C., Maxwell, H., Birch, F., Illingworth, D.V., Jackson, D.W., and Telfer, S.B., 1999, A comparison of the efficacy of proprietary products in the treatment of molybdenum induced copper deficiency. Proceedings of TEMA-10, in press. Vézina, D., Mauffette, F., Roberts, K.D., and Bleau, G., 1996, Selenium-vitamin E supplementation in infertile men—effects on semen parameters and micronutrient levels and distribution, Biological Trace Element Research, 53:65–83.

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THE EFFECT OF LEAD ON THE RATE OF FERMENTATION OF HERBAGE BY RUMEN MICRO-ORGANISMS

S. T. Strojan and C. J. C. Phillips University of Cambridge Department of Clinical Veterinary Medicine Cambridge, CB3 OES

INTRODUCTION Lead from car exhausts persists on roadside verges, and ruminants grazing close to a busy road are likely to consume contaminated herbage. However, cattle can detect lead acetate sprayed onto herbage when it is at concentrations of at least 170 mg Pb/kg DM and have been observed to avoid grazing pasture with 304 and 462 mg Pb/kg DM (Strojan and Phillips, 1997). Herbage 7.5m from a busy dual carriageway has been recorded with 454 mg Pb/kg DM (Motto et al., 1970). The avoidance of lead-contaminated herbage in our previous experiment may be the result of low digestibility of the lead-contaminated herbage, so an experiment was conducted with samples of herbage from the experiment to determine the effects of the lead on rumen bacteria.

MATERIALS AND METHODS Using 15 and 10 replicates/treatment from the conditioning period (Experiment 1) and the grazing preference test (Experiment 2) respectively that are referred to above, 200 mg DM of each herbage sample was incubated in sheep rumen liquor in syringe pipettes, following the technique of Menke and Steingass (1988). Gas production was recorded at intervals of 0, 2, 4, 6, 8, 10, 12, 16, 24, 32 and 48 hours. Sigmoidal curves, y = a0 + a1/(1 + exp(–(x – a2)/a3)), were fitted using SlideWrite Plus, and the total gas production (a0 + a1) and the inflexion point of x (a2) were compared statistically using anova, except a2 in experiment 2, which was analysed by the Mood’s median test as it was not normally distributed. 772

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RESULTS Total gas production was reduced by lead in experiment 1 (132 and 102 1/kg DM, SED for 2.5 and 347 mg Pb/kg DM respectively) but not in experiment 2 (165, 155 and 1481/kg DM, SED 8.4 for 10, 304 and 462 mg Pb/kg DM respectively). The absence of effect in experiment 2 may be due to the high baseline level of lead. However, the point of inflexion of x was later when herbage was contaminated with lead in Experiment 2 (–13, 18 and for 10, 304 and 462 mg Pb/kg DM respectively). In Experiment 1 it was not affected by lead (0.9 and 1.6 h, SED 0.90 for 2.5 and 347 mg Pb/kg DM respectively). Thus in experiment 2 lead reduced the bacterial growth rate but not total gas production.

CONCLUSION Contamination of herbage by lead reduces its rate of fermentation by rumen microorganisms, which may provide feedback to enable cattle to avoid herbage with lead.

REFERENCES Strojan, S.T. and Phillips, C.J.C. 1997. The detection and avoidance of lead contaminated pasture by cattle. Proceedings of the British Society of Animal Science, 1997, p. 148. Menke, K.H. and Steingass, H. 1988. Estimation of the energetic feed value obtained from chemical analysis and in vitro gas production using rumen fluid., Animal Research and Development 28:7. Motto, H.L., Daines, R.H., Chilko, D.M., and Motto, C.K. 1970. Lead in soils and plants: its relationship to traffic volume and proximity to highways. Environmental Science and Technology 4:232.

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EFFECTS OF INTRARUMINAL MONENSIN AND SELENIUM CAPSULES ON GLUTATHION PEROXIDASE, CALCAEMIA, PHOSPHATEMIA AND LIVE WEIGHT GAIN IN HEIFERS ON PASTURE WITH SOLANUM MALACOXYLON

Bruna E. Ruksan, Laura Marangunich, and L. Barry Lowe* Instituto de Patobiología CICV, INTA, cc 77, CP 1708, Morón Bs. As. Argentina *Elanco Animal Health 112 Warf Rd. West Ryde NSW Australia

The ingestion of Solanum malacoxylon, Sendtner, (SM) has been identified as the cause of hipercalcaemia, hiperphosphatemia, soft tissue mineralization with loss of weight known as “Enteque Seco”. The cattle from areas where it occurs have subclinical selenium (Se) deficiency. Monensin inhibits the multiplication of protozoo and certain bacteria in the rumen, thus reducing fermentation and production of gas and viscosity that may cause bloat. The purpose of this study was to correct the Se deficiency and low weight gain in heifers with intraruminal controlled release capsule containing hexaglycerol distearate core containing monensin, monensin Se and Se injectios in heifers grazing the low area with improve pasture contaminated with Solanum malacoxylon. One hundred Aberdeen Angus heifers of 277.4 ± 20.0 Kg/animal were allocated to one of the four treatments, Group I control, Group II monensin capsule, Group III monensin-Se capsule and Group IV Se injection. Animals were weighed at the beginning of the trial and blood samples obtained from 10 animals/group and repeated during the experimental trial in order to evaluate the changes in live weight and biochemical parameters. Comparisons of profiles were done by repeated measures analyses of variance with the Green House-Grisser corrections on levels that were significant. At the end of the study the increase in live weight gain was higher in heifers from Group IIII treated with monensinSe 52.7(a) versus 47.6 (a,b) for Group II, 42.1 (b) Group IV and 40.1 (b) Kg/animal Group I. The activity of glutathion peroxidase increased from 11.4 to 48.9 ± 13.6, and 47.1 ± 14.1 U/g Hb in the animals from Groups III and 18.5 ± 4.9 and 17.1 ± 5.5 U/g Hb 775

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in Group IV and maintained in the same levels in the Group I and II. There where no diferences amont treatments for hemoglobine, copper and zinc concentration. But the concentrations of calcium, phosphorous and their product after thirty days of treatments, where as follow: calcium concentration 9.79 (a), 10.4 (a), 11.4 (b) and 12.6 (c); phosphorus concentration 6.4 (a), 7.3 (b), 7.1 (ab) and 7.0 (ab) and the product CaxP 63.0 (a), 75.2 (b, 81.1(b) and 90.5(c) for the animals with monensin-Se capsules, monensin capsules Se injection and control respectively. From the results obteining it is possible to see significant decrease in calcium (p < 0.01) and phosphorous (p < 0.06) concentrations in serun and their products (p < 0.01) in the animals treated with monensin-Se capsules. On the bases of previous results where Se increased the number of neutrophils and prevented soft tissues calcifications in sheep doseged with SM, may be by the action of glutation peroxidase system on free radicals and/or selenio-protein P an extracelular oxidant defense. Monensin by inhibition protozoan and certain bacteria in the rumen inhibits the formation of 1, 24, 25, (OH)3D3 and/or pontetiate the convertions to 19-nor-10 keto forms, wich posible represents a detoxication mechanism. Preleminary findings appear to indicate that Se and monensin may prevent “Enteque Seco” and more research is warranted.

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TRACE ELEMENT CONTENT IN NATURAL AND COMMERCIAL CAT’S FOOD AND TRACE ELEMENT STATUS OF CATS DEPENDING ON SEX AND DISEASE S. Anke, H. Gürtler, and M. Anke1 Leipzig University Veterinary-Medical Faculty Semmelweisstr. 4, D-04103 Leipzig 1 Friedrich Schiller University Jena Institute of Nutrition and Environment Dornburger Str. 24, D-07743 Jena Germany

The natural nutrition of the European domestic cat mainly consists of different species of real mice and voles. 45 animals of five different species from 5 different habitats were caught and analyzed. Furthermore, 12 commercially produced cat’s foods were analyzed in sixfold repetition (Table 1). Since the Zn requirement of cats is 50 mg/kg ration dry matter, this requirement is always met by the consumption of mice or commercially produced cat’s foods. The Mn requirement of cats was not determined experimentally but derived from other species. It is indicated with 0.1 mg/kg cat. Although some commercially produced cat’s foods do not contain this Mn amount, Mn deficiency symptoms did not occur in animals fed on them. Mice deliver sufficient Mn to cats, even during pregnancy and lactation. Most of the commercially produced cat’s foods deliver many times more Mn than the natural Mn offer. The surplus is reached by Mn supplementation which is not necessary to such an extent. The titanium content in many commercially produced cat’s foods is also increased by the supplementation with titanium oxide in order to brighten the dark colour of the meat. This Ti offer is not dangerous, but not necessary. The Cd status of mice is determined by their Cd offer and can be extremely abundant. It varied between 0.6 and The latter value is extremely high and can be damaging to health. The animals fed on commercially produced cat’s foods live less dangerous. 777

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The mean Zn content in the organs of healthy cats increased in the order of cerebrum (63 mg + 6.8 mg/kg dry matter), kidneys (65 + 11 mg/kg dry matter), femur (91 + 17mg/kg dry matter), liver (105 + 29 mg/kg dry matter), ribs (109 + 14 mg/kg dry matter) and top hair (201 + 47mg/kg dry matter). The cerebrum, kidneys and liver of animals suffering from feline infectious peritonitis and the kidneys of cats with leucosis contained significantly more Zn than those of healthy animals. The Zn content of ribs and femur, of ribs and liver and of ribs and hair correlated significantly. Liver, top hair, femur and ribs can be used as indicator organs for the determination of the zinc status of cats.

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VALIDATION AND NORMAL RANGES OF PLASMA CERULOPLASMIN CONCENTRATION IN CATS AND DOGS

C. J. Charlton, S. L. Wattam, and N. D. Skinner WALTHAM Centre for Pet Nutrition Waltham-on-the-Wolds Melton Mowbray Leicestershire, LE14 4RT UK

The cuproenzyme, ceruloplasmin has a number of functional roles within the body. As a copper storage protein, ceruloplasmin helps protect against the catalytic ability of free copper ions. Free copper can accelerate autoxidation reactions through singleelectron (radical) transfer, as well as react with hydrogen peroxide to form highly reactive hydroxyl radicals which can lead to cellular disruption. At times of trauma, ceruloplasmin also promotes the conversion of iron from its pro-oxidant ferrous form to ferric iron. As an acute phase protein, ceruloplasmin can be indicative of inflammation or infection and thus be used in conjunction with serum ferritin as a measure of iron stores. A colorimetric method to determine ceruloplasmin oxidase activity in cats and dogs was validated based on the method of Sunderman et al. (1970) using p-phenylenediamine as the substrate. Two dog and two cat serum samples were analysed ten times within a single run. Intra-assay coefficients of variation of 1.94% and 2.95% were determined for the dog samples, and 1.81% and 3.49% for the cat. Analysing the same samples on ten separate days, inter-assay coefficients of variation of 8.21% and 7.01% for dog, 6.88% and 9.35% for cat samples were determined. Hence an acceptable level of intra- and interassay variability was achieved. Following this, the difference between the ceruloplasmin concentration of serum and plasma samples was evaluated. No significant difference was determined between plasma and serum samples for either species. Hence thereafter plasma samples were collected in order to reduce the total volume of sample required. In order to establish normal ranges, plasma samples were obtained from 102 healthy dogs (mixed breed, age and sex) and 54 healthy domestic short-haired cats (mixed age and sex) from the WALTHAM Centre for Pet Nutrition. The mean plasma ceruloplasmin concentrations determined for dog and cat were 9.28 IU/L (SD 3.03 IU/L) and 10.90 IU/L (SD 3.34 IU/L) respectively. Using these values, normal ranges (mean ± 2SD) of 779

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3.22 IU/L to 15.35 IU/L for dogs, and 4.22 IU/L to 17.58 IU/L for cats were established. This normal range determined for dogs is consistent with that previously reported (Solter et al., 1991). A normal range for cats has not been previously established, however its similarity to that of the dog suggests that there is little difference in circulating ceruloplasmin levels between these two species. The normal ranges established during this study may be used to evaluate the health status of dogs and cats.

REFERENCES 1. Sunderman, F.W. and Nomoto, S. (1970) Clin Chem 16, 903. 2. Solter, P.F. et al. (1991) Am J Vet Res 52, 1738.

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CURRENT AAFCO AND NRC RECOMMENDATIONS FOR SELENIUM ARE TOO LOW FOR PUPPIES

K. J. Wedekind, J. DeBraekeleer, and G. F. Combs Hill’s Pet Nutrition, Inc. Topeka, KS, USA 66601 and Division of Nutritional Sciences Cornell University, Ithaca, NY USA 14853

The purpose of this study was to determine the selenium (Se) requirement in dogs. Current recommendations are based on extrapolation from other species (0.11mg/kg diet) without concern for bioavailability from ingredients unique to petfoods. Previously we have shown low bioavailability of Se in petfoods (30% average in canned diets; 50% average in extruded diets). Thus, we felt it was critical to determine experimentally the Se requirement in dogs in order to assure Se adequacy in canine petfoods. Thirty beagle puppies (8 wk old) were utilized in a randomized complete block design (RCBD) with age, litter and gender used as blocking criteria. Puppies were depleted of Se by feeding a low Se (0.014mg/kg Se) torula yeast/dextrose based diet for 14 d after which this same diet was supplemented with 5 levels of for 21 d to construct a standard curve (0, 0.05, 0.10, 0.15, and 0.20mg/kg Se). Response parameters measured included Se concentrations and Se-dependent glutathione peroxidase activities [GSHpx] in serum as well as serum total and serum total No significant changes in feed intake, weight gain or clinical signs were observed. However, a broken-line, two-slope response in serum Se occurred with a breakpoint at 0.06mg/kg. increased linearly, whereas decreased linearly in response to supplemental Se. The application of a bioavailability factor for Se to the serum Se breakpoint (e.g., 0.06/0.3 = 0.2), suggested an optimal value of 0.2mg/kg Se for canine diets. Thus, it is our recommendation that the diets for growing dogs contain a minimum of 0.2mgSe/kg diet.

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THE EFFECT OF A ZINC, COBALT, AND SELENIUM BOLUS ON SHEEP FROM NINE UPLAND SCOTTISH FARMS N. R. Kendall1, A. M. Mackenzie2, D. V. Illingworth1, D. W. Jackson1, I. M. Gill3, and S. B. Telfer1 1

Centre for Animal Sciences Leeds Institute of Biotechnology and Agriculture School of Biology University of Leeds Leeds LS2 9JT, United Kingdom 2 Harper Adams Animal Science Research Centre Harper Adams University College Newport, Shropshire, TF10 8NB UK 3 Thrums Veterinary Group 1 Morrison Street, Kirriemuir Angus DD8 5DB, UK

Trace element supplementation of upland grazing sheep can prove difficult, especially as supplementing with in-feed mineral requires feeding of these extensively produced animals. A convenient method of supplementation for sheep would be to provide trace element supplements in the form of a long lasting intra-ruminal bolus. A bolus has been developed to supply zinc, cobalt and selenium to sheep (Zincosel©, Telsol Ltd). The trials reported here investigate the use of this bolus in nine different flocks of overwintered gimmer sheep in the Kirriemuir area of Scotland. Six flocks had fifty sheep bolused and fifty left unbolused as controls whilst the other three flocks had thirty five bolused and thirty five controls. The sheep were blood sampled prior to bolusing and then resampled between 100 and 133 days later. The blood samples were analysed for zinc status (plasma zinc concentration (PlZn)), cobalt status (serum vitamin B12 concentration (B12)), selenium status (erythrocyte glutathione peroxidase activity (GSHPx)) and copper status (serum Caeruloplasmin activity (CP), plasma copper concentration (PlCu), T.C.A. copper concentration (TCA), erythrocyte superoxide dismutase activity (SOD) and the calculated ratio between the CP and PlCu (CP/PlCu)), using the methods of Mackenzie et al. (1997). 782

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All nine flocks showed a significant response to the selenium content of the bolus with increased GSHPx. Six of the flocks could be classed as being selenium deficient, one as marginal and two as adequate from an assessment of the control groups. In seven of the nine flocks both the plasma zinc concentrations and cobalt status were significantly higher for the bolused sheep. However, in one of the two farms not showing a zinc or cobalt response the second sampling was at 133 days after bolusing. The other flock not exhibiting a response was sampled at 119 days after bolusing but was adequate in cobalt status, although these sheep had also lost an average of 3 kg per head over the trial period. The data from these flocks therefore indicates that the plasma zinc and vitamin B12 falls away between 119 and 133 days (4 months) and this is likely to be the time at which this bolus formulation has dissolved away. A selenium response is found to continue beyond the physical lifetime of the bolus due to the re-utilisation of the selenium in the body. There was no depression in copper status in any of the flocks. In conclusion the zinc, cobalt and selenium soluble glass bolus was able to supply these trace elements to extensively grazed animals for a period of 4 months.

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FIELD TRIALS OF A COPPER, COBALT, AND SELENIUM SOLUBLE GLASS BOLUS N. R. Kendall, A. M. Mackenzie1, and S. B. Telfer Centre for Animal Sciences Leeds Institute of Biotechnology and Agriculture School of Biology University of Leeds Leeds LS2 9JT, United Kingdom

Three field trials were carried out to evaluate the performance of a sintered soluble glass copper, cobalt and selenium bolus (Cosecure®, Telsol Ltd) for maintaining adequate levels of the three trace elements. The first trial used 34 growing lambs, trial 2 used 36 growing lambs whilst trial 3 used fifty gimmer sheep. In each trial, the sheep were split into two groups by restricted randomisation according to day 0 live weight and one group bolused and the other group left untreated. Blood samples were taken at day 0 immediately prior to bolusing and then at regular intervals throughout each trial. The samples were analysed for copper status (serum caeruloplasmin activity (CP), plasma copper concentration (PlCu), erythrocyte superoxide dismutase activity (SOD) and the ratio between the CP and PlCu (CP/PlCu)), cobalt status (serum vitamin B12 concentration (B12)) and selenium status (erythrocyte glutathione peroxidase activity (GSHPx)) using the methods of Mackenzie et al. (1997). Liver samples were also collected from the first trial when lambs were slaughtered at day 86 and 121 and these were analysed for their copper and zinc concentrations. Statistical analysis was carried out using ANOVA with day 0 as a covariate (where applicable) using GLM on MINITAB 11. Trial 1 blood samples were taken on days 20, 42 and 63 and at all three samplings there were significant increases in both the cobalt and selenium status (B12 and GSHPx) (p < 0.001), but there were no other significant differences in any other blood parameter. There was a significant increase in liver copper concentration for the bolused lambs irrespective of slaughter date (p < 0.001). The second trial gave similar results for four samplings (28, 51, 69, 91 days) with there being significant increases in both selenium and cobalt status (p < 0.001). In

1 Current address: Harper Adams Animal Science Research Centre, Harper Adams University College, Newport, Shropshire, TF10 8NB, UK

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trial three the selenium status was significantly increased at all three samplings (21, 51, 105 days), although this was only at p < 0.05 for day 21 and at p < 0.001 for the 51 and 105 samples. The cobalt status was significantly increased (p < 0.001) for days 21 and 51. The day 105 vitamin B12 was not statistically significant although the concentrations were still higher in the treated group even though the controls had an adequate cobalt status. The copper status of all of the sheep in all three trials was adequate whether they had been bolused or left untreated. The bolus was able to correct deficiencies of selenium and cobalt consistently throughout these trials. The bolus had little measured effect on the already adequate copper status, although the liver copper concentrations of the bolused sheep were higher in the trial in which they were analysed.

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EVALUATION OF THE TRACE MINERAL STATUS OF LACTATING DAIRY CATTLE IN TRANS NZOIA DISTRICT WESTERN KENYA

F. D. O. Oduor, I. O. Jumba, and S. O. Wandiga Department of Chemistry University of Nairobi P.O. Box 3019, Nairobi, Kenya

A study was conducted to determine the micromineral nutrition status of lactating dairy cattle in three regions in Trans Nzoia District of Western Kenya. Soil, forage and serum samples were collected from a total of twelve different farms. The farms in each region were categorized into those that fed common salt or iodized salt and those that fed complete mineral supplements, respectively. Sampling was done at the end of the dry season (April 1994) and at the end of rainy season (October 1994). Except for Cu concentrations in serum, Fe, Mn and Zn concentrations in soil, forage and serum were affected by season (P < 0.05). although Soil, Fe, Mn and Cu showed no deficiencies at the end of both seasons, percentage deficiencies for Zn were higher at the end of the dry season. Forage Cu and Zn concentrations were higher at the end of the dry season, however, the percentage deficiencies were higher at the end of the rainy season, with forage Cu having 100% deficiency for all the three regions. Significant differences (P < 0.05) were observed between regional trace mineral concentrations. Serum trace mineral concentrations were higher (P < 0.05) for the mineral unsupplemented farms at the end of the rainy season, however, at the end of the dry season Cu and Zn concentrations were higher for the mineral supplemented farms. Trace minerals most likely to be deficient at least in one season are Cu and probably Zn in most areas. Based on forage and serum analysis, it was concluded that Cu was the most deficient trace element. This plus Zn should be continually supplemented in Trans Nzoia District. There was no direct relationship between soil and trace mineral concentrations, however mineral imbalance problems in lactating cattle could be predicted from forage and serum analyses. Occurrences of microelement toxicity are not likely in any of the regions.

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CANINE FERRITIN Assay Validation and Normal Range for Serum

N. D. Skinner, C. J. Charlton, and S. L. Ebbrell WALTHAM Centre for Pet Nutrition Waltham-on-the-Wolds, Melton Mowbray Leicestershire, LE14 4RT. UK

Ferritin plays an important role in the antioxidant defence system within the body. As a high affinity storage protein for iron, ferritin maintains iron in a safely bound form preventing the reactive ferrous ion from participating in Fenton reactions, which can lead to oxidative damage. In normal health ferritin, a species-specific protein, is found in the blood at concentrations that reflect body iron stores and in conjunction with other parameters can be used to assess in vivo iron status. In order to determine circulating ferritin levels in the dog, an enzyme-linked immunoassay (ELISA) was developed, adapted from the method of Weeks et al. (1988) using monoclonal antibodies. Following the assay validation, a normal range for dogs was established. In the range of 0–40ng/ml the ferritin standards were linear (least squares regression analysis, r = 0.997) and the recovery of purified ferritin added to canine sera was 97.7%. The intra-assay coefficient of variations derived from determining the ferritin concentration in two serum samples 12 times were 8.2% and 6.6%. The inter-assay coefficient of variations of two serum samples assayed 10 times on separate days were 16.6% and 16.2%. Serum samples were obtained from 96 healthy dogs from the WALTHAM Centre for Pet Nutrition of mixed sex and five different breeds for ferritin determination in order to establish a normal range. Each sample was assayed either in triplicate or quadruplicate and the mean of these values was used as the ferritin concentration for that sample. The serum ferritin concentrations varied from 67.20 to 621.07 with a mean value of 371.62ng/ml (SD 102.85ng/ml). The data was normally distributed. These results demonstrate that serum ferritin can be determined with good repeatability and reproducibility 787

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for dogs. The values obtained will be used as a normal range for future studies and may provide a useful method of determining iron storage levels in dogs.

REFERENCE Weeks, B.R. et al. (1988) Am J Vet Res 49, 1193.

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IRON IN INFANCY Absorption, Erythrocyte Incorporation, Loss

Robert E. Serfass Division of Human Nutrition Department of Preventive Medicine and Community Health University of Texas Medical Branch Room 3.102 Ewing Hall 700 Harborside Dr.; Galveston TX, 77555-1109 USA

Iron deficiency is the most commonly recognized nutritional deficiency worldwide, estimated to affect more than 2 billion people. In the U.S.A., iron deficiency anemia is still a common nutritional problem in all age groups, with prevalence near 10% in 12-month-old infants, higher than any other age-sex group except pregnant women. Evidence is accumulating for a relationship between iron deficiency anemia in infancy and subsequent, irreversible deficits in psychomotor and cognitive development (Pollitt, 1999). The major factor affecting iron requirements during infancy is rate of growth. Infants have higher iron requirements per unit of body weight than other age groups. Requirement for absorbed iron are higher for babies with lower birth weights, hence more rapid growth rates (Fomon, 1993). The most critical period for term infants is during weaning between 4 and 12 months of age. Unless there is a substantial increase in whole body iron during this interval, the rapid expansion of hemoglobin mass will deplete body iron stores (Dallman, 1986). In addition to requirements for absorbed iron to support growth, iron losses must be replaced. Occult gastrointestinal blood loss is medically significant in a sizable minority of younger infants fed cow milk. Smith and Hunter (1970) observed remarkable differences between indices of iron status in groups of 21–24 infants fed infant formula (with or without added iron) or cow milk from birth to 18 months of age. Iron intakes met recommendations of 1 mg per kilogram of body weight per day in the groups fed

Telephone: 409-747-4575; fax: 409-772-6287; email: [email protected] Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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cow milk or unfortified infant formula and were over 2mg per kilogram per day in the group fed iron-fortified infant formula. Plots of the time course of concentration of serum iron, blood hemoglobin, and of transferrin saturation over the eighteen months showed that the means for the groups began to differ at 6 months of age. At eighteen months of age, 22 of the 24 infants in the cow-milk fed group were anemic or sideropenic, and the benefits of iron fortification of infant formula were demonstrated. However, explanations for the adverse effects of cow milk were not addressed. Subsequent investigators established that the presence of hemoglobin in stools of infants fed cow milk is common, but the amounts of lost hemoglobin were not quantified. A more recent study determined blood lost in stool to be medically significant in 20% of the infants fed cow milk (Ziegler et al., 1990). From 168 through 252 days of age, 26 infants were fed cow milk and 26 infants were fed a low-iron milk-based formula. Quantification of fecal hemoglobin demonstrated that ten of the cow-milk fed infants showed appreciably higher fecal hemoglobin concentrations than the others. One of these infants had such massive hemoglobin loss that anemia developed within 4 weeks and necessitated withdrawal of the infant from the study. Five of the 26 cow-milk fed infants had daily fecal iron loss from hemoglobin in excess of 0.1 mg per day, an amount considered medically significant by comparison with the estimated requirements for absorbed iron of 0.55 to 0.7 mg per day (Fomon, 1993). The Institute of Medicine (1993) guidelines for preventing iron deficiency in term infants and children, based on the foregoing results, can be summarized: 1) Infants should be breast-fed or fed iron-fortified infant formulas until one year of age; 2) Cow milk should be avoided for the first year; 3) Complementary foods such as iron-fortified cereals or meat, or supplementary iron, should be started at four months of age in term, breastfed infants; 4) Ascorbic acid rich foods, meat, or both with meals are also recommended. However, when these recommendations were made, fresh fluid cow milk was fed to 55% and breast milk to less than 10% of all 10-month-old infants in the United States (Fomon, 1993). Dietary iron requirements of normal infants also depend upon extent of iron absorption and incorporation into hemoglobin and iron stores. Recently, enriched stable isotopes of iron have proven their utility for the evaluation of alternative strategies to improve these aspects of iron nutrition in infants. The value of iron supplements given in drops with vitamins A, D, and C between feedings to 8-week-old well, term infants fed human milk or infant formula has been assessed (Fomon et al., 1995). Two groups of 14–15 infants were given iron-containing drops daily for four weeks by their parents before drops labeled with as sulfate were given on day 56 of age. Incorporation of into erythrocyte hemoglobin two and four weeks later was measured. Mean serum ferritin concentration in the breast-fed group was significantly higher even though mean oral intake (6.2mg/d) was somewhat lower than in the formula-fed group (9.6mg/d). The percentage of oral incorporated into hemoglobin of breast-fed infants was significantly higher than in formula-fed infants, and was inversely correlated to serum ferritin concentration in the breast-fed group. Iron-containing drops contributed significant amounts of iron toward the iron requirements in breast-fed infants and may be beneficial when introduced at two months of age. Iron-fortified formulas contain different amounts of iron in different countries. Incorporation of as sulfate into hemoglobin of five-month-old infants from formulas fortified with 8 or 12mg of Fe per liter has been assessed (Fomon et al., 1997). Each group of infants was fed its level or iron fortification for six weeks before the stable isotopically labeled iron was substituted for a portion of the fortification iron. The mean

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percentage of incorporated into hemoglobin by infants fed formula fortified with 8mg Fe per liter was higher than that of the group fed formula fortified with 12mg Fe per liter. However, the mean amount of iron in micrograms per day incorporated into hemoglobin did not differ between the two groups. Six weeks later, hemoglobin and serum ferritin concentrations also did not differ between groups. The percentages of iron incorporated into erythrocytic hemoglobin from iron-fortified cow-milk formulas are low. Hurrell and his collaborators (1990) have shown that phosphopeptides from bovine casein and the higher levels of calcium and phosphorus are partly responsible for the low percentage of iron absorbed from cow-milk formulas. Iron must be added to cow-milk formulas in amounts high enough that these inhibitory effects are not clinically important but not so high that adverse interactions of iron with zinc or copper occur. Iron absorption, as calculated from erythrocyte incorporation of stable and radioisotopically labeled iron, has been assessed for soy formulas of two phytate and ascorbate concentrations (Hurrell et al., 1998). In their mean-feeding studies of infants, percentage iron absorption was increased substantially by dephytinization, but approximately the same increase was achieved by doubling the ascorbic acid concentration of soy formula without dephytinization. The effect of addition of 25g of meat (lean beef) on nonheme iron absorption from a vegetable puree meal was studied in a cross-over design in 8 healthy infants at 43–49 weeks of age. Incorporation of and from ferrous sulfate was measured. Nonheme iron absorption was significantly higher from the test meal that contained meat (means: 15.0 vs. 9.9%). No significant correlations between age, weight, hemoglobin, plasma ferritin and nonheme iron absorption were found (Englemann et al., 1998). In preterm infants, potential effects of erythropoietin (Epo) treatment on iron absorption and incorporation into hemoglobin have been studied (Widness et al., 1997). Oral iron (6mg/k-d) was given as ferrous sulfate along with ascorbic acid (30mg/kg-d) between feedings while recombinant human erythropoietin (rHuEpo, 500 Units/kg-wk) or placebo was administered for six weeks to very low birth weight (VLBW) infants (<1250g, 14 or 15 per group) of stable respiratory status starting at 7–42 days after birth. After one and four weeks of the study, as sulfate was given in place of part of the iron supplement. Iron absorption was measured by fecal monitoring of unabsorbed isotope, and incorporation into hemoglobin was also measured. Iron incorporation was significantly higher in the Epo-treated group than in the placebo-treated group after one week, but not after four weeks of treatment. No significant differences in iron absorption with treatment or over time were found. Nonheme iron absorption as ferrous sulfate was high (~35%) in these VLBW infants compared with values typically observed in term infants. Preliminary results of a subsequent study by Widness and collaborators (Wu et al., 1998) in which both oral and intravenous stable isotopic iron labels were administered to VLBW infants showed no significant differences between treatment groups (placebo, Epo, Epo plus intravenous iron) in any of the quantities assessed with isotopic labels (incorporation, absorption, utilization, retention). Again, absorption of nonheme iron from supplement measured by the fecal monitoring method was very high (~50%), and erythrocyte incorporation of intravenous iron label as iron sucrose was low after two weeks (~30–35%) compared with adult values of 80–90%. At what stage infants develop the ability to control or regulate nonheme iron absorption, and how partitioning of iron between tissue stores and erythropoiesis changes during development are unknown. The beneficial effect of human milk on iron bioavailability to human infants is not completely explained by its lower content, compared with cow milk, of casein

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phosphopeptides, calcium, and phosphorus. It is likely that factors in human milk act to enhance iron absorption. Recently, Dr. M.S. Hussain in my laboratory has been fractionating human milk by ultracentrifugation and ultrafiltration to identify and characterize such factors with the aid of a Caco-2 cell monolayer system developed by Professor Manju Reddy for use in iron bioavailability studies. Currently, we are concentrating our efforts on ultrafiltrates of human milk whey that contain components with molecular weights (MW) between one and three kilodaltons because they have elicited the largest enhancement of iron flux across Caco-2 cell monolayers (Hussain et al., 1999). The largest portion of the iron in human milk, and the highest iron/protein ratio, is found in the fraction of whey ultrafiltered through a membrane with a 10 kilodalton cut-off. This fraction contains components of MW less than 10 kilodaltons, what we call the l0kDa filtrate, or 10KF. The fraction retained on the membrane with a 10 kilodalton cutoff is called the l0kDa retentate, or 10KR. It contains components of MW greater than 10 kilodaltons. The 10KF is subsequently ultrafiltered through a membrane with a 3 kilodalton cutoff, giving a 3 kilodalton retentate (3 KR) that contains components of MW 3–10 kilodaltons and a 3 kilodalton filtrate (3 KF) with MW less than 3 kilodaltons. The 3 KF is then ultrafiltered through a membrane with a 1 kilodalton cutoff, to give a 1 kilodalton retentate (1 KR) with MW 1–3 kDa and a 1 kilodalton filtrate (1 KF) with MW less than 1 kDa. We simulate gastric digestion of these milk fractions by exposing them to gastric pH for 15min, followed by intestinal pH for 12min. This simulation is based on reports of rapid stomach emptying with inappreciable gastric proteolysis in breast-fed infants. After simulated digestion, each fraction is mixed with radioisotopic iron in buffered cell culture medium for l0min before adding the mixtures to the apical (luminal) sides of Caco-2 cell monolayers grown to confluency in iron-adequate medium on porous membranes in multiple wells of transwell culture plates. The preparations are incubated for one hour, after which the radioactivity in the cell monolayer and in the basolateral (serosal) medium is counted and expressed as a percentage of the initial radioactivity in apical (luminal) medium. Transmonolayer flux is represented by the radioactivity in the basolateral (serosal) medium. Transmonolayer (TM) flux of radioactive iron in the presence of various fractions of human milk previously exposed to pH change is shown in Figure 1. TM flux of

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iron is much greater in the presence of whey fractions that contain components between 1 and 3 kDa (10 KF, 3 KF, 1KR) than for fractions that contain only components of molecular weight below 1 kDa (1KF) or above 3 or l0 kDa (3 KR, 10 KR). Note that TM flux of iron in the presence of milk or outer fat globular membrane is comparable to that for iron ascorbate and greater than that in the presence of blank or casein. The 10 KR fraction is the lactoferrin-containing fraction. If the milk fractions are not exposed to pH change, TM flux of iron after one hour incubation is small, although it is significantly greater than flux for blank or in the presence of ascorbic acid (data not shown). If cell monolayers are loaded with iron by preincubation with ferrous ascorbate for one hour in the presence of iron label, subsequent incubations with milk fractions for one hour reveal that flux from the cell interior to basolateral medium is enhanced by whey fractions under 10 kDa molecular weight in the apical medium (3KF > 3KR > iron ascorbate). These initial experiments point the way to identification of enhancers of iron absorption in human milk and studies of their mechanisms of action. Such enhancers might become useful as future components of more effective iron fortification vehicles for infant formulas or other iron-fortified foods.

REFERENCES Dallman, P.R., 1986, Iron deficiency in the weanling: a nutritional problem on the way to resolution, Acta. Paediatr. Scand. 323:59–67. Engelmann, M.D.M., Davidsson, L., Sandstroem, B., Walczyk, T., Hurrell, R.F., and Michaelsen, K.F., 1998, The influence of meat on nonheme iron absorption in infants, Pediatr. Res. 43:768–773. Fomon, S.J., 1993, Nutrition of Normal Infants, Mosby-Year Book Inc., St. Louis. Fomon, S.J., Ziegler, E.E., Nelson, S.E., Serfass, R.E., and Frantz, J.A., 1995, Erythrocyte incorporation of iron by 56-day-old infants fed a 58Fe-labeled supplement, Pediatr. Res. 38:373–378. Fomon, S.J., Ziegler, E.E., Serfass, R.E., Nelson, S.E., and Frantz, J.A., 1997, Erythrocyte incorporation of iron is similar in infants fed formulas fortified with 12mg/L or 8mg/L of iron, J. Nutr. 127:83–88. Hurrell, R.F., Berrocal, R., Lynch, S.R., Dassenko, S.A., and Cook, J.D., 1990, The influences of bovine milk proteins on iron absorption in man, in: Recent Knowledge on Iron and Folate Deficiencies in the World, Colloque INSERM Volume 197 (S. Hercberg, P. Galan, and H. Dupin, eds.). pp. 265–273, INSERM, Paris. Hurrell, R.F., Davidsson, L., Reddy, M., Kastenmayer, P., and Cook, J.D., 1998, A comparison of iron absorption in adults and infants consuming identical infant formulas, Brit. J. Nutr. 79:31–36. Hussain, M.S., Serfass, R.E., Reddy, M.B., 1999, Low molecular weight fraction from human milk enhances iron bioavailability in Caco-2 cells, FASEB J. 13(4):A241 (abstr. 216.2). Institute of Medicine, 1993, Iron Deficiency Anemia: Recommended Guidelines for the Prevention, Detection, and Management Among U.S. Children and Women of Childbearing Age, National Academy Press, Washington, D.C. Pollitt, E., 1999, Early iron deficiency anemia and later mental retardation, Am. J. Clin. Nutr. 69:4–5. Smith, N.J. and Hunter, R.E., 1970, Iron requirements during growth, in: Iron Deficiency: Pathogenesis, Clinical Aspects, Therapy, (L. Hallberg, H.G. Harwerth, and A. Vannotti, eds.). pp. 199–211, Academic Press, New York. Widness, J.A., Lombard, K.A., Ziegler, E.E., Serfass, R.E., Carlson, S.J., Johnson, K.J., and Miller, J.E., 1997, Erythrocyte incorporation and absorption of 58Fe in premature infants treated with erythropoietin, Pediatr. Res. 41:416–423. Wu, C, Widness, J., Lombard, K., and Serfass, R.E., 1998, Recombinant human erythropoietin (EPO) and parenteral iron supplementation in anemia of prematurity, FASEB J. 12(5):A846(abstr. 4901). Ziegler, E.E., Fomon, S.J., Nelson, S.E., Rebouche, C.J., Edwards, B.B., Rogers, R.R., and Lehman, L.J., 1990, Cow milk feeding in infancy: further observations on blood loss from the gastrointestinal tract, J. Pediatr. 116:11–18.

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TRANSPORT OF IRON IN HEALTH AND DISEASES

Andreas Rolfs and Matthias A. Hediger Membrane Biology Program and Renal Division Department of Medicine Brigham & Women’s Hospital and Harvard Medical School and Department of Biological Chemistry & Molecular Pharmacology Harvard Medical, Boston, Massachusetts 02115 USA

Iron is required in all organisms for growth and crucial metabolic pathways. The two redox states of iron, and make it a versatile cofactor in biological redox reactions. The classical concept for iron uptake by mammalian cells has been the pathway which involves endocytosis of transferring (Tf)-bound via the transferrin receptor (TfR). Considering that there is no available apo-transferrin in the intestinal lumen, except that arising from biliary excretion, other non-receptor-mediated uptake systems must exist in the intestine (Green et al., 1968; Iancu et al., 1995). Furthermore, the process of transferrin receptor-mediated endocytosis, thought to be the principle mechanism for iron uptake into cells, did not explain how iron actually crosses the endosomal membrane. Recent progress in the molecular identification of intestinal iron transporters revealed two distinct transporters for the apical and basolateral membranes. The studies provided novel insight into the regulation of intestinal iron absorption and its dysfunction in iron overload and deficiency disorders. The activity of two distinct iron transporters in luminal and basolateral membranes of enterocytes of the mammalian intestine was promoted by vesicle studies (Eastham et al., 1977) as well as findings of two genetic mouse models: 1) Microcytic hypochromic anemia (mk) mice exhibit defects in apical intestinal iron absorption and also erythroid iron utilization (Fleming, 1997). This anemia is indistinguishable from iron deficiency anemia but is unresponsive to increased dietary iron. In mk mice, a transporter located in the brush border membrane of micro villus cells was thought to be defective, thereby

Corresponding address: Matthias A. Hediger, Harvard Institutes of Medicine, Room 570, 77 Avenue Louis Pasteur, Boston, Massachusetts 02115, USA, Tel: (617) 525 5820, Fax: (617) 525 5821, E-mail: [email protected] Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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blocking iron uptake into these cells. 2) In sex-linked anemia (sla) mice, iron uptake into the micro villus cells is detectable, but the iron is not released into the serum (Anderson et al., 1998). In recent years, various iron transporters have been identified for lower organisms, including the transporters feoB from E. coli (Kammler et al., 1993), FET4 from yeast (Dix et al., 1994), the plant IRT1 (Eide et al., 1996), and the transporter FTR1 from yeast (Stearman et al., 1996). Nelson and colleagues furthermore provided evidence that the yeast SMF1 transporter, a homologue of the mammalian Nramp family, functions as a manganese transporter (Supek et al., 1996). Until recently, the transporters which mediate direct uptake of iron and metal ions into mammalian cells remained elusive.

CLONING AND CHARACTERIZATION OF THE METAL ION TRANSPORTER DCT1 Using expression cloning with Xenopus laevis oocytes, our laboratory isolated the divalent cation/metal ion transporter DCT1 (also called Nramp2) from a duodenal cDNA library, prepared from mRNA from rats fed a low-iron diet (Gunshin et al., 1997). DCT1 was isolated by screening this library using a radiotracer assay of uptake in Xenopus oocytes. The isolated cDNA clone encodes a 561 amino acid polypeptide, which, when expressed in oocytes, increases the uptake of more than 200-fold compared with control (water injected) oocytes. Interestingly, we found that DCT1 transports a variety of divalent metal ions but not and DCT1 expression was found to be widespread in rat tissues, based on in situ hybridization and Northern analysis. Comparison of DCT1 expression in tissues from rats fed a normal diet or an iron deficient diet for 3 weeks showed that iron deprivation triggers a strong increase in DCT1 mRNA levels in the intestine and to some extent in all tissues examined. The finding of an iron responsive element (IRE) in the 3' untranslated region of the cloned DCT1 cDNA indicates regulation of DCT1 at the RNA stability level, in analogy to the transferrin receptor mRNA. Recent studies in our laboratory and by others on the function of the DCT1-IRE support this hypothesis and suggest that the iron response element binding protein 1 (IRP1) binds to this IRE (see Wardrop and Richardson, 1999). Our recent data show that upregulation of DCT1 is associated with the pathogenesis of hereditary hemochromatosis (HH), a common genetic disorder (Feder et al., 1998). This upregulation is likely a result of abnormal iron sensing in crypt cells, in conjunction with iron-dependent regulation of DCT1 mRNA via RNA degradation. Andrews and colleagues (Fleming et al., 1997) searched for the mk gene by positional cloning. This approach resulted in the isolation of the mouse DCT1/Nramp2 gene, confirming our speculation that the DCT1 metal ion transporter is the principal luminal iron uptake mechanism in the intestine.

THE BASOLATERAL IRON TRANSPORTER The mutated sla gene in sex linked anemia mice has been recently cloned and was shown to encode a membrane bound ceruloplasmin homologue, named “hephaestin” (Anderson et al., 1999). Hephaestin itself is probably not capable of mediating iron transport itself since it codes for only one transmembrane domain. In analogy to the oxidase

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activity of ceruloplasmin, the function of hephaestin is most likely to act as a ferrioxidase to convert to which binds instantly to transferrin in the serum. The protein mediating the transfer of from the cytosol to the cell outside is currently still missing.

REFERENCES Anderson, G.J., Murphy, T.L., Cowley, L., Evans, B.A., Halliday, J.W., and Mclaren, G.D., 1998, Mapping the gene for sex-linked anemia—An inherited defect of intestinal iron absorption in the mouse, Genomics 48, 34–39. Dix, D.R., Bridgham, J.T., Broderius, M.A., Byersdorfer, C.A., and Eide, D.J., 1994, The FET4 gene encodes the low affinity Fe(II) transport protein of Saccharomyces cerevisiae, Journal of Biological Chemistry 269, 26092–26099. Eastham, E.J., Bell, J.I., and Douglas, A.P., 1977, Iron-transport characteristics of vesicles of brush-border and basolateral plasma membrane from the rat enterocyte, Biochemical Journal 164, 289–294. Eide, D., Broderius, M., Fett, J., and Guerinot, M.L., 1996, A novel iron-regulated metal transporter from plants identified by functional expression in yeast, Proceedings of the National Academy of Sciences USA 93, 5624–5628. Feder, J.N., Penny, D.M., Irrinki, A., Lee, V.K., Lebron, J.A., Watson, N., Tsuchihashi, Z., Sigal, E., Bjorkman, P.J., and Schatzman, R.C., 1998, The hemochromatosis gene product complexes with the transferrin receptor and lowers its affinity for ligand binding, Proceedings of the National Academy of Sciences USA 95, 1472–1477. Fleming, M.D., Trenor, C.C., Su, M.A., Foernzler, D., Beier, D.R., Dietrich, W.F., and Andrews, N.C., 1997, Microcytic anaemia mice have a mutation in Nramp2, a candidate iron transporter gene, Nature Genetics 16, 383–386. Green, R., Charlton, R., Seftel, H., Bothwell, T., Mayet, K, Adams, B., Finch, C., and Layrisse, M., 1968, Body iron excretion in man: a collaborative study, American Journal of Medicine 45, 336–353. Gunshin, H., Mackenzie, B., Berger, U.V., Gunshin, Y., Romero, M.F., Boron, W.F., Nussberger, S., Gollan, J.L., and Hediger, M.A., 1997, Cloning and characterization of a mammalian proton-coupled metalion transporter, Nature 388, 482–188. Iancu, T.C., Shiloh, H., Raja, K.B., Simpson, R.J., Peters, T.J., Perl, D.P., Hsu, A., and Good, P.F., 1995, The hypotransferrinaemic mouse: ultrastructural and laser microprobe analysis observations, Journal of Pathology 177, 83–94. Kammler, M., Schon, C., and Hantke, K., 1993, Characterization of the ferrous iron uptake system of Escherichia coli, Journal of Bacteriology 175, 6212–6219. Stearman, R., Yuan, D.S., Yamaguchi-Iwai, Y., Klausner, R.D., and Dancis, A., 1996, A permease-oxidase complex involved in high-affinity iron uptake in yeast, Science 271, 1552–1557. Supek, F., Supekova, L., Nelson, H., and Nelson, N, 1996, A yeast manganese transporter related to the macrophage protein involved in conferring resistance to mycobacteria, Proce Natl. Acad. Sci. USA 93, 5105–5110. Wardrop, S.L. and Richardson, D.R., 1999, The effect of intracellular iron concentrationand nitrogen monoxide on Nramp2 expression and non-transferrin-bound iron uptake, Eur. J. Biochem. 263, 41–49.

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BENEFITS AND PROBLEMS ASSOCIATED WITH IRON SUPPLEMENTATION AND FORTIFICATION

Serge Hercberg Institut Scientifique et Technique de la Nutrition et de l’Alimentation/CNAM 2 rue Conté, F-75003 Paris

As in the story of Dr Jekyll and Mr Hyde, iron (like most nutrients and particularly micronutrients) possesses both the best and the worst possible qualities. Both an inadequate supply of iron to body tissues and excessive iron accumulation within the body lead to significant morbidity. So, iron is an intriguing compound, with a wide range of benefits when it enables covering iron requirements and preventing and controlling iron deficiency (that is the Dr Jekyll’s side), but also toxicity, as it is able to induce tissue damage with formation of toxic oxygen species (that is Mr Hyde’s side). This paper focuses on results of epidemiological data related to two fundamental points in the heart of the debate, as we are faced in developed country: – are preventive measures against iron deficiency (such as iron supplementation and iron fortification) justified and are they efficient? – do they present any risks for the general or specific populations?

ARE PREVENTIVE MEASURES AGAINST IRON DEFICIENCY (SUCH AS IRON SUPPLEMENTATION OR IRON FORTIFICATION) JUSTIFIED AND ARE THEY EFFICIENT? The answer is related to different kinds of data: why do we find iron deficiency in the context of industrialized countries? what is the prevalence of iron deficiency in the context of these countries? what are the consequences of iron deficiency on health? is it possible to prevent iron deficiency by iron supplementation and/or iron fortification? Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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1.1. Why Do We Find Iron Deficiency in the Context of Industrialized Countries? It is important to keep in mind that, according to the WHO, iron, among all the nutrients is probably one that raises the most practical problems in terms of covering physiological requirements in humans (WHO, 1992). This explains why iron deficiency is considered to be the most common nutritional deficiency worldwide. Although iron deficiency is mainly a problem in developing countries, it also seems to affect large fractions of the populations in the industrialized world. The existence of iron deficiency in the context of the abundance existing in developed countries may be related to rapid evolution of the diet and modifications in the way of life over the last few decades (Hercberg et al., 1990). Indeed, in the last several generations, we have observed, in industrialized countries, a reduction in total calorie intake (due to a reduction in physical activity) which has led to a decrease in iron intake. Moreover, the increase in consumption of foods containing only energy, without trace elements or vitamins, has contributed to a decrease in the micronutrient density per unit of energy in diet, and particularly iron density. For these reasons, the usual diets in developed countries may no longer meet the iron requirements of a number of people, particularly those with high iron requirements including children and menstruating or pregnant women, who may be considered at high risk of iron deficiency. This is confirmed by epidemiologic data using pertinent markers of iron status.

1.2. What Is the Prevalence of Iron Deficiency in Industrialized Countries? Nutritional studies performed in developed countries in adults using serum ferritin as a marker of iron stores show (Table 1) that iron depletion concerns 10–30% of menstruating women and iron deficiency anemia 1.5 to 14%. Other factors such as type of contraception (use of intrauterine devices), blood donation or minor pathological blood loss (hemorrhoids, gynecological bleedings, etc) may considerably increase the difficulty in covering iron needs and the risk of iron deficiency. In pregnant women (Table 2), 25 to 77% present a total depletion of iron stores at the end of gestation and, the prevalence of iron deficiency anemia is 6 to 30% (the highest levels are observed in countries where routine iron supplementation is not usually given during pregnancy). While iron deficiency anemia concerns small fractions of the population (particularly pregnant women), depleted iron stores are widely observed in populations in developed countries. Although there is no evidence that an absence of iron stores has any adverse consequences, it does indicate that iron nutrition is borderline, since any further reduction in body iron is associated with a decrease in the level of functional compounds such as hemoglobin.

1.3. What Are the Consequences of Iron Deficiency on Health? In the human body, iron is present in all cells and has several vital functions—as a carrier of oxygen to the tissues from the lungs in the form of hemoglobin, as a facilitator of oxygen use and storage in the muscles as myoglobin, as a transport medium for electrons within the cells in the form of cytochromes, as an integral part of enzyme reactions in various tissues (Hallberg and Asp, 1996). Too little iron can interfere with these vital functions and lead to morbidity and mortality.

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In infants and children, iron deficiency anemia results in developmental delays and behavioral disturbances (decreased motor activity, social interaction, and attention to tasks), in pregnant women it increases the risk for a preterm delivery and delivering a low-birthweight baby, and lower working capacity in adults (Allen, 1997; Viteri, 1994). While we do not have conclusive evidence to recommend that depleted iron stores be the objects of public health measures, excepted dietary recommandations, it is clear that iron-deficiency anemia needs to be prevented.

1.4. Is It Possible to Prevent Iron Deficiency by Iron Supplementation and/or Iron Fortification? This includes primary prevention through appropriate dietary intake and secondary prevention through detecting and treating iron-deficiency anemia. 1.4.1. Iron Supplementation. Iron supplementation (with adapted doses) during pregnancy seems particularly useful, because a large proportion of women have difficulty maintaining iron stores during pregnancy and are at risk for anemia; iron deficiency anemia during pregnancy is associated with adverse outcome, and supplementation during pregnancy is not associated with a substantial risk health (Allen, 1997; Viteri, 1994). There is little doubt that iron supplementation in pregnancy improves maternal iron status in both developed and developing countries. Increases in hemoglobin, hematocrit, mean corpuscular volume, serum ferritin, serum iron and transferrin saturation, compared with unsupplemented controls, are usually apparent within 3 months, and the usual depletion of maternal iron stores is reduced or eliminated when assessed by maintenance of serum ferritin or bone marrow (De Benazé et al., 1989; Milman et al., 1991; Doyle et al., 1990; Thanangkul et al., 1994; Milman et al., 1994; Milman et al., 1997; Preziosi et al., 1997; Gaspar et al., 1997). Iron supplementation during pregnancy also improves maternal iron status postpartum (De Benazé et al., 1989). Differences in maternal iron stores as a result of pregnancy supplementation may persist to at least 6 months postpartum (De Benazé et al., 1989; Allen, 1997). Data currently available from supplementation studies fail to provide conclusive evidence that iron supplementation is beneficial to fetal growth or pregnancy outcome. Nevertheless, the decision to recommend iron supplementation is based on a sufficient number of considerations: – dietary intake is unlikely to provide the additional absorbed iron needed for use and deposition by the fetus and mother and to replace blood losses at parturition; – iron deficiency is common in pregnant women; – supplements have been shown to improve the iron status of pregnant women. The only negative effects of iron supplementation that may be observed under certain conditions during pregnancy are some classical side effects. These occur much less frequently at doses of 30–45 mg per day, but with higher intakes, there is an increase in heartburn, nausea, constipation or diarrhea and upper abdominal discomfort. Curently there is interest in the possibility that iron supplementation once a week might be as effective as daily supplementation in improving iron status with less side effects in pregnant women.

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Effects of iron supplements on zinc absorption are not really documented: some studies have found an adverse effect on zinc absorption, especially when the ratio of iron to zinc is greater than 2. Other did not report any modification in plasma zinc concentration. 1.4.2. Iron Fortification. The fortification of foods has been often regarded as the most cost-effective long-term approach to reducing the prevalence of iron deficiency (Hurrell, 1997). This can be in the form of “mass medication”, by fortifying foods such as cereals, milk, salt, sugar and condiments that are widely consumed by both at-risk populations and others who have little or no need for extra iron. Alternatively, a targeted fortification program in which a food product preferentially consumed by one of the atrisk groups is fortified can be considered. Although targeted fortification is relatively easy to design for infant foods such as formulas and commercial infant cereals, or for schoolchildren through school feeding programs including such foods as fortified drinks or cookies, it is more difficult to target a fortified food specifically for adult fertile women. For this group, the fortification of a widely consumed product would seem the best way to provide extra food iron, but other groups such as adult men and postmenopausal women, who do not require extra iron, will also consume the fortified food. In industrialized countries, there is concern that this excess iron may be detrimental and lead to an increased incidence of atherosclerosis or cancer due to increased oxidative stress. Although widespread iron deficiency has been recognized for more than 50 years, intervention strategies including food fortification have met with only limited success. The only clear success story has been in industrialized countries, such as Sweden or the United States where the steady drop in the prevalence of iron deficiency in infants and preschool children over the last 30 years is related to iron-fortified infant formulas (Hurrell, 1997). The current low incidence of iron deficiency anemia in fertile US women, reported as 2.9% in the NHANES II survey could also be due in part to the high consumption of iron-fortified foods in the United States which represent about 20% of the total iron intake.

1.2. Do Preventive Measures Against Iron Deficiency, Such As Iron Supplementation and Iron Fortification, Present any Risks for the General or a Specific Population? Are There Potential Adverse Effects Involved in Increasing Dietary Iron Intake? While iron deficiency is widespread in some groups of populations in developed countries, about 10% of Caucasians carry the mutation for hereditary hemochromatosis (Hallberg et al., 1995; Milman et al., 1991). Based on extensive screening studies in Europe (Table 3), the pre-valence of hereditary hemochromatosis is probably below rather than above 0.1% in, the general population. Some areas may be considered as genetic superisolates showing a higher prevalence, such as those noted in Brittany (in France), Iceland and Jämtland (in Sweden). Although increases in iron intake would seem to be contraindicated in persons with hemochromatosis, there is no evidence that iron fortification of foods or the use of a recommended iron supplementation regimen during pregnancy is associated with increased risk of clinical disease due to hemochromatosis. The observation that iron overload increases myocardial damage caused by anoxia and reperfusion on animal experiments, and its potential rôle in lipoprotein oxidation,

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have provoked renewed interest in the hypothesis that high body iron may be a risk factor for coronary heart disease. The ability of iron to catalyze the production of free radicals, which may increase oxidative stress and damage DNA, has also raised concern about the possibility that excess body iron may increase the risk of cancer. However, the results of epidemiological studies have been conflicting, and do not provide convincing evidence of an adverse effect of iron (Table 4). In 1992, Salonen reported (Salonen et al., 1992), in a prospective study in Finland, a twofold increase in acute myocardial infarction among men with serum ferritin levels above compared to those below that level. However, conflicting findings were later reported from two other investigations among US male physicians and Icelandic

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men, that also used serum ferritin as a marker of body iron stores (Stampfer et al., 1993). No association was found between serum ferritin or serum iron and the risk of myocardial infarction. In a Canadian cohort with a follow up of 17 years (Magnusson et al., 1994), an increased risk of fatal myocardial infarction was found among men and women with abnormally high serum iron concentrations or above) presumed to represent iron overload. Since serum iron concentrations above are likely to occur in individuals who are genetically predisposed, it is the genotype (i.e. the hemochromatosis gene or other genes covarying with it) or accumulated iron, which increases the risk of fatal acute myocardial infarction. Other studies, such as NHANES I (Morrison et al., 1994) and the Kaiser-Permanente Multiphasic study (Sempos et al., 1994) did not find a relationship between transferrin saturation and risk of acute myocardial infarction. A primary reason for the interest in the association of iron stores with the risk of CVD is the possibility that higher intake may increase risk; this has been addressed in the Finnish cohort but not in others. However in a large cohort study among US males, no association was found between intake of non-heme iron (from foods and supplements) and the risk of CVD or myocardial infarcion (Sempos et al., 1994). However, heme iron (mainly from red meat) was directly related to the risk of myocardial infarction. Since Finnish men have a high meat intake, total iron intake may reflect heme iron and meat intake to a greater degree in the Finnish cohort, whereas it reflects largely non-meat sources and supplements in the US (Ascherio et al., 1994). Concern remains that the small fractions of persons homozygous for hemochromatosis (about 1 in 300) might experience elevated risk of CVD due to iron intake within the range of US diets. The available studies do not exclude such an effect in a small subgroup. Concerning cancer, in 1986 Stevens et al. reported a positive association between baseline serum ferritin and risk of death from cancer or development of primary hepatocellular carcinoma in a prospective study of Chinese male government workers in Taiwan. This relation was found in some other studies, and for some cancer localisations. In several studies, transferrin saturation was used to measure iron status. Although transferrin saturation is a poor indicator of body iron stores, when these are within the normal range, high levels of transferrin saturation (60%) are suggestive of iron overload and are frequently associated with abnormal metabolism. Some of these studies provide indirect evidence that iron overload is associated with an increased risk of cancer, although whether this is due to the effect of iron itself rather than a genetic predisposition remains unsettled (Table 5). Finally, iron deficiency affects large fractions of populations in developed countries, particularly such physiological groups as children, menstruating women and pregnant women. Primary health-care providers can help prevent and control iron deficiency by counseling individuals and families about sound iron nutrition during infancy and beyond and about iron supplementation during pregnancy, by screening persons on the basis of their risk for iron deficiency, and by treating and following up persons with presumptive iron deficiency. This may help to reduce manifestations of iron deficiency and thus improve public health. Evidence linking iron status with risk of cardiovascular disease or cancer is unconvincing and does not justify changes in food fortification or medical practice, particularly because the benefits of assuring adequate iron intake during growth and development are well established. But stronger evidence is needed before rejecting the hypothesis that greater iron stores increase the incidence of CVD or cancer. Body iron in excess offers no health benefit, and there is evidence that it may be detrimental. Therefore, an important question is whether a moderate elevation in body iron levels leads to an increased risk of cancer or cardiovascular diseases.

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Further research, including basic research and large-scale epidemiologic studies, is needed to fully assess the association between iron status and the risk of cardiovascular diseases, cancers and other adverse outcomes. At present, currently available data do not support radical changes in dietary recommendations.

REFERENCES Allen, L.H., 1997, Pregnancy and iron deficiency: unresolved issues, Nutr. Rev. 55:91–101. Ascherio, A., Willet, W.C., Rimm, E.B., Giovannui, E,, and Stampfer, M.J., 1994, Dietary iron intake and risk of coronary heart disease, Circulation 89:969–974. Baer, D.M., Tekawa, I.S., and Hutley, L.B., 1994, Iron stores arenot associated with acute myocardial infarction. Circulation 89:2915–2918. Brussaard, J.H., Brants, H.A., Bouman, M., and Löwik, M.R.H., 1997, Iron intake and iron status among adults in the Netherlands, Eur. J. Clin. Nutr. 51:S52–SS8. Centers for Disease Control, 1989, CDC criteria for anemia: in childbearing aged women. MMWR. 38:400–104. De Benazé, C., Galan, P., Wainer, R., and Hercberg, S., 1989, Prevention de l’anemie ferroprive au cours de la grassesse par un supplementation maritale precoce: un essaie contrôle, Rev. Epidemiol. Sante Publique 27:109–119. Doyle, W., Crawford, M.A., Wynn, A.H.A., and Wynn, S.W., 1990, The association between maternal diet and birth dimensions, J. Nutr. Med. 1:9–17. Fogelholm, M., Alopaeus, K., Silvennoinen, T., and Teirilä, J., 1993, Factors affecting iron status in nonpregnant women from urban South Finland, Europ. J. Clin. Nutr. 47:567–574. Galan, P., Hercberg, S., and Soustre, Y, 1985, Factors affecting iron stores in French females, Human Nutr. Clin. Nutr. 39C:279–287. Galan, P., Yoon, H.C., Preziosi, P., Viteri, F, Valeix, P., Fieux, B., Briançon, S., Malvy, D., Roussel, A.M., Favier, A., and Hercberg, S., 1998, Determining factors in the iron status of adult women in the SU.VI.MAX Study, Europ. J. Clin. Nutr. 52:383–388.

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Gaspar, M.J., Ortega, R.M., and Moreiras, O, 1993, Relationship between iron status in pregnant women and their newborn babies: Investigation in Spanish population, Acta Obstet. Gynecol. Scand. 72:534–537. Godel, J.C., Pabst, H.F., Hodgers, P.E., and Johson, K.E., 1992, Iron status and pregnancy in a northern Canadian population: relationship to diet and iron supplementation, Canadian J. Public Health 339–343. Hallberg, L. and Asp, N.G., 1996, Iron nutrition in health and disease, John Libbey, London, p. 364. Hallberg, L., Hultén, L., Bengtsson, C, Lapidus, L., and Lindstedt, G., 1995, Iron balance in menstruating women, Europ. J. Clin. Nutr. 49:200–207. Hercberg, S., Galan, P., and Dupin, H., 1990, Recent knowledge on iron and folate deficiency in the world, Eds INSERM, Paris, Vol 197, p. 700. Hercberg, S., Galan, P., Soustre, Y., Dop, M.C., Devanlay, M., and Dupin, H., 1985, Prevalence of iron deficiency duiring pregnancy in a French area, Nutr. Rep. Int. 32:719–726. Hercberg, S., Bichon, L., Galan, P., Christides, J.P., Carroget, C., and Potier de Courcy, G., 1987, Iron and folacin status of pregnant women: relationship with dietary intakes, Nutr. Rep. Int. 56:915–930. Hurell, R., 1997, Fortification, Nutrition Rev. 55:210–222. Knekt, P., Reunanen, A., Takkunen, H., Aromaa, A., and Hakulinen, T., 1994, Body iron stores and the risk of cancer, Int. J. Cancer 56:379–382. Knottnerus, J.A., Delgado, L.R., Knipschild, P.G., Essed, G.G., and Smits, F., 1990, Haematologic parameters and pregnancy outcome, J. Clin. Epidemiol. 43:461–466. Magnusson, M.K., Sigfusson, N., Sigvaldason, H., Johannesson, G.M., Magnusson, S., and Thorgeirsson, G., 1994, Low iron-binding capacity as a risk factor for myocardial infarction, Circulation 89:102–108. Milman, N., Graudal, N., Nielsen, O.J., and Agger, A.O., 1997, Serum erythropoietin during normal pregnancy: relationship to hemoglobin and iron status markers and impact of iron supplementation in a longitudinal, placebo-controlled study, Int. J. Hematol. 66:159–168. Miiman, N., Agger, A.O., and Nielsen, O.J., 1991, Iron supplementation during pregnancy: effect on iron status markers, serum erythropoietin and human placental lactogen: a placebo controlled study in 207 Danish women, Danish Med. Bull. 38:471–176. Milman, N, Agger, A.O., and Nielsen, O.J., 1994, Iron status markers and serum erythropoietin in 120 mothers and newborn infants: effect of iron supplementation in normal pregnancy, Acta Obstet. Gynecol. Scand. 73:200–204. Morrison, H.I., Semenciw, R.M., Mao, Y., and Wigle, D.T., 1994, Serum iron and risk of fatal acute myocardial infarction. Epidemiology 5:243–246. Perry, G.S., Yip, R., and Zyrkowski, C., 1995, Nutritional risk factors among low-income pregnant US wromen: the Centers for Disease control and Prevention (CDC) Pregnancy Nutrition Surveillance System, 1979 through 1993, Semin. Perinatal. 19:211–221. Preziosi, P., Prual, A., Galan, P., Daouda, H., Boureima, H., and Hercberg, S., 1997, Effect of iron supplementation on the iron status of pregnant women: consequences for newborns, Am. J. Clin. Nutr. 66:1178–1182. Preziosi, P., Hercberg, S., Galan, P., Devanlay, M., Cherouvrier, E, and Dupin, H., 1994, Iron status of a healthy French population: factors determining biochemical markers, Ann. Nutr. Met. 38:192–202. Robinson, S., Godfrey, K., Denne, J., and Cox, V, 1998, The determinants of iron status in early pregnancy, Br. J. Nutr. 79:249–255. Salonen, J.T., Nyyssönen, K., Korpela, H., Tuomilehto, J., Seppären, J., and Salonen, R., 1992, High stores iron levels are associated with excess risk of myocardial infarction in eastern Finnish men, Circulation 86:803–811. Scholl, T.O. and Hediger, M.L., 1994, Anemia and iron-deficiency anemia: Compilation of data on pregnancy outcome, Am. J. Clin. Nutr. 59:S492–S501. Selby, J.V. and Friedman, G.D., 1988, Epidemiological evidence of an association of body iron stores and risk of cancer, Int. J. Cancer 41:677–682. Sempos, C.T., Looker, A.C., Gillum, R.F., and Makuc, D.M., 1994, Body iron stores and the risk of coronary heart disease, N. Engl. J. Med. 330:1119–1124. Soustre, Y, Dop, M.C., Galan, P., and Hercberg, S., 1986, Dietary determinants of the iron status in menstruating women, Int. J. Vit. Nutr. Res. 56:281–286. Stampfer, M.J., Grodstein, F., Rosenberg, I., Willet, W.C., and Hennekens, C., 1993, A prospective study of plasma ferritin and risk of myocardial infarction in US physicians, Circulation 87:688 (Abstract). Stevens, R.G., Beasley, R.P., and Blumberg, B.S., 1986, Iron-binding proteins and risk of cancer in Taiwan, J. Natl. Cancer Inst. 76:605–610. Stevens, R.G., Jones, D.Y., Micozzi, M.S., and Taylor, P.R., 1988, Body iron stores and risk of cancer, N. Engl. J. Med. 319:1047–1052.

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Strain, J.J., Barker, M.E., Livingstone, M.B.E., and Mc Kenna, P.G., 1990, The Northern Ireland dietary surveys and related studies, Proc. Nutr. Soc. 49:289–296. Thanangkul, O., Amatayakul, K., and Kulapongs, P. et al., 1994, Iron and folate supplementation duringpregnancy: maternal and fetal consequences. In: Allen, L.H., King, J.C., and Lonnerdal, B. eds. Nutrient regulation during pregnancy, lactation and infant growth, Adv. Exp. Biol. Med. 352:151–156. Van de Vijver, L.P.L., Kardinaal, A.P.M., Charzewska, J., Rotily, M., Charles, P., Maggiolini, M., Ando, S., Väänänen, K., Wajszczyk, B., Heikkinen, J., Deloraine, A., and Schaafsma, G., 1999, Calcium intake is weakly but consistently negatively associated with iron status in girls and women in six European countries, J. Nutr. 129:963–968. Viteri, F.E., 1994, The consequences of iron deficiency and anemia in pregnancy. In: Nutrient Regulation during Pregnancy, Lactation and Infant Growh, Allen, L., King, J., and Lönnerdal, B. ed. Plenum Press, New York. pp. 127–139. WHO, 1992, The prevalence of anaemia in women: a tabulation of available information, 2nded. Geneva: World Health Organizatio.

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LOW IRON DIET AND CADMIUM EXPOSURE DISRUPT STEROIDOGENESIS IN THE RAT Martina Piasek1, John W. Laskey2, Krista Kostial1, Maja Blanuša1, and Janet M. Ferrell2 1

Institute for Medical Research and Occupational Health HR-10001 Zagreb, Croatia 2 United States Environmental Protection Agency Research Triangle Park, NC 27711 USA

1. INTRODUCTION The existing evidence in humans is not sufficient to consider female reproductive effects as critical effects of cadmium (Cd) exposure (Järup et al., 1998). Further facts argue for Cd as an element of concern as a potential reproductive toxicant in women. It accumulates in internal organs during a lifetime, including tissues of a reproductive axis; more in smokers (Varga et al., 1993) and women habitually eating a diet rich in Cd (shellfish and high fiber diet) (Moberg Wing et al., 1992). Cadmium interferes with trace element homeostasis. Iron (Fe) status has been shown to influence the gastrointestinal absorption of Cd both in experimental animals and humans (Berglund et al., 1994). Women are high at risk group for Cd effects; 1) they usually show greater tissue Cd concentrations than men; 2) they are prone to Fe deficiency during childbearing age; 3) as evidenced by animal studies, gastrointestinal absorption of Cd is increased during gestation and lactation (Järup et al., 1998; Kostial et al., 1991a,b). To date only limited information is available on the effects of Cd exposure on ovarian steroid production (Paksy et al., 1989; Piasek and Laskey, 1994). No data on Cd effect on placental steroidogenesis are available. We found that acute Cd exposure in rats appeared to cause endocrine disruption in the ovary with target organ Cd accumulation and concomitant decrease in tissue Fe, which were related to the reproductive stage. The objective of this work was to evaluate the effect(s) of low Fe diet and concurrent subchronic Cd exposure during gestation on both ovarian and placental steroidogenesis in rats. Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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2. MATERIAL AND METHODS The study was conducted on timed-pregnant 60-days-old Sprague-Dawley rats fed semisynthetic pelleted laboratory diets (Teklad, Madison, WI, USA) with either high Fe (240mg/kg) or low Fe (10mg/kg) content. From gestation day one, the dams were continuously exposed to Cd (chloride) at a total dose of 0 (control), 3, or 5 mg/kg body weight during 19 days (minimum six animals per group) by subcutaneously implanted osmotic pumps (MP 2ML4 Alzet, Alza Co., Palo Alto, CA, USA). On gestation day 19, the dams were exsanguinated by cardiac puncture in anesthesia and blood was taken for serum. Right ovaries and two placentas (one from each extreme lateral position in the uterine horn) were removed. Placentas were separated into maternal and fetal portions. Data of the maternal portions of the placentas are presented here. Progesterone and estradiol serum concentrations and progesterone, testosterone and estradiol productions in the minced whole ovary cultures were analysed as described earlier (Piasek and Laskey, 1994). Placental progesterone and testosterone productions were assessed in the cultures of minced maternal placentas. Specific radioimmunoassay (RIA kits Los Angeles, CA) was used for steroid hormone analyses. Cadmium was analysed in the placental tissue (composite samples) by electrothemal atomic absorption spectrometry (Varian SpectrAA 300A, Australia). The data were statistically evaluated using both one-way and two-way analysis of variance (ANOVA) and test for linearity in the general linear models procedure (PROC GLM) available in 1985 edition of the Statistical Analysis System (SAS) with dietary Fe and Cd exposure as two independent variables.

3. RESULTS AND DISCUSSION No Cd effects were found on maternal general health and fetal viability. Low Fe diet itself was associated with lower maternal body weights, decreased maternal and fetal hematocrits, reduced number of viable fetuses and increased late resorptions (data not presented). Summary effects of diet with low Fe and concomitant parenteral Cd exposure during 19 days of pregnancy are presented in the Table 1. The data show that all dams fed low Fe diet had reduced serum progesterone. With subchronic Cd exposure, serum estradiol concentrations decreased and the effect was linear. The effects of low Fe diet and concurrent Cd exposure on placental steroidogenesis were additive and linear with significant reduction in maternal placental progesterone production at 5 mg/kg Cd dose. No effect was observed on either testosterone production in the maternal portions of the placentas or steroid production in whole ovary cultures (data not shown). With both high and low Fe diets, increase in Cd concentrations in the maternal placentas of exposed dams was dose-related. Average placental Fe concentrations were greater in the rats fed high Fe feed comparing to the low Fe diet group, no matter the Cd exposure (85 ± 3 and 52 ± 2. g/g wet tissue wt., respectively). The results of our previous studies have suggested that, with acute exposure, Cd appears to interfere with normal rat streoidogenesis at several sites in the biosynthetic pathway, with serum estradiol concentration and ovarian estradiol production most affected in proestrus and in the first third of pregnancy (Piasek and Laskey, 1994). Findings of this study imply that low Fe diet with concomitant subchronic Cd exposure during pregnancy disrupt placental steroid production and concentrations of circulating steroid

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hormones near the full term. This, together with decreased maternal and fetal Fe body stores and increased placental Cd, may pose a risk for growth and development of the young.

ACKNOWLEDGMENT The authors are grateful for expert technical help of Marija Ciganovi_ in tissue element analyses.

and Ms.

REFERENCES Berglund, M, Åkesson, A., Nermell, B., and Vahter, M., 1994, Intestinal absorption of dietary cadmium in women is dependent on body iron stores and fiber intake, Environ. Health Perspect. 102:1058–1066. Järup, L., Berglund, M., Elinder, C.G., Nordberg, G., and Vahter, M., 1998, Health effects of cadmium exposure—a review of the literature and risk estimate, Scand. J. Work Environ. Health 24(suppl. 1): 1–52. Kostial, K., Blanuša, M., Karga_in, B., Piasek, M., Maljkov_, T., and Kello, D., 1991a, Sex-related differences in genetic susceptibility to toxic metals, in: Ecogenetics: genetic predisposition to the toxic effects of chemicals (P. Grandjean, ed.), pp. 111–122, Chapman & Hall, London, UK.

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Kostial, K., Blanuša, M., Maljkovi_, T., Karga_in, B., Piasek, M., Mom_ilovi_, B., and Kello, D., 1991b, Age and sex influence the metabolism and toxicity of metals, in: Trace Elements in Man and Animals 7 (B. Mom_ilovi_, ed.), pp. 11.1–11.5, IMI, Zagreb, Croatia. Moberg Wing, A., Wing, K., Tholin, K., Sjöström, R., Sandström, B., and Hallmans, G., 1992, The relation of the accumulation of cadmium in human placenta to the intake of high-fibre grains and maternal iron status, Eur. J. Clin. Nutr. 46:585–595. Paksy, K., Varga, B., Horváth, E., Tátrai, E., and Ungváry, G., 1989, Acute effects of cadmium on preovulatory serum FSH, LH, and prolactin levels and on ovulation and ovarian hormone secretion in estrous rats, Reprod. Toxicol. 3:241–247. Piasek, M. and Laskey, J.W., 1994, Acute cadmium exposure and ovarian steroidogenesis in cycling and pregnant rats, Reprod. Toxicol. 8:495–507. Varga, B., Zsolnai, B., Paksy, K., Naray, M., and Ungváry, G, 1993, Age dependent accumulation of cadmium in the human ovary, Reprod. Toxicol. 7:225–228.

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PECULIARITIES OF NONHEME IRON METABOLISM UPON EXPERIMENTAL MODELLING OF RAT GLIAL BRAIN TUMOUR. PERSPECTIVES FOR DIAGNOSIS AND TREATMENT

Olga M. Mykhaylyk, Natalie A. Dudchenko, Eugene A. Lebedev, Bogdan S. Shurunov, A. P. Cherchenko, and Yu. A. Zozulya Institute for Applied Problems in Physics and Biophysics National Academy of Sciences of the Ukraine P.O. Box 355, Kyiv 252001 Institute of Neurosurgery Academy of Medical Sciences of the Ukraine Manuil’skogo 32, 254050 Kyiv Ukraine

1. INTRODUCTION Recently, the role of excess iron in some central nervous system disorders has been recognized (Connor, 1993). High plasma iron levels are shown to be positively associated with increased risk of death from cancer (Stevens et al., 1988). Iron is found to be a limiting nutrient for tumor cell growth, and the iron withholding that gives rise to hypoferremia (Huges, 1972) has proved to be a defense against neoplasia (Weinberg, 1984). Recent data confirm that labile iron concentration is an essential factor that determines the relationship between cell proliferation and apoptosis (Richardson and Milnes, 1997). The various brain tumors (especially glioblastomas) show much greater immunoreactivity for TR than the normal brain tissue does (Recht et al., 1990). However, much less is known about the shifts in the content of nonheme iron species in brain tumour growth. The aim of the present study was to determine the indices of noheme iron exchange upon experimental modelling of glial brain tumour in rats using the ESR evidence.

email [email protected] Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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2. EXPERIMENTAL METHODS The suspension of the tumour cells A.101.08 was implanted into the left hemisphere (cortex) of week rats (males) (n = 14). After 10 days the tissue samples were taken from the tumour region (t), from the near tumour region, i.e. perifocal zone (nt), from a zone symmetrical to the tumour region (st) and from the hypothalamus (hy). Samples of the blood (bl) and liver (lv) were also collected and frozen. The tissue samples were taken from the blood, liver, cortex and hypothalamus brain regions for the reference group of animals (n = 5). The rats with glial brain tumour were divided into two groups: rats with small (n = 8) and large (n = 6) brain tumours, respectively. The index of iron in the form of iron storage protein ferritin [Ft-Iron] in the whole blood and tissues, the concentration of iron in the form of iron transport protein transferrin [Tf-Iron], the concentration of transferrin [Tf], the transferrin saturation with iron %Tf in the whole blood, the concentration of iron available for chelation with desferrioxamine B in tissues [Df-iron] were determined by our procedures developed using the ESR technique (Mykhaylyk and Dudchenko, 1998; Mykhaylyk et al., 1997). The data are given as mean ± statistic deviation. The value of Fischer’s distribution function was treated as a normalized probabilistic measure of distinctions between experimental and references data sets. The method was realized as a dialogue system for PC and applied to the treatment of the statistical data on nonheme iron status in rats with glial brain tumour.

3. RESULTS AND DISCUSSION The data obtained indicate that iron status of rats was strongly affected by the glial brain tumour development already at the early stage of its growth. It is noteworthy, that highly saturated transferrin can be lost during plasma preparation. Therefore the analysis of the whole blood is preferable for an adequate estimation of iron exchange indices in transferrin pool. The transferrin iron concentration values in the whole blood of the rats with small (29.6 ± 10.7 mkM) and large (27.9 ± 6.7 mkM) tumours are markedly lower than the reference data (33.8 ± 5.8 mkM) (Fig. 1). The transferrin protein concentration values in the whole blood of rats with small (68.8 ± 29.7 mkM) and large (55.7 ± 26.2 mkM) tumours are greater than the reference data (43.9 ± 14.3 mkM). The transferrin saturation determined in the whole blood decreases substantially and reliably in animals with both small and large tumours compared to the reference data (40.9 ± 13.7%). These findings are in close agreement with the observed correlation of depression of serum iron and transferrin saturation upon increasing severity of neoplastic disease (Weinberg, 1984). The ferritin iron indices increased considerably (twofold-to-threefold) and reliably in the tumour tissue and in the cortex symmetric to the tumour and also in hypothalamus and perifocal zone, i.e. the brain as a whole is enriched in ferritin iron at the initial stage of glial tumour growth, as well as in the case of large tumours (Fig. 2A). The ferritin iron indices in the blood and liver tissues are 1.5 and 2 times as much as those in the case of reference group of rats, respectively. It is well known that ferritin protein accumulates in plasma during malignancies (Weinberg, 1984). It follows from our observations that ferritin iron content in the whole blood and liver increased in the case of glial brain tumours.

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Quite recently it has been realized that ferritin iron is the primary determinant of MRI contrast in normal brain tissues and MRI is a useful noninvasive tool for evaluation of brain ferritin iron levels despite difficulties associated with unique magnetic ordering in the ferritin cores complicated with clustering and ordering of ferritin/hemosiderin in tissues (Vymazal et al., 1996). It should be noted that a much greater contribution of components displaying magnetic anisotropy into the ESR spectra of the blood and tumour tissues of rats bearing glial brain tumour compared to the reference spectra was revealed The fact that brain tissues were enriched in ferritin iron during glial neoplasia enabled us to expect that iron chelators might be effective as antiproliferative agents as it is shown in the case of some solid tumors and hematological malignancies (Estrov et al., 1987). The hypothesis has to be tested. The chelatable iron concentration increases reliably in the liver tissue and in all brain regions under study except hypothalamus upon tumour growth (Fig. 2B). The concentration of chelatable iron in tumour tissue is especially high at early stage of tumour growth, which is consistent with the need in iron for tumour growth. Table 1 shows the selected data on Machalonobis distance and normalized distance between samplings for combinations of seven parameters of iron exchange with maximal values. The high values (close to unit) and an increase in distance between samplings upon combined parameters treatment indicate that the parameters are informative ones for glial brain tumour diagnosis in rats. The normalized distance as a function of combination of nonheme iron exchange parameters can be introduced as an essential parameter in the designing a risk model of glial brain tumour in rats.

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The distance between the experimental and reference data sets increased with tumour growth.

4. CONCLUSIONS Our results have shown that nonheme iron status of rats is strongly affected already at the early stage of glial brain tumour growth. The ferritin iron indices increase essentially in the blood, liver, in the brain tumour and in the cortex symmetric to the tumour, i.e. the brain as a whole is enriched in ferritin iron in rats bearing glial brain tumour. A further work implies the estimation of the nonheme iron exchange parameters determined in the blood and brain tissues taken from patients bearing glial brain tumours and evaluation of their diagnostic power as well as testing of iron chelators as antiproliferative agents in the case of glial brain tumours.

REFERENCES Connor, J.R., 1993, Cellular and Regional maintenance of iron homeostasis in the brain: normal and diseased states, in: Iron in central nervous system disorders (P. Riederer and M.B.H. Youdim, eds.), pp. 1–18, Springer-Verlag, New York. Estrov, Z., Tawa, A., Wang, X.H., Dube, I.D., Sulh, H., Cohen, A., Gelfand, E.W., and Freedman, M.H., 1987, In vitro and in vivo effects of deferoxamine in neonatal acute leukemia, Blood 69:757–761. Huges, N.R., 1972, Serum transferrin and ceruloplasmin concentrations in patients with carcinoma, melanoma, sarcoma and cancers of haematopoietic tissues, Aust. J. Exp. Biol. Med. Sci. 50:97–107. Mykhaylyk, O.M., Razumov, O.N., Dudchenko, A.K., Pankratov, Yu. V., Dobrinsky, Ed. K., Sosnitsky, V.N., and Bakai, Ed. A., 1997, Use of ESR, Mossbauer spectroscopy and SQUID-magnetometry for the characterization of magnetic nanoparticles on the base of metal iron and its implications in vivo, in: Scientific and Clinical Applications of Magnetic Carriers (U. Hafeli, W. Schutt, J. Teller, and M. Zborowski, eds.), pp. 177–204, Plenum Press, New York. Mykhaylyk, O.M. and Dudchenko, N.A., 1998, Nonheme iron determination in biological samples on evidence derived from electron spin resonance data, in: Metal Ions in Biology and Medicine, Volume 5 (P. Collery, P. Bratter, V.N. de Bratter, L. Khassanova, and J.-C. Etienne, eds.), pp. 3–7, John Libbey Eurotext, Paris. Recht, L., Torres, C.O., Smith, T.W., Raso, V, and Griffin, T.W., 1990, Transferrin receptor in normal and neoplastic brain tissue: implications for brain-tumor immunotherapy, J. Neurosurg. 72:941–945. Richardson, D.R. and Milnes, K., 1997, The potential of iron chelators of the pyridoxal isonicotinoyl hydrazone class as effective antiproliferative agents II: the mechanism of action of ligands derived from salicylaldehyde benzoyl hydrazone and 2-hydroxy-1-naphthylaldehyde benzoyl hydrazone, Blood 89:3025–3038. Stevens, R.G., Jones, D.Y., Micozzi, M.S., and Taylor, PR., 1988, Body iron stores and the risk of cancer, New Eng. J. Med. 319:1047–1051. Vymazal, J.A., Brooks, R.A., Baumgarner, C.A., Tran, V.A., Katz, D.A., Bulte, J.W.A., Bauminger, R.A., and Di Chiro, G.T., 1996, The relation between brain iron and NMR relaxation times: an in vitro study, J. Magn. Reson. Med. 35:56–61. Weinberg, E.D., 1984, Iron withholding: a defence against infection and neoplasia, Physiol. Rev. 64:65–102.

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IRON BIOAVAILABILITY FROM IRON AMINO ACID CHELATE OR FERROUS SULFATE FOR TREATMENT OF IRON DEFICIENCY ANEMIA

Oscar Pineda, H. De Wayne Ashmead, and Alain Bourdonnais Albion Laboratories, Inc. Clearfield Utah, U.S.A

Iron deficiency anemia frequently occurs in infants and young children as a result of rapid growth. Often, iron (Fe) supplementation becomes an issue due to bioavailability. In a double blind study, 40 Fe deficient anemic infants (6 months to 3 years) were paired for age, weight, and hemoglobin (Hb). Hb ranges were 5–7, 8–9, and 10–11 g/dL. Two blood samples—one at commencement and the other at termination—were obtained from a dorsal vein in the hand of each child. All samples were assayed for Hb and serum ferritin (SF). Daily for 28 days, each child was given a syrup dose containing 5 mg Fe/kg body weight as either iron amino acid chelate (FeAAC) or ferrous sulfate plus of folic acid with earch Fe source. Both treatments resulted in significant (p < 0.001) Hb increases, although the FeAAC group’s increase was greater. The increase in SF was significant (p < 0.005) for the FeAAC group, but not for the group, suggesting greater bioavailability for FeAAC. The bioavailability for each Fe source was calculated using changes in Hb and Sf, computed total blood volume (75 mg/kg body weight) and total Fe dose ingested during the 28 days. The facts that 1 g Hb contains 3.4 mg Fe, and a change equals 10 mg of deposited Fe were incorporated into the calculations. Thus, Fe bioavailability from was calculated at 27.8%, compared to 75.0% for FeAAC. Regression analysis demonstrated that Hb changes were inversely proportional to basal Hb regardless of Fe source. The regression slopes for both Fe sources were similar, even though FeAAC bioavailability was greater. It was concluded that FeAAC was a superior treatment for Fe deficiency anemia because this source demonstrated greater Fe bioavailability than from but was equally well regulated by body Fe stores.

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THE EFFECT OF A HIGH INTAKE OF WHOLE WHEAT BREAD PRODUCED WITH AND WITHOUT PHYTASE ON IRON STATUS IN YOUNG WOMEN

M. Hansen, B. A. Jorgensen, and S. Sandström Research Department of Human Nutrition Centre for Advanced Food Studies The Royal Veterinary and Agricultural University Rolighedsvej 30. DK 1958 Frederiksberg C Denmark

A high intake of whole grain cereals is considered beneficial for health in Western countries due to the high content of fibre and other carbohydrates. Whole wheat bread is also a rich source of minerals including iron, however utilization is often restricted due to a high phytate content. Activating the endogenous activity of phytase or addition of industrially prepared phytase during bread making reduces the phytate content of whole wheat bread. As marginal or deplete iron stores is a common problem in Western countries among children and young women, the effect of a high intake of whole wheat bread produced with and without phytase on iron status was studied. Fourty-one women (19–38 y) with hemoglobin >7.4 mmol/L and serum ferritin were given 300 g of brown bread daily with their habitual diet for 4 months. The subjects were divided in two groups given either bread produced with phytase (n = 23) or the same bread produced without phytase (n = 18). The breads were produce commercially in a large scale. The phytase content was (phytase-bread) and (control bread). Iron status was evaluated from blood hemoglobin and serum ferritin concentrations in a total of 7 blood samples taken before (n = 3), during (n = 1) and after the intervention (n = 3). While hemoglobin was unchanged in both groups, there was a significant reduction in serum ferritin during the four month in the group given phytase bread by (p < 0.001) and in the control group by (p < 0.001). There was, however, no significant difference in change of serum ferritin between the groups suggesting that phytate was not sufficiently reduced. These results show that a high intake of whole meal bread reduces iron stores in young women. Studies are needed to investigate the applicability of methods to improve iron bioavailability in high-phytate bread types. 820

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ZINC STATUS IN PREGNANCY ASSESSED BY HAIR ANALYSIS—NUTRITIONAL INFLUENCES

U. Heins, C. Koebnick, and C. Leitzmann Institute of Nutritional Science Wilhelmstr. 20, 35392 Giessen Germany

INTRODUCTION For the prevention of nutrition-related diseases a reduction of meat consumption is recommended internationally. However meat is an important nutritional source of zinc in western diets. An adequate supply with zinc in pregnancy is necessary for fetal growth and development.

OBJECTIVES To investigate the zinc status of pregnant women following recommendations for an overall healthy diet (in the following called Wholesome Nutrition, WN) in comparison to an average mixed diet.

METHOD Pregnant women adhering to WN for about 2 years (preference of foods of plant origin, little processing of food e.g. whole grain products, up to two portions of meat per week) (n = 56) were compared to women practising an average western diet (control group, n = 32). In each trimenon of pregnancy data were collected. The nutritional zinc intake was assessed by an estimated dietary food record (3 × 4d) and calculated with the German Nutrient Data Bank (Bundeslebensmittelschlüssel BLS II.2). For the assessment of each trimenon zinc status scalp hair was washed with solutions of Triton-X and EDTA to eliminate contaminations and dissolved in concentrated Zinc concentration was analysed by atomic absorption spectrophotometry. 821

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RESULTS The nutrient density of zinc is higher in the WN group than in the control group (p = 0.000). Zinc concentration in hair decreases during pregnancy for both study groups (p = 0.000 and p = 0.003 respectively). In all three trimenons the WN group show higher zinc concentrations in the hair than the control group (p = 0.002),

DISCUSSION The supply with zinc for both groups is adequate during pregnancy, while the zinc content in hair is higher in WN group than in the control group. Because of the more favourable zinc status of those consuming more whole grain products the current discussion of the lower zinc bioavailiability due to phytate cannot be supported. This study shows that a well balanced low-meat or ovo-lacto vegetarian diet during pregnancy may lead to a better supply with zinc.

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IN VITRO PROPERTIES OF FERROUS AND FERRIC IONS WITH PHYTATE

D. Oberleas Food and Nutrition College of Human Sciences Texas Tech University Lubbock, Texas USA

Ferric iron has been utilized in the analysis of phytate since early in this century. Others have studied the properties of iron and phytate in vitro utilizing buffering agents which confounded their data. This is an attempt to study ferrous and ferric ions independently in the absence and presence of phytate at pHs from 1 to 7 and without the use of a confounding buffer. Both 1:1 and 2:1 molar ratios of iron ions to phytate were studied. All solutions were prepared from analytical reagents. Several concentrations of HCl and NaOH were utilized to adjust the pH of these solutions. With 1:1 molar ratio of ferrous iron and phytate, white precipitate formed increasingly to a peak at pH 6. With 1:1 ferric phytate solutions, dense white precipitates formed at pHs 1 and 2. At pH 3, a light rusty-brown precipitate formed, an obvious mixture of ferric phytate and ferric hydroxide. At pH 4, more ferric hydroxide formed mixed with some white ferric phytate. At pH 5 and above, a brown colloidal suspension formed with some brown precipitation at pH 7. Ferric ion was soluble at the higher pHs. At 2:1 molar ratio, ferrous ion reacted similarly to other divalent cations except over a slightly broader pH range. Even at pH of 1, 40% of the ferrous iron was contained in the precipitate. This increased to over 90% at pHs 4 and 5 and declined at pHs 6 and 7. Phytate was detectable in the solutes of samples with pH of 1 and 2 and from the precipitates formed at pHs 2 through 7. The precipitate formed was white at all pHs. For ferric ion, over 98% of the ferric ion was found in the precipitate at pHs 1 and 2. This declined almost linearly from pH 3 to 7 with 72% of the ferric iron in the precipitate at pH 7. Phytate was not detectable in any solute from the ferric ion series at any pH. Phytate was detectable in slightly decreasing amounts in the precipitates as the pH increased from 1 to 7. Visually, white precipitates were present at pHs 1–3 whereas pH 4 precipitate had a slight brown cast and 5 to 7 was increasingly darker brown. This indicates that ferric iron complexes with phytate at all of the pHs studied gradually dissociating to form ferric hydroxide above pH 3. It is impossible to discern whether any of the complexed ferric iron may form hydroxides while still complexed with phytate. In conclusion, ferrous and ferric ions react differently with 823

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phytate. At pHs 5–7 ferrous ion forms the more stable complex with more of the ferric ions forming hydroxides. Ferric hydroxide is more soluble than ferric phytate. Both ferrous and ferric ions may compete favorably for phytate complexation or secondary ion synergism decreasing the absorption of essential elements.

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IRON METABOLISM IN RATS CONSUMING OLIVE OIL OR SUNFLOWER OIL UNUSED OR USED IN REPEATED POTATO FRYING

A. M. Pérez-Granados, M. P. Vaquero, and M. P. Navarro Instituto de Nutrición y Bromatología (CSIC-UCM) Facultad de Farmacia Ciudad Universitaria 28040 Madrid, Spain

The aim of this study was to investigate the influence of the consumption of olive or sunflower oil, unused or after being used in frying, on several aspects of iron metabolism. Olive oil and sunflower oil were used in repeated frying of potatoes without turnover until the oils presented a polar compound content near the limit allowed by law for human consumption (25%). Such value was obtained after 69-time-repeated fryings with olive oil and 48 times with sunflower oil. Olive oil from 48-time-repeated-fryings was also used to compare olive and sunflower oils with the some number of fryings. This oil showed 19.02% of polar compounds. Five groups of weanling rats (initial body weight: 40.0 ± 0.3 g) were fed over 28 days semipurified diets containing, as the only source of dietary fat, 8% of: olive oil (O), olive oil from 48 fryings (O-48 F), olive oil from 69 fryings (O-69 F), sunflower oil (S) and sunflower oil from 48 fryings (S-48 F). Body weight and food intake were monitored weekly and during the last week faecal and urinary excretions were collected. On day 28 the animals were sacrificed and blood, liver and spleen were stored. Serum iron and iron concentrations in erythrocytes were determined. Haemoglobin (Hb) and total iron binding capacity (TIBC) were measured in blood and serum respectively. Food intake and body weight did not vary significantly among groups. Urinary iron excretions were higher in both groups that consumed oil from 48 fryings, either olive oil or sunflower oil, but iron absorption and retention were not affected. Iron contents and concentrations in liver and spleen did not differ between groups. Hb and TIBC values did not show significant differences. The animals consuming sunflower oil, unused or used in frying, showed total erythrocytic iron concentrations higher than the animals consuming the olive oils. Hb and TIBC values did not vary between groups. It was concluded that: 1) olive oil vs sunflower oil is more stable during frying; 2) the consumption of a polyunsaturated oil, such as sunflower oil, compared to olive oil could affect intra and extracellular hematic distribution of iron probably associated to changes 825

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in the membrane permeability; 3) the higher urinary iron excretion caused by the intake of the two oils from 48-time-repeated fryings should be related with alteration compounds in the oil capable of binding iron. Finally, since very small variations were observed by the differents treatments, it is suggested that the consumption of either unused or used frying oils have no deletereous effects on iron metabolism.

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THE FERRITIN SECRETED BY CULTURED HEPATOMA CELLS IN RESPONSE TO IRON AND INFLAMMATORY CYTOKINES STRONGLY RESEMBLES THE FERRITIN IN SERUM AND HAS HOMOLOGY TO INTRACELLULAR FERRITINS AS WELL AS IMMUNOGLOBULINS

L. Butcher, M. Hazegh-Azam, A. Nguyen, P. Nguyen, K. Vu, L. Rezaee, C. Juska, K. Schaffer, M. Zamany, L. Estevez, M. Hallock, and M. C. Linder California State University Fullerton, California 92834-6866 USA

Intracellular ferritins are well characterized iron storage and detoxification proteins, translationally regulated by iron and inflammatory cytokines. Serum ferritin is not well characterized, although its measurement by immunoassays (employing antibodies against intracellular liver and spleen ferritins) is widely employed to assess iron status. We have isolated ferritin from the serum of horses and found that it differs substantially from intracellular ferritin in overall molecular weight as well as in the size and composition of its subunits, which also contain N-linked carbohydrate. We have also found that it resembles low molecular weight heart ferritin, which has a similar subunit composition (Linder et al., Arch Biochem Biophys 273: 34–41, 1989); and we have identified a rat hepatoma cell line that secretes ferritin and responds to treatments with iron and/or IL-1 and TNF by secreting more ferritin without eliciting cell damage (Tran et al., Blood 90: 4979–4986, 1997). In contrast to intracellular ferritin, regulation by iron and IL-1 or TNF appears to be transcriptional. In the studies here described, the secreted ferritin was purified and characterized in preparation for cloning. Secreted ferritin was purified from conditioned medium of rat hepatoma cells that had been grown to confluence in serum containing medium, then acclimated to serumfree and protein-free medium, and finally cultured in the same medium to which either iron [as the Fe(III)-nitrilotriacetate complex; 1:1, mol:mol] or combinations of IL-1 and TNF had been added. Purification involved 70° heat and pH 4.8 acid treatments, as used in intracellular ferritin purification, as well as ammonium sulfate precipitation, and combinations of affinity chromatography involving concanavalin A and protein G. Ferritin 827

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was detected by immunoblotting and quantitated by rocket immunoelectro-phoresis, using antibody against horse spleen ferritin that contained only H and L subunits. The secreted ferritin was composed of subunits of the same sizes as those in horse serum and low molecular weight heart ferritins, primarily 28, 57 and 66 kDa. The larger subunits contained sialic acid and mannose, as determined by blotting with specific lectins. Portions of amino acid sequence were obtained after digestion of subunits with modified trypsin to obtain peptides that were N-terminally sequenced by standards methods. Amino acid sequence showed homology to intracellular ferritin as well as to immunoglobulins. Sequences obtained for serum and low molecular weight heart ferritins also gave homology to immunoglobulins, and about half of human serum ferritin (measured by clinical assays) bound to protein G. The rat liver cell line did not express mRNA for rat immunoglobulins G nor secrete IgG detectable with monoclonal antibodies against these immunoglobulins. These results indicate that serum ferritin is secreted by hepatic cells, and that while having some homology to intracellular ferritin, the secreted serum ferritin is a distinct and different protein that also has homology to the superfamily of immunoglobulins. Supported in part by NSF Grant No. MCB 973745, and USPHS Grants R01 DK53080, SO6 GM 08258, and R25 GM 56820

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TWO LEVELS OF IRON SUPPLEMENTATION AND DEVELOPMENTAL OUTCOME, IRON NUTRITION, AND ADVERSE RESULTS IN LOW BIRTH WEIGHT INFANTS

J. K. Friel, P. F. Kwa, B. Simmons, K. Aziz, C. Mercer, A. MacDonald, and W. L. Andrews Departments of Biochemistry & Pediatrics Memorial University Janeway Child Health Centre St. John’s, Newfoundland Canada, A1B 3X9

Conflicting opinions exist over the optimal amount of iron that low birth weight infants (LBW <2,500g birth weight) should receive. To investigate the effect of increased dietary iron intakes we randomly assigned 58 infants at 2,000 ± 100gm study entry to receive either a premature formula with 12mg iron/L (iron) or 21 mg/L (high iron) for a period of one year. Birth weight (high iron 1,432 ± 390; iron 1,491 ± 339 g) and gestational age (high iron (32 ± 2; iron 32 ± 3 wks) did not differ between groups. At baseline, hospital discharge, 3, 6, 9 and 12 months corrected age, the following measurements were completed: weight, infection rate; RBC hemoglobin (HGB), catalase, hydrogen peroxide fragility and superoxide dismutase (in progress); plasma malondialdehyde (MDA), ferritin, transferrin, zinc and copper; Griffiths Mental Development Scales (GMD) and 3 day diet records. There were no differences at any time for weight, catalase, MDA, or GMD. HGB (High iron 123 ± 9; iron 118 ± 8) showed a trend at 3 months (p = 0.07). Ferritin (High iron 17 ± 8; iron 12 ± 5) was greater in the high iron group at 6 months. Transferrin was greater in the high iron group at 3 months (High iron 320 ± 32; iron 297 ± 43). Plasma zinc (High iron 71 ± 13; iron 94 ± 26) and plasma copper (High iron 107 ± 24; iron 131 ± 29; P < 0.05) at 12 months indicated possible inhibition of zinc and copper absorption with increased iron intake as there was no difference in zinc or copper intakes at 9 or 12 months. Infections other than respiratory, tended (p = 0.01) to be greater in the high iron group at both 9 months (0.06 ± 0.05 vs 0.03 ± 0.05) and 12 months (0.07 ± 0.06 vs 0.03 ± 0.06). There appears to be no advantage to iron intakes above current recommendation for LBW infants. Supported by the Medical Research Council of Canada and Ross Laboratories. 829

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SOME FUNCTIONS OF THE ESSENTIAL TRACE ELEMENT, SELENIUM

Thressa C. Stadtman* Laboratory of Biochemistry National Heart, Lung and Blood Institute National Institutes of Health Bethesda, MD 20892-0320

In recent years there have been impressive increases in information concerning selenium biochemistry. After the initial discoveries that bacteria (Pinsent, 1954) and animals (Patterson et al., 1957; Schwarz and Foltz, 1957) require this trace element, almost 20 years elapsed before selenium was demonstrated to be an essential component of a mammalian enzyme, glutathione peroxidase,(Flohe et al., 1973; Rotruck et al., 1973) and a protein component of the Clostridial glycine reductase complex (Turner and Stadtman, 1973). Selenocysteine was determined to be the chemical form of selenium present in glycine reductase (Cone et al., 1976). Other prokaryotic selenoenzymes, several formate dehydrogenases (Stadtman, 1979, 1980a, 1980b, 1990 and 1996), a hydrogenase of Methanococcus vannielii (Yamazaki, 1982) and a clostridial nicotinic acid hydroxylase (Dilworth, 1982) were discovered. Mammalian glutathione peroxidase isoenzymes (Ursini et al., 1995) and selenoprotein P, a glycoprotein of unknown function, were isolated (Hill et al., 1991). Selenoprotein P is unusual in that it contains 10 selenocysteine residues in the polypeptide chain. Selenoprotein W, a 10 kDa muscle protein of unknown function was purified from normal muscle and partially characterized (Vendeland et al., 1993; Vendeland et al., 1995). The function of selenoprotein W is unknown but the occurrence of the selenocysteine residue in a Cys-x-x-Secys motif is suggestive of a redox role. The importance of selenium in mammalian physiology was magnified by the discovery of its occurrence in a 5' deiodinase that converts the prothyroid hormone, tetraiodothyronine, to the active hormone, 3,5,3'-tri-iodothyronine (Berry et al., 1991) and this established a direct role of selenium in eukaryotic developmental processes. Several years after the identification of selenocysteine in selenoproteins this unusual amino acid was shown to be inserted cotranslationally. An in-frame TGA codon was

Address all corespondence: Laboratory of Biochemistry, NHLBI, NIH, Building 3, Room, 106, MSC-0320 Center Drive, Bethesda, MD 20892-0320. Tel: (301)-496-3002. Fax: (301)-496-0599 Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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found in a mouse glutathione peroxidase gene (Chambers et al., 1986) in a position corresponding to the selenocysteine residue in bovine glutathione peroxidase (Ursini et al., 1995) and TGA in the E. coli formate dehydrogenase gene was shown to direct selenocysteine insertion (Bock and Stadtman, 1988; Zinoni et al., 1986) in the isolated gene product (Stadtman et al.,1991). An important clue came from Roger Sunde’s finding that the carbon chain of serine is the precursor of the selenoamino acid in mammalian glutathione peroxidase (Sunde and Evenson, 1987). Elucidation of the exact mechanism of selenocysteine incorporation was facilitated by availability of a collection of E. coli mutants isolated by Marie-Andree MandrandBerthelot (Graham et al., 1980; Haddock and Mandrand-Berthelot, 1982) that were deficient in ability to synthesize formate dehydrogenase. Genes complementing four of these mutants (Leinfelder et al., 1988a) established requirements for the corresponding gene products later identified as, (SELC) a specific that is aminoacylated by serine (Leinfelder et al., 1988b), (SELA) a pyridoxal phosphate-dependent enzyme that catalyzes the of the serine hydroxyl group forming (Forchhammer et al., 1991), (SELD) an enzyme that synthesizes selenophosphate, the activated selenium donor required for formation (Leinfelder et al., 1990) and (SELB) a specialized elongation factor that after interaction with the selenocysteyl-tRNA and the selenocysteine insertion element (SECIS hairpin) promotes ribosome binding and delivery of selenocysteine as specified by UGA in the mRNA (Bock, 1999; Heider and Bock, 1993; Heider, Baron and Bock, 1992). The elegant studies of August Bock and his associates have unraveled this sequence of events in E. coli and related bacterial species (Bock, 1999) but in eukaryotes there are several pieces of missing information, particularly regarding the mechanism of formation from and the final steps of the process. Whereas the SECIS stem loop structure in E. coli is located adjacent to the UGA in the open reading frame, in eukaryotes the corresponding stem loop structure occurs in the 3' non-translated region of the message (Berry et al., 1993). The occurrence of additional SECIS element variations is suggested by the finding that the putative stem-loop structure in the glycine reductase selenoprotein A gene of Clostridium sticklandii was not recognized by the E. coli translation system (Garcia and Stadtman, 1992). Anaerobic expression of this gene in E. coli resulted in the formation of a full length immunologically reactive protein indicating readthrough of the in-frame UGA codon. However, synthesis of this gene product was independent of the presence of selenium in the growth medium and both wild type and SelD mutant host strains expressed similar amounts of protein. Although catalytic activity was completely dependent on selenium availability, this activity was 10% or less that of normal fully active selenoprotein A isolated from C. sticklandii. The fact that catalytic activity was affected by the Se to S ratio in the culture medium and was independent of selenophosphate availability indicated that UGA in the message was translated as cysteine and to a lesser extent as selenocysteine, both carried by The reactive selenium donor compound that is required both by prokaryotes and eukaryotes for UGA directed specific selenocysteine insertion into selenoproteins is selenophosphate (Ehrenreich et al., 1992; Glass et al., 1993; Veres et al., 1992) an oxygen labile compound formed from ATP and selenide by selenophosphate synthetase, the selD gene product (Veres et al., 1994). Selenophosphate also is the selenium donor for synthesis of 2-selenouridine from 2-thiouridine in the anticodons of certain tRNAs (Veres and Stadtman, 1994; Wittwer and Stadtman, 1986). By site directed mutagenesis it was shown that cysteine-17 and lysine-20 in a glycine rich amino-terminal region of the E. coli selenophosphate synthetase protein are

Some Functions of the Essential Trace Element, Selenium

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essential for enzyme activity (Kim, Veres and Stadtman, 1992; Kim, Veres and Stadtman, 1993). Several isoenzyme forms, mouse and human SPS-2 containing selenocysteine in place of Cys-17 and human SPS-1 with a threonine replacement, have been isolated or predicted from genomic sequences (Alsina et al., 1998; Guimaraes et al., 1996; Lacourciere and Stadtman, 1999). Bacterial homologs of SPS-2 occur in Methanococcus jannaschii and Haemophilus influenzae. A Drosophila potential homolog (Alsina et al., 1998; Persson et al., 1997) with arginine in place of Cys-17 did not exhibit selenophosphate synthetase activity in vitro and failed to complement an E. coli selD mutant in vivo (Persson et al., 1997). A carrier mediated form of selenium analogous to a sulfane sulfur, rather than free selenide per se which is very toxic, may be the actual substrate of the enzymes in vivo (Lacourciere and Stadtman, 1999). In the E. coli selenophosphate synthetase reaction a group on the enzyme is phosphorylated initially by ATP and then the gamma- phosphoryl group present in the phosphoenzyme intermediate is transferred to selenide to form selenophosphate (Mullins et al., 1997; Walker, Ferretti and Stadtman, 1998). The tightly bound ADP moiety remaining on the enzyme is hydrolyzed releasing orthophosphate and AMP. In this process solvent oxygen is incorporated exclusively in the orthophosphate derived from the beta-phosphoryl group of ATP. In the absence of selenide ATP is converted in a very slow reaction completely to AMP and 2 orthophosphate (Veres et al., 1994). AMP is a competitive inhibitor of ATP in the overall reaction but neither orthophosphate nor selenophosphate inhibit except at high concentrations indicating lack of reversibility of the reaction. The bond energy of the gamma phosphoryl group of ATP is conserved in the selenophosphate product as indicated by the ability of selenophosphate to phosphorylate alcohols and amines in aqueous solution (Kaminski et al., 1997). An in vivo role of selenophosphate as a phosphoryl group donor is an intriguing possibility. The most recent addition to the list of known selenoproteins in eukaryotes is mammalian thioredoxin reductase (Tamura and Stadtman, 1996). Two isoenzyme forms, a human cytosolic (TrxRl) and a rat and human mitochondrial (TrxR2) form have been identified (Gasdaska et al., 1995; Gladyshev, Jeang and Stadtman, 1996; Lee et al., 1999; Miranda-Vizuete et al., 1999). Regions involved in nucleotide and FAD binding, the redox active cysteine pair and the peptide sequence -Gly-Cys- Secys-Gly at the Cterminus of TrxR1 and TrxR2 are highly conserved (Gasdaska et al., 1995; Gladyshev, Jeang and Stadtman, 1996; Lee et al., 1999; Miranda-Vizuete et al., 1999). In studies of thioredoxin reductase from cultured human HeLa cells we could show that activity of the enzyme is directly proportional to the amount of selenium present in the protein (Gorlatov and Stadtman, 1998) and selective alkylation at low pH of the selenocysteine residue in fully active enzyme inhibited enzyme activity completely. Fully reduced enzyme in the absence of bound pyridine nucleotide is highly oxygen sensitive and undergoes rapid loss of selenium and loss of catalytic activity upon exposure to air. The selenocysteine residue near the C-terminus of each TrxR subunit clearly is essential for ability of the enzyme to reduce its disulfide substrate with NADPH as electron donor. However, the exact pathway of electron flow from the NADPH substrate via the two previously known redox centers, enzyme-bound FAD and the redox active disulfide, and this third selenocysteine center remains to be established. Thioredoxin reductase has been reported to function as a peroxidase (Bjornstedt et al., 1995) but the very low turnover rate and the rapid loss of catalytic activity (Gorlatov and Stadtman, 1998) in the presence of peroxides suggest that in vivo this enzyme probably does not have a major peroxidase function. The requirement of added

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selenols for appreciable peroxidase activity in vitro (Bjornstedt et al., 1995) is an example of the well-known rate enhancement properties of selenols (Jacob, Maret and Vallee, 1999).

REFERENCES Alsina, B., Serras, F., Baguna, J., and Corominas, M., 1998, patufet, the gene encoding the Drosophila melanogaster homologue of selenophosphate synthetase, is involved in imaginal disc morphogenesis. Mol. Gen Genet 257:113–123. Berry, M.J., Banu, L., and Larsen, P.R., 1991, Type I iodothyronine deiodinase is a selenocysteine-containing enzyme. Nature 349:438–440. Berry, M.J., Banu, L., Harney, J.W., and Larsen, P.R., 1993, Functional characterization of the eukaryotic SECIS elements which direct selenocysteine insertion at UGA codons. EMBO J. 12:3315–3322. Bjornstedt, M., Hamberg, M., Kumar, S., Xue, J., and Holmgren, A., 1995, Human thioredoxin reductase directly reduces lipid hydroperoxides by NADPH and selenocystine strongly stimulates the reaction via catalytically generated selenols. J. Biol. Chem. 270:11761–11764. Bock, A., 1999, Biosynthesis of selenoproteins—an overview. BioFactors 9:(in press). Bock, A. and Stadtman, T.C., 1988, Selenocysteine, a highly specific component of certain enzymes, is incorporated by a UGA-directed co-translational mechanism. BioFactors 1:245–250. Chambers, I., Frampton, J., Goldfarb, P., Affara, N. McBain, W, and Harrison, P.R., 1986, The structure of the mouse glutathione peroxidase gene: the selenocysteine in the active site is encoded by the “termination” codon, TGA. EMBO J. 5:1221–1227. Cone, J.E., del Rio, M., Davis, J.N., and Stadtman, T.C., 1976, Chemical characterization of the selenoprotein component of clostridial glycine reductase: Identification of selenocysteine as the organoselenium moiety. Proc. Nat. Acad. Sci. USA. 73:2659–2663. Dilworth, G.L., 1982, Properties of the selenium-containing moiety of nicotinic acid hydroxylase from Clostridium barkeri. Arch. Biochem. Biophys. 219:30–38. Ehrenreich, A., Forchhammer, K., Tormay, P., Veprek, B., and Bock, A., 1992, Selenoprotein synthesis in E. coli. Purification and characterization of the enzyme catalyzing selenium activation. Eur. J. Biochem. 206:767–773. Flohe, L., Gunzler, W.A., and Schock, H.H., 1973, Glutathione peroxidase: A selenoenzyme. FEBS Lett. 32:132–134. Forchhammer, K., Leinfelder, W., Boesmiller, K., Veprek, B., and Bock, A., 1991, Selenocysteine synthase from Escherichia coli. J. Biol. Chem. 266:6318–6323. Garcia, G.E. and Stadtman, T.C., 1992, Clostridium sticklandii glycine reductase selenoprotein A gene: Cloning, sequencing and expression in Escherichia coli. J. Bacteriol. 174:7080–7089. Gasdaska, P.Y., Gasdaska, J.R., Cochran, S., and Powis, G., 1995, Cloning and sequencing of human thioredoxin reductase. FEBS Lett. 373:5–9. Gladyshev, V.N., Jeang, K.-T., and Stadtman, T.C., 1996, Selenocysteine, identified as the penultimate Cterminal residue in human T-cell thioredoxin reductase, corresponds to TGA in the human placental gene. Proc. Nat. Acad. Sci. USA 93:6146–6151. Glass, R.S., Singh, W.P., Jung, W., Veres, Z., Scholz, T.D., and Stadtman, T.C., 1993, Monoselenophosphate: Synthesis, characterization and identity with the prokaryotic biological selenium donor, Compound SePX. Biochemistry 32:12555–12559. Gorlatov, S.N. and Stadtman, T.C., 1998, Human thioredoxin reductase from HeLa cells: Selective alkylation of selenocysteine in the protein inhibits enzyme activity and reduction with NADPH influences affinity to heparin. Proc. Nat. Acad Sci. USA 95:8520–8525. Graham, A., Jenkens, H.E., Smith, N.H., Mandrand-Berthelot, M.-A., Haddock, B.A., and Boxer, D.H., 1980, The synthesis of formate dehydrogenase and nitrate reductase proteins in various fdh and chl mutants of Escherichia coli. FEMS Microbiol. Lett. 7:145–151. Guimaraes, M.J., Peterson, D., Vicari, A., Cocks, B.G., Copeland, N.G., Gilbert, D.J., Ferrick, D.A., Kastelein, R.A., Bazan, J.F., and Zlotnik, A., 1996, Identification of a novel selD homolog from eukaryotes, bacteria and archae: is there an autoregulatory mechanism in selenocysteine metabolism? Proc. Nat. Acad. Sci USA 93:15086–15091. Haddock, B.A. and Mandrand-Berthelot, M.-A., 1982, Escherichia coli formate-to-nitrate respiratory chain: genetic analysis. Biochem. Soc. Trans. 10:478–480. Heider, J. and Bock, A., 1993, Selenium metabolism in microorganisms. Adv. Microbial Phys. 35:71–109.

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Heider, J., Baron, and Bock, A., 1992, Coding from a distance: dissection of the mRNA determinants required for the incorporation of selenocysteine into a protein. EMBO J. 11:3759–3766. Hill, K.E., Lloyd, R.S., Yang, J.G., Read, R., and Burk, R.F., 1991, The cDNA for rat selenoprotein P contains 10TGA codons in the open reading frame. J. Biol. Chem. 266:10050–10053. Jacob, C., Maret, W., and Vallee, B.L., 1999, Selenium redox biochemistry of zinc-sulfur coordination sites in proteins and enzymes. Proc. Nat. Acad. Sci. USA 96:1910–1914. Kaminski, R., Glass, R.S., Schroeder, T.B., Michalski, J., and Skowronska, A., 1997, Monoselenophosphate: Its hydrolysis and its ability to phosphorylate alcohols and amines. Bioorganic Chem. 25:249–259. Kim, I.Y., Veres, Z., and Stadtman, T.C., 1992, Escherichia coli mutant SELD enzymes. J. Biol. Chem. 267:10650- 19654. Kim, I.Y., Veres, Z., and Stadtman, T.C., 1993, Biochemicl analysis of Escherichia coli selenophosphate synthesis mutants. J. Biol. Chem. 268:27020–27025. Lacourciere, G.M. and Stadtman, T.C., 1999, Catalytic properties of selenophosphate synthetases: Comparison of the selenocysteine-containing enzyme from Haemophilus influenzae with the corresponding cysteine-containing enzyme from Escherichia coli. Pro. Nat. Acad. Sci. USA 96:44–48. Lee, S.-R., Kim, J.-R., Kwon, K.-S., Yoon, H.W., Levine, R.L., Ginsburg, A., and Rhee, S.G., 1999, Molecular cloning and characterization of a mitochondrial selenocysteine-containing thioredoxin reductase from rat liver. J. Biol. Chem. 274:4722–4734. Leinfelder, W., Forchhammer, K., Zinoni, F., Sawers, G., Mandrand-Berthelot, M.-A., and Bock, A., 1988a, Escherichia coli genes whose products are involved in selenium metabolism. J. Bact. 170:540–546. Leinfelder, W., Forchhammer, K., Veprek, B., Zehelein, E., and Bock, A., 1990, In vitro synthesis of selenocysteyl-tRNAuca from seryl-tRNAuca: Involvement and characterization of the selD gene product. Proc. Nat. Acad. Sci. USA 87:543–547. Leinfelder, W., Zehelein, E., Mandrand-Berthelot, M.-A., and Bock, A., 1988b, Gene for a novel tRNA species that accepts L-serine and cotranslationally inserts selenocysteine. Nature 331:723–725. Liu. S.-Y. and Stadtman, T.C., 1997, Heparin-binding properties of selenium-containing thioredoxin reductase from HeLa cells and human lung adenocarcinoma cells. Proc. Nat. Acad. Sci. USA 94:6138–61141. Miranda-Vizuete, A., Damdimopoulos, A.E., Pedrajas, J.R., Gustafsson, J.-A., and Spyrou, G., 1999, The human mitochondrial thioredoxin reductase. cDNA cloning, expression and genomic organization. Eur. J. Biochem. 261:405–412. Mullins, L.S., Hong, S.-B., Gibson, G.E., Walker, H., Stadtman, T.C., and Raushel, F.M., 1997, Identification of a phosphorylated enzyme intermediate in the catalytic mechanism for selenophosphate synthetase. J. Am. Chem. Soc. 119:6684–6685. Patterson, E.L., Milstrey, R., and Stokstad, E.L.R., 1957, Effect of selenium in preventing exudative diathesis in chicks. Proc. Soc. Exp. Biol. Med. 95:617–620. Persson, B.C., Bock, A., Jackie, H., and Vorbruggen, G., 1997, SelD Homolog from Drosophila lacking selenide-dependent monoselenophosphate synthetase activity. J. Mol. Biol. 274:174–180. Pinsent, J., 1954, The need for selenite and molybdate in the formation of formic dehydrogenase by members of the coli-aerogenes group of bacteria. Biochem. J. 57:10–16. Rotruck, J.T., Pope, A.L., Ganther, H.E., Swanson, A.B., Hafeman, D.G., and Hoekstra, W.G., 1973, Selenium: Biochemical role as a component of glutathione peroxidase. Science 179:588-590. Schwarz, K. and Foltz, C.M., 1957, Selenium as an integral part of factor 3 against dietary necrotic liver degeneration. J. Am. Chem. Soc.79:3292–3293. Stadtman, T.C., 1979, Some selenium-dependent biochemical processes. Advances in Enzymology 48:1–28. Ed. A. Meister, John Wiley & sons, Inc. N.Y. Stadtman, T.C., 1980a, Biological functions of selenium. Trends Biochem. Sci. (TIBS) 5:203–206. Stadtman, T.C., 1980b, Selenium-dependent enzymes. Ann. Rev. Biochem. 49:93–110. Stadtman, T.C., 1990, Selenium biochemistry. Ann. Rev. Biochem. 59:111–127. Stadtman, T.C., 1996, Selenocysteine. Ann. Rev. Biochem. 65:83–100. Stadtman, T.C., Davis, J.N., Ching, W.-M., Zinoni, F., and Bock, A., 1991, Amino acid sequence analysis of Escherichia coli formate dehydrogenase (FDHH) confirms that TGA in the gene encodes selenocysteine in the gene product. BioFactors 3:21–27. Sunde, R.A. and Evenson, J.K., 1987, Serine incorporation into the selenocysteine moiety of glutathione peroxidase. J. Biol. Chem. 262:933-937. Tamura, T. and Stadtman, T.C., 1996, A new selenoprotein from human lung adenocarcinoma cells: Purification, properties and thioredoxin reductase activity. Proc. Nat. Acad. Sci USA 93:1006–1011. Turner, D.C. and Stadtman, T.C., 1973, Purification of protein components of the clostridial glycine reductase system and characterization of protein A as a selenoprotein. Arch. Biochem. Biophys. 154:366–381.

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Ursini, F., Maiorino, M., Brigelius-Flohe, R., Aumann, K.D., Roveri, A., Schomburg, D., and Flohe, L., 1995, The diversity of glutathione peroxidases. Methods Enzymol. 252:38–53. Vendeland, S.C., Beilstein, M.A., Chen, C.L., Jensen, O.N., Barofsky, E., and Whanger, P.D., 1993, Purification and properties of selenoprotein W from rat muscle. J. Biol. Chem. 268:17103–17107. Vendeland, S.C., Beilstein, M.A., Yeh, J.-Y, Ream, W., and Whanger, P.D., 1995, Rat skeletal muscle selenoprotein W: cDNA clone and mRNA modulation by dietary selenium. Proc. Nat. Acad. Sci. USA 92:8749–8753. Veres, Z., Kim, I.Y., Scholz, T.D., and Stadtman, T.C., 1994, Selenophosphate synthetase: enzyme properties and catalytic reaction. J. Biol. Chem. 269:10597–10603. Veres, Z. and Stadtman, T.C., 1994, A purified selenophosphate-dependent enzyme from Salmonella typhimurium catalyzes the replacement of sulfur in 2-thiouridine residues in t-RNAs with selenium. Proc. Nat. Acad. Sci. USA 91:8092–8096. Veres, Z., Tsai, L., Scholz, T.D., Politino, M., Balaban, R.S., and Stadtman, T.C., 1992, Synthesis of 5methylaminomethyl-2-selenouiridine in tRNAs: 31P NMR studies show the labile selenium donor synthesized by the selD gene product contains selenium bonded to phosphorus. Proc. Nat. Acad. Sci. USA 89:2975–2979. Walker, H., Ferretti, J.A., and Stadtman, T.C., 1998, Isotope exchange studies on the Escherichia coli selenophosphate synthetase mechanism. Proc. Nat. Acad. Sci. USA 95:2180–2185. Wittwer, A.J. and Stadtman, T.C., 1986, Biosynthesis of 5-methylaminomethyl-2-selenouridine, a naturally occurring nucleoside in Escherichia coli. Arch. Biochem. Biophys. 248:540–550. Yamazaki, S., 1982, A selenium-containing hydrogenase from Methanococcus vannielii. J. Biol. Chem. 257:7926–7929. Zinoni, F., Birkman, A., Stadtman, T.C., and Bock, A., 1986, Nucleotide sequence and expression of the selenocysteine-containing polypeptide of formate dehydrogenase (formate-hydrogen-lyase-linked) from Escherichia coli. Proc. Nat. Acad. Sci. USA 83:4650–4654.

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CHARACTERISTICS AND FUNCTION OF SELENOPROTEIN P

Raymond F. Burk and Kristina E. Hill Division of Gastroenterology Department of Medicine and Clinical Nutrition Research Unit Vanderbilt University School of Medicine Nashville, Tennessee 37232-2279 USA

1. INTRODUCTION Selenoprotein P was so named because it was first identified in the plasma of the rat, where it accounts for more than 60% of the selenium (Read et al., 1990). It is relatively abundant with a peptide concentration of plasma (Read et al., 1990), and its selenium (presumably the protein also) has a half-life in plasma of about 4h (Burk et al., 1991). Based on these data, selenoprotein P can be calculated to have a synthesis rate that is around 4% that of albumin. Thus, maintenance of this selenoprotein has a significant cost to the organism.

2. CHARACTERIZATION Selenoprotein P is the only selenoprotein that has been shown to contain more than one selenium atom per polypeptide chain. Analysis of purified selenoprotein P indicates cDNA has 10 TGAs in the open reading frame, implying a content of 10 selenocysteine residues (Hill et al., 1991). This discrepancy could be explained by several selenocysteine residues being derivatized (see below), but it remains to be clarified. Figure 1 shows the amino acid sequence implied by rat selenoprotein P cDNA. Verification of approximately 80% of the sequence has been accomplished using Edman chemistry and verified residues are underlined in the figure (Himeno et al., 1996). The protein contains several disulfide bridges but the cysteines involved in them have not been determined (Read et al., 1990). cDNAs of selenoprotein P have been cloned from several species and there is 62–87% identity of their implied amino acid sequences (Hill et al., 1991; Hill et al., 1993; Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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Saijoh et al., 1995; Steinert et al., 1997). Comparison of deduced amino acid sequences of selenoprotein P from different species (Burk and Hill, 1999) indicates almost complete conservation of cysteine and selenocysteine residues when these amino acids are considered together. The apparent interchangeability of these two amino acids at some positions in selenoprotein P mirrors the presence of cysteine analogs of selenocysteinecontaining glutathione peroxidases (Perry et al., 1992). A cDNA implying a protein with over 60% identity of amino acid residues with selenoprotein Ps was obtained from bovine brain by a cerebellum minus cerebrum subtraction cloning procedure (Saijoh et al., 1995). It contains 12TGAs in the open reading frame, of which 8 are identical with TGAs in the rat and human cDNAs. Northern analysis demonstrated that most regions of the bovine brain expressed the corresponding mRNA but the paper did not report whether other organs of this species were examined for expression of selenoprotein P. Thus, the possibility remains open that this species expresses a different selenoprotein P in brain from the form expressed in other tissues. This appears unlikely, however, because open reading frames of selenoprotein P cDNAs from rat liver and brain are identical (Yang et al., 1998). Study of selenoprotein Ps from different species will undoubtedly provide additional insights into the metabolism and function of this protein. Selenoprotein P was characterized in 1977 as a heparin-binding protein (Herrman, 1977), and later efforts to study it took advantage of this property (Åkesson and MÂrtensson, 1991; Deagen et al., 1993). Proteins typically bind heparin through interactions of the positively-charged amino acids arginine and lysine with negatively-charged sulfates on the heparin. Histidine can also be positively charged at pHs in the lower physiological range (pKa of histidine is 6.5). Analysis of the primary structure of selenoprotein P revealed 2 dense stretches of histidine residues (residues 185–198 and 225–234 in Fig. 1). It was reasoned that these might serve as pH-dependent heparin-binding sites.

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Therefore, purified selenoprotein P was applied to a heparin-Sepharose column at pH 7 and it was observed that the protein bound to the column. When the column was eluted with a pH gradient, selenoprotein P eluted as several peaks. A short form of the protein that migrated at 45kDa on SDS-PAGE and a long form that migrated at 57 kDa were identified and separated (Chittum et al., 1996). Both these forms appear to be present in plasma because they are detectable in an autoradiograph of SDS-PAGE of plasma (Himeno et al., 1996). The short form (45 kDa) was found in 2 heparin-Sepharose peaks and the long form (57 kDa) was found in 3 peaks. The short form was taken from a peak in which it appeared to be the only protein present and the long form from a peak in which it appeared to be the only protein present. These apparently pure forms of the 2 size isoforms of selenoprotein P were further characterized. N-terminal sequencing revealed that the 2 forms had the same N terminus. Carboxy terminal sequencing was performed using carboxypeptidase P and HPLC detection of released amino acids. Results obtained indicated that the short form terminated precisely at the second TGA in the open reading frame (second selenocysteine in the protein) (Himeno et al., 1996). The long form yielded results that indicated it terminated at the TAA after readthrough of all 10 TGAs. However, the stoichiometry of amino acids recovered from the long form with carboxypeptidase P digestion indicated that one or more of the selenocysteine residues in the carboxy terminus was not detectable by conventional means. This suggests that one or more selenocysteine residues at the C terminus is modified or derivatized. Amino acid analysis of both forms yielded results that corresponded well with the predicted sequences. It was considered that proteolysis might yield the short form from the long form. However, the presence of short and long forms in plasma and the lack of known proteolytic enzymes that cut where the short form terminates are against this explanation. Thus, it was concluded that 2 isoforms of selenoprotein P are present in plasma. They share the same sequence and probably the same mRNA. However, they appear to terminate at different stop codons in the mRNA. This finding suggests that a cell can regulate the amount of the short form it makes and the amount of the long form it makes. It implies that the short and long forms have different functions or mechanisms. Two groups have isolated the promoter region of the selenoprotein P gene (Dreher et al., 1997; Fujii et al., 1997). One showed that a metal response element is present, but expression of selenoprotein P mRNA was not increased by addition of metals to the culture medium.

3. LOCALIZATION Selenoprotein P is an extracellular protein and therefore might be expected to be found in extracellular fluids. Indeed, it is present in plasma. However, it has heparinbinding properties and on this basis it could be expected to associate with heparan sulfate proteoglycans on cell surfaces or in the interstitial matrix. To evaluate the distribution of selenoprotein P, immunohistochemistry with a polyclonal antibody that had been raised against purified rat selenoprotein P was used to examine a number of rat tissues (Burk et al., 1997). Selenoprotein P was heavily concentrated on endothelial cells in all tissues. There was striking localization of selenoprotein P to glomerular capillaries in the kidney and to sinusoid-lining endothelial cells in the liver. Brain capillaries and arterial endothelial cells also stained.

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4. EFFECT OF SELENIUM DEFICIENCY Selenoprotein P plasma concentration falls in selenium deficiency. In low-selenium areas of China, plasma selenoprotein P is 10–20% of control values (Hill et al., 1996). Selenoprotein P is better conserved in selenium deficiency than is glutathione peroxidase (Yang et al., 1989). This appears to be related to preservation of selenoprotein P mRNA in selenium deficiency while liver GSHPx-1 mRNA falls. This has the effect of diverting selenium from GSHPx-1 to selenoprotein P under deficiency conditions and indicates the importance of selenoprotein P to the organism. Induction of selenium deficiency in the rat causes the plasma concentration of selenoprotein P to decline to around 5% of control concentration (Yang et al., 1989). Differences could not be demonstrated in selenoprotein P immunostaining of glomerular capillaries between kidneys taken from selenium-deficient rats and from control rats (Burk et al., 1997). This suggests that bound selenoprotein P is not depleted by selenium deficiency as much as is plasma selenoprotein P. Thus, the possibility exists that plasma selenoprotein P is a reserve pool of the protein and that the functioning protein is that bound to cells.

5. FUNCTION The biochemical function of selenoprotein P has not yet been established. Because of its high selenium content and extracellular location, selenoprotein P has been suggested to be a selenium transport protein. Selenium is covalently bound in the protein so its release would require breakdown of the protein. Thus, a general transport role is unlikely. A study of uptake of selenium administered intravenously as selenoprotein P showed that most tissues took up the same amount of selenium regardless of the selenium status of the animal (Burk et al., 1991). However, the brain was an exception, being much more efficient in selenium uptake from selenoprotein P in selenium deficiency than in the selenium-replete state. Thus, the possibility that selenoprotein P transports selenium (or itself) to the brain remains open. An in vivo model linking selenoprotein P with protection against tissue injury has been reported (Burk et al., 1980). In the model, selenium-deficient rats administered doses of diquat much lower than the LD50 for selenium-replete rats die within hours. Massive lipid peroxidation (measured by plasma F2 isoprostanes) occurred in the minutes following diquat administration to the selenium-deficient rats. Massive liver necrosis ensued at 2–4 hours. However, accumulation of F2 isoprostanes did not occur in the liver, even after it was injured (Burk et al., 1995). Several interpretations of these results are possible. One is that the injury of the hepatocyte occurred at its plasma membrane and that internal hepatocyte membranes did not peroxidize. This was consistent with the demonstration that injection of selenium protected against the injury only after selenoprotein P had risen. Selenoprotein P is outside the cell and would have access only to plasma membranes of endothelial cells and hepatocytes. Thus, we postulated that diquat led somehow to a radical attack on the endothelial cells or the hepatocyte plasma membrane that was defended against by selenoprotein P (from outside the cell). Our more recent results point to Se-P protecting the liver endothelial cells against oxidant injury (Atkinson et al., 1998). These clues suggest that anchored locations in the extracellular

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space are sites of selenoprotein P function and made our studies of its localization important (Burk et al., 1997). Because selenoproteins are redox proteins, it is likely that selenoprotein P serves a redox function in the extracellular space. Association of the protein with endothelial cells has led us to postulate that selenoprotein P protects endothelial cells against oxidant molecules such as those produced during inflammation.

6. CONCLUSIONS Selenoprotein P has several characteristics that may be clues to its function. It contains many selenium atoms and therefore probably has a redox function. It binds heparin in a pH-dependent manner that would predict its attraction to areas of inflammation where pH is low. It is associated with endothelial cells which produce nitric oxide. It appears to protect against diquat toxicity in the rat liver. Diquat produces superoxide which can react with nitric oxide to yield peroxynitrite. Thus, it seems possible that selenoprotein P may protect cell membranes against peroxynitrite.

ACKNOWLEDGMENT The research of the authors is supported by NIH ES 02497.

REFERENCES Åkesson, B. and Martensson, B., 1988, Heparin interacts with a selenoprotein in human plasma, J. Inorg. Biochem. 33:257–261. Atkinson, J.B., Hill, K.E., and Burk, R.F., 1998, Sinusoidal endothelial cell injury: An early lesion in diquatinduced massive liver necrosis of selenium-deficient rats, Hepatology 28:324A, 1998. Burk, R.F. and Hill, K.E., 1999, Orphan selenoproteins, BioEssays 21:231–237. Burk, R.F., Hill, K.E., Awad, J.A., Morrow, J.D., Kato, T., Cockell, K.A., and Lyons, P.R., 1995, Pathogenesis of diquat-induced liver necrosis in selenium-deficient rats. Assessment of the roles of lipid peroxidation and selenoprotein P, Hepatology 21:561–569. Burk, R.F., Hill, K.E., Boeglin, M.E., Ebner, F.F., and Chittum, H.S., 1997, Selenoprotein P associates with endothelial cells in rat tissues, Histochem. Cell Biol. 108:11–15. Burk, R.F., Hill, K.E., Read, R., and Bellew, T., 1991, Response of rat selenoprotein P to selenium administration and fate of its selenium, Am. J. Physiol. 261:E26–E30. Burk, R.F., Lawrence, R.A., and Lane, J.M., 1980, Liver necrosis and lipid peroxidation in the rat due to paraquat and diquat: Effect of selenium deficiency, J. Clin. Invest. 65:1024–1031. Chittum, H.S., Himeno, S., Hill, K.E., and Burk, R.F, 1996, Forms of selenoprotein P in rat plasma, Arch. Biochem. Biophys. 325:124–128. Deagen, J.T., Butler, J.A., Zachara, B.A., and Whanger, P.D., 1993, Determination of the distribution of selenium between glutathione peroxidase, selenoprotein P, and albumin in plasma, Anal. Biochem. 208:176–181. Dreher, I., Jakobs, T.C., and Köhrle, J., 1997, Cloning and characterization of the human selenoprotein P promoter. Response of selenoprotein P expression to cytokines in liver cells, J. Biol. Chem. 272:29364–29371. Fujii, M., Saijoh, K., Kobayashi, T., Fujii, S., Lee, M.J., and Sumino, K., 1997, Analysis of bovine selenoprotein P-like protein gene and availability of metal responsive element (MRE) located in its promoter, Gene 199:211–217. Herrman, J.L., 1977, The properties of a rat serum protein labelled by the injection of sodium selenite, Biochim. Biophys. Acta 500:61–70.

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Hill, K.E., Lloyd, R.S., and Burk, R.F., 1993, Conserved nucleotide sequences in the open reading frame and 3'-untranslated region of selenoprotein P, Proc. Natl. Acad. Sci. 90:537–541. Hill, K.E., Lloyd, R.S., Yang, J.-G., Read, R., and Burk, R.F., 1991, The cDNA for rat selenoprotein P contains ten TGA codons in the open reading frame, J. Biol. Chem. 266:10050–10053. Hill, K.E., Xia, Y., Åkesson, B., Boeglin, M.E., and Burk, R.F., 1996, Selenoprotein P concentration in plasma is an index of selenium status in selenium-deficient and selenium-supplemented Chinese subjects, J. Nutr. 126:138–145. Himeno, S., Chittum, H.S., and Burk, R.F., Isoforms of selenoprotein P in rat plasma. Evidence for a fulllength form and another form that terminates at the second UGA in the open reading frame, J. Biol. Chem. 271:15769–15775. Perry, A.C.F., Jones, R., Niang, L.S.P., Jackson, R.M., and Hall, L., 1992, Genetic evidence for an androgenregulated epididymal secretory glutathione peroxidase whose transcript does not contain a selenocysteine codon, Biochem. J. 285:863–870. Read, R., Bellew, X, Yang, J.-G, Hill, K.E., Palmer, I.S., and Burk, R.F., 1990, Selenium and amino acid composition of selenoprotein P, the major selenoprotein in rat serum, J. Biol Chem. 265:17899–17905. Saijoh, K., Saito, N., Lee, M.J., Fujii, M., Kobayashi, T., and Sumino, K., 1995, Molecular cloning of cDNA encoding a bovine selenoprotein P-like protein containing 12 selenocysteines and a (His-Pro) rich domain insertion, and its regional expression, Mol. Brain Res. 30:301–311. Steinert, P., Ahrens, M., Gross, G., and Flohé, L., 1997, cDNA and deduced polypeptide sequence of a mouse selenoprotein P, Biofactors 6:311-–19. Yang, J.-G., Hill, K.E., and Burk, R.F., 1989, Dietary selenium controls rat plasma selenoprotein P concentration, J. Nutr. 119:1010–1012. Yang, X.G., Hill, K.E., and Burk, R.F., 1998, Secretion of selenoprotein P by rat astrocytes and by other cell types in primary cell culture, FASEB J. 12:A824.

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SOME BIOCHEMICAL FUNCTIONS OF SELENIUM IN ANIMALS AND MAN J. R. Arthur1 and G. J. Beckett2 1

Rowett Research Institute Greenburn Road, Bucksburn Aberdeen, AB21 9SB UK 2 University Department of Clinical Biochemistry The Royal Infirmary Edinburgh, EH3 9YW UK

1. INTRODUCTION Differing dietary intakes of selenium have been associated with a range of biochemical functions and diseases in animals and humans. Many of these diseases and biochemical responses can be associated with changes in expression and activity of proteins that contain selenium as selenocysteine at the active site (Arthur and Beckett, 1994; Foster and Sumar, 1997). At physiological pH the selenol residue of selenocysteine is very reactive; consequently it can facilitate many biochemical reactions which involve oxidation and reduction of the selenium. The biochemical reactions catalysed by mammalian selenoproteins fall into three broad categories, namely antioxidant defence systems, thyroid hormone metabolism and redox control of cell reactions (Arthur, 1999). These three processes are not necessarily exclusive in their requirements for the different selenoproteins. For example, the glutathione peroxidases are necessary for both antioxidant systems and the control of thyroid hormone synthesis (Howie et al., 1995; 1998).

2. SELENOPROTEINS The central role of selenium in essential areas of cell metabolism is reflected in the ever increasing number of selenoproteins, which have been identified, cloned and characterised. Approximately 30 selenoproteins can be detected in mammalian tissues by in vivo labelling with and nearly half of these can now be identified. In order to demonstrate the mechanisms, which underlie selenium responsive diseases it is essential to Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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understand how these different selenoproteins respond to changes in selenium levels in the diet (Arthur and Beckett, 1994). This is not a straightforward task since there is selective expression of selenoproteins in different tissues, which also varies from species to species. For example, type I deiodinase which converts thyroxine to the more biologically active triiodothyronine is highly expressed in thyroids of humans, rats, mice and guinea pigs whereas the expression is very low in most ruminant species and pigs (Beech et al., 1993). The type I deiodinase is also expressed in the brown adipose tissue of ruminant animals in the first 3 weeks of life (Trayhurn et al., 1993; Nicol et al., 1994). However, in rodents and humans the brown adipose tissue contains type II deiodinase. Although both these deiodinases are selenium-containing proteins there is a fundamental difference between their metabolic effects. Type I deiodinase provides the main source of plasma T3 which can then enter a range of tissues and interact with nuclear T3 receptors and thus modify the expression of T3 responsive genes (Larsen and Berry, 1995). In contrast the type II deiodinase produces T3 which has only a local action and makes little contribution to the circulating pool of T3 (Larsen and Berry, 1995; Mitchell et al., 1998). Tissues which contain the type II deiodinase show little response to changes in circulating concentrations of T3 unless plasma levels are increased into the concentration range found in hyperthyroidism: such tissues however respond to only modest changes in circulating T4 (Mitchell et al., 1998).

3 INVESTIGATION OF SELENOPROTEIN FUNCTION Taking into account the species-specific and organ-specific expression of different selenoproteins, the conventional way to investigate the biochemical functions of selenium is to study animals or humans consuming different amounts of selenium in the diet (Arthur and Beckett, 1994). The selenium proteins, which are most sensitive to the low levels of the micronutrients in the diet, are those that are most likely to be implicated in any disease process. In such experiments, the levels of selenium intake, which are needed to retain normal health may vary in response to the intake of other nutrients in the diet. Many debilitating conditions that are prevented by selenium supplementation occur only when there is combined selenium and vitamin E deficiencies (Arthur and Beckett, 1994). These tend to be degenerative diseases such as skeletal and cardiac myopathies (white muscle disease) which occur particularly in cattle and sheep. In pigs, such myopathies also occur in addition to liver necrosis. Such degenerative diseases are likely to be a consequence of the loss of glutathione peroxidase activities, which interact with vitamin E deficiency to allow free radical-mediated reactions that damage cell membranes. In particular, phospholipid hydroperoxide glutathione peroxidase, which is associated with cell membranes is closely associated with the functions of vitamin E (Maiorino et al., 1991). This membrane associated peroxidase can metabolise phospholipid hydroperoxides in membranes which cytosolic glutathione peroxidase cannot react with. Although loss of phospholipid hydroperoxide glutathione peroxidase activity, coupled with vitamin E deficiency, can explain degenerative disease under these conditions, selenium deficiency also causes significant decreases in expression of other selenoproteins (Bermano et al., 1995). Thus loss of deiodinase activities and lowered thioredoxin reductase activities have the potential to affect many aspects of metabolism in the cell (Mitchell et al., 1997; Howie et al., 1998). In addition, in the presence of both adequate or deficient dietary vitamin E, selenium deficiency can impair thyroid hormone metabolism, impair immune responses and increase cardiotoxicity of Coxsackie B3 virus (Levander and Beck, 1997).

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4 SELENIUM IN HUMANS There is a wide range of selenium intakes in human populations. The lowest levels of blood selenium occur in populations in China, Tibet and Zaire (Morenoreyes et al., 1998). In these countries, very different conditions have been associated with low selenium status. In China, Keshan disease, a cardiomyopathy particularly prevalent in growing children and pregnant females has been almost completely eliminated by selenium supplementation. A similar very low selenium status in Tibet has been associated with Kaschin Beck disease, which may also be exacerbated by iodine deficiency (Morenoreyes et al., 1998). The low selenium status in Zaire does not seem to cause conditions similar to Keshan disease or Kaschin Beck disease. Iodine deficiency is common in the low selenium areas of Zaire and the two deficiencies may interact to cause the severest forms of myxoedematous cretinism, a debilitating disease involving impaired neurological development and growth (Ngo et al., 1997). Low selenium status in humans has also been associated with increased incidence of cancer and coronary heart disease although such epidemiological studies should always be interpreted with caution. However, many studies with selenium deficient rats and mice have shown that they are more susceptible to chemical carcinogenesis (Nelson et al., 1996). In humans, selenium supplementation over several years has decreased the incidence of colon, prostate and lung cancers in subjects with previously diagnosed skin cancer (Clark et al., 1996). This selenium supplementation was carried out in an American population with a dietary selenium intake of at least twice that found in most European countries. Further research is required to determine whether such effects are a consequence of modulation of selenoprotein expression or a pharmacological consequence of a high selenium intake. In order to understand the biochemical functions of selenium in humans it is possible to study the expression of selenoproteins in a number of cell lines. Human umbilical vein endothelial cells (HUVECs), thyroid and skin cells all have very different patterns of selenoprotein expression revealed by labelling with In each cell type the predominant protein labelled with is different, and in HUVECs it is notable that a 58 kDa protein accounts for approximately 40% of protein labelling (Howie et al., 1998; Rafferty et al., 1998). This protein has been identified as thioredoxin reductase by Western blotting against antibodies raised against the selenoprotein purified from rat liver or human placenta (Anema et al., 1999). This form of thioredoxin reductase is distinct from the 54 kDa thioredoxin reductase which is associated with mitochondria in liver (Lee et al., 1999). In human thyrocytes the 58 kDa protein accounts for a much smaller proportion of total radioactivity after in vitro labelling with However, treatment of human thyrocytes in culture with a calcium ionophore, (A23187) causes a remarkable induction of thioredoxin reductase labelling and protein identified by Western blotting. Phorbol 12-myristate 13-acetate (PMA) also induces expression of thioredoxin reductase, but is much less effective than A23817. In addition, the TSH-cyclic AMP pathway does not affect expression of thioredoxin reductase in thyrocytes (Howie et al., 1998). Thus the regulation of thioredoxin reductase and extracellular glutathione peroxidase expression are regulated in similar ways in thyrocytes. An important function of thioredoxin reductase is to regulate DNA-binding activity of transcription factors through maintaining reduction of thioredoxin in the cell cytosol. In addition thioredoxin reductase can act as an antioxidant in an analogous way to glutathione peroxidases thus protecting cells from hydrogen peroxidase and lipid hydroperoxides. Either a redox regulatory or antioxidant

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function of thioredoxin reductase would be important to the thyrocyte. In particular, hydrogen peroxide is produced during thyroid hormone synthesis and any system, which guards against the potentially injurious effects of peroxide induced free radical damage would be of potential importance to the cell. Additionally, the second messenger systems, which induce thioredoxin reductase expression would also be involved in changes in expression of many cellular proteins and effects that would be mediated by transcription factors. Selenium deficiency can induce a large number of enzymes involved in xenobiotic metabolism in the liver (McLeod et al., 1997). Glutathione S-transferases and aldehyde reductases involved in aflatoxin metabolism and activation of potential carcinogens, all increase in activity in the selenium-deficient rat liver. This effect could be mediated by selenium-induced changes in transcription factor activity. There are several instances where selenium has been hypothesised to have a general effect on cell function and metabolism and the mechanism underlying these effects could well be modulation of thioredoxin reductase activity (McLeod et al., 1997). Future research should address the question of whether the potential beneficial anti-cancer effects and immunestimulating effects of selenium are mediated through modulation of thioredoxin reductase activity.

5. CONCLUSIONS Thus there is considerable evidence for an essential role of selenium in many processes in the cell and a major challenge is to relate this biochemical knowledge to a potential for low dietary selenium to be involved in the development of diseases. It is difficult to relate determination of single, selenium-containing enzyme activities or total selenium content of blood to the many selenoenzymes in tissues of humans. However, in animal studies selenoenzyme activities in different tissues are very consistent and show some correlation with blood selenium concentrations. Thus in animals it is possible to correlate a particular blood selenium concentration with metabolic effects in specific tissues. Diets consumed by humans are much more variable than those consumed by animals in investigations of selenium status and there is little data to relate particular blood selenium concentrations to activities of selenoproteins in different organs. Thus, there is much circumstantial evidence, which relates to inadequate supply of dietary selenium to optimal health in human subjects. Further experiments are needed to relate to different dietary selenium intakes to health problems such as cancer and impaired immune function. This work is critically facilitated by detailed studies of selenium biochemistry and function in cell culture and experimental animals. JRA is grateful to the Scottish Office Agriculture, Environment and Fisheries Department for financial support. We are grateful to our collaborators whose work has been extensively cited in the reference list.

REFERENCES Anema, S.M., Walker, S.W., Howie, A.F., Arthur, J.R., Nicol, R, and Beckett, G.J., 1999, Thioredoxin reductase expression in human umbilical vein endothelial cells. J. Endocrinol. 160:P165. Arthur, J.R., 1999, Functional indicators of iodine and selenium status. Proc. Nutr. Soc. In press. Arthur, J.R. and Beckett, G.J., 1994, New metabolic roles for selenium. Proc. Nutr. Soc. 53:615–624.

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Beech, S.G., Walker, S.W., Dorrance, A.M., Arthur, J.R., Nicol, F., Lee, D., and Beckett, G.J., 1993, The role of thyroidal type-I iodothyronine deiodinase in tri- iodothyronine production by human and sheep thyrocytes in primary culture. J. Endocrinol. 136:361–370. Bermano, G., Nicol, F., Dyer, J.A., Sunde, R.A., Beckett, G.J., Arthur, J.R., and Hesketh, J.E., 1995, Tissuespecific regulation of selenoenzyme gene expression during selenium deficiency in rats. Biochem. J. 311:425–430. Clark, L.C., Combs, G.F., Turnbull, B.W., Slate, E.H., Chalker, O.K., Chow, J., Davis, L.S., Glover, R.A., Graham, G.F., Gross, E.G., Krongrad, A., Lesher, J.L., Park, H.K., Sanders, B.B., Smith, C.L., and Taylor, J.R., 1996, Effects of selenium supplementation for cancer prevention in patients with carcinoma of the skin: A randomized controlled trial. JAMA 276:1957–1963. Foster, L.H. and Sumar, S., 1997, Selenium in health and disease: A review. Crit. Rev. Food Sci. Nutr. 37:211–228. Howie, A.F., Arthur, J.R., Nicol, R, Walker, S.W., Beech, S.G., and Beckett, G.J., 1998, Identification of a 57-kilodalton selenoprotein in human thyrocytes as thioredoxin reductase and evidence that its expression is regulated through the calcium-phosphoinositol signaling pathway. J. Clin. Endocrinol. Metab. 83:2052–2058. Howie, A.F., Walker, S.W., Akesson, B., Arthur, J.R., and Beckett, G.J., 1995, Thyroidal extracellular glutathione peroxidase: A potential regulator of thyroid-hormone synthesis. Biochem. J. 308:713–717. Larsen, P.R. and Berry, M.J., 1995, Nutritional and hormonal regulation of thyroid hormone deiodinases. Ann. Rev. Nutr. 15:323–352. Lee, S.R., Kim, J.R., Kwon, K.S., Yoon, H.W., Levine, R.L., Ginsburg, A., and Rhee, S.G., 1999, Molecular cloning and characterization of a mitochondrial selenocysteine-containing thioredoxin reductase from rat liver. J. Biol. Chem. 274:4722–4734. Levander, O.A. and Beck, M.A., 1997, Interacting nutritional and infectious etiologies of Keshan disease— Insights from Coxsackie virus B-induced myocarditis in mice deficient in selenium or vitamin E. Biol. Tr. Elem. Res. 56:5–21. Maiorino, M., Thomas, J.P., Girotti, A.W., and Ursini, F., 1991, Reactivity of phospholipid hydroperoxide glutathione peroxidase with membrane and lipoprotein lipid hydroperoxides. Free Rad. Res. Commun. 12:131–135. McLeod, R., Ellis, E.M., Arthur, J.R., Neal, G.E., Judah, D.J., Manson, M.M., and Hayes, J.D., 1997, Protection conferred by selenium deficiency against aflatoxin B- 1 in the rat is associated with the hepatic expression of an aldo- keto reductase and a glutathione S-transferase subunit that metabolize the mycotoxin. Cancer Res. 57:4257–4266. Mitchell, J.H., Nicol, F., Beckett, G.J., and Arthur, J.R., 1997, Selenium and iodine deficiencies: effects on brain and brown adipose tissue selenoenzyme activity and expression. J. Endocrinol. 155:255–263. Mitchell, J.H., Nicol, F., Beckett, G.J., and Arthur, J.R., 1998, Selenoprotein expression and brain development in preweanling selenium- and iodine-deficient rats. J. Molecular. Endocrinol. 20:203–210. Morenoreyes, R., Suetens, C., Mathieu, F., Begaux, F., Zhu, D., Rivera, M.T., Boelaert, M., Neve, J., Perlmutter, N, and Vanderpas, J., 1998, Kashin-Beck osteoarthropathy in rural Tibet in relation to selenium and iodine status. N. Engl. J. Med. 339:1112–1120. Nelson, R.L., Abcarian, H., Nelson, T.M., Misumi, A., Kako, H., Rizk, S., and SkyPeck, H., 1996, The effect of dietary selenium deficiency on acute colorectal mucosal nucleotoxicity induced by several carcinogens in the rodent. Am. J. Surg. 172:85–88. Ngo, B., Dikassa, L., Okitolonda, W., Kashala, T.D., Gervy, C., Dumont, J.E., Yanovervelt, N., Contempre, B., Diplock, A.T., Peach, S., and Vanderpas, J.B., 1997, Selenium status in pregnant women of a rural population (Zaire) in relationship to iodine deficiency. Trap. Med. Internat. Health 2:572–585. Nicol, F., Lefranc, H., Arthur, J.R., and Trayhurn, P., 1994, Characterization and postnatal development of 5'deiodinase activity in goat perirenal fat. Am. J. Physiol. 267:R144–R149. Rafferty, T.S., McKenzie, R.C., Hunter, J.A., Howie, A.F., Arthur, J.R., Nicol, F., and Beckett, G.J., 1998, Differential expression of selenoproteins by human skin cells and protection by selenium from UVBradiation-induced cell death. Biochem. J. 332:231–236. Trayhurn, P., Thomas, M.E.A., Duncan, J.S., Nicol, F., and Arthur, J.R., 1993, Presence of the Brown FatSpecific Mitochondrial Uncoupling protein and iodothyronine 5'-deiodinase activity in subcutaneous adipose tissue of neonatal lambs. FEBS Lett. 322:76–78.

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THE SECIS ELEMENT IN SELENOPROTEIN MRNAS. A ROSETTA STONE FOR DECODING THE UGA SELENOCYSTEINE CODON AND A MOLECULAR TAG FOR UNVEILING NEW SELENOPROTEINS 1

2

1,3

1

Alain Lescure , Daniel Gautheret , Robert Walczak , Philippe Carbon , 1 and Alain Krol 1

UPR 9002 du CNRS Structure des Macromolécules Biologiques et Mécanismes de Reconnaissance Institut de Biologie Moléculaire et Cellulaire 67084 Strasbourg, France 2 EP CNRS 91 Information GÈnÈtique et Structurale Marseille, France 3 Present address: Institut de Biologie Animale UniversitÈ de Lausanne Switzerland

1. INTRODUCTION Selenocysteine is the major biological form of selenium in cells, in which selenium replaces sulfur. It is cotranslationally incorporated into selenoproteins, and for those selenoenzymes for which a function has been ascribed, it was found in the active center (Stadtman, 1996). Until now, seven selenoprotein families have been described. What characterizes them is their implications in the different oxidation-reduction reactions encountered in various pathways. In effect, one can cite the glutathione peroxidase family for scavenging free radicals, the thioredoxin reductase-thioredoxin couple acting in essential functions such as synthesis of deoxynucleotides and maintaining the redox status of the cell, and lastly the iodothyronine deiodinases involved in developmental

Address all correspondence to: Dr. Alain Krol; UPR 9002 du CNRS Structure des Macromolécules Biologiques et Mécanismes de Reconnaissance. Institut de Biologie Moléculaire et Cellulaire. 67084 Strasbourg Cedex. France; telephone: 33-3-88-41-70-50; fax: 33-3-88-60-22-18; email: [email protected] Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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processes (Burk and Hill, 1999). Containing itself selenocysteine, especially appealing is selenophosphate synthetase SPS2, the enzyme implicated in selenocysteine biosynthesis by converting selenite to phosphoselenoate (Guimaraes et al., 1996). Aside from these proteins, three others of unknown functions have been characterized, SelW, SelP and the 15 kDa selenoprotein (Gladyshev et al., 1998; reviewed by Burk and Hill, 1999). The selenocysteine biosynthesis pathway is remarkable (reviewed by Hüttenhofer and Böck, 1998). The first step consists in the charging of serine on a specialized tRNA, the Selenocysteine synthase subsequently ensures the seryl- to selenocysteylconversion on the Selenocysteine being encoded by an in-frame UGA codon, its cotranslational incorporation into proteins appeals to an original mechanism in order to discriminate UGA selenocysteine from UGA stop codons. In prokaryotes, this is readily achieved through the intervention of a stem-loop structure immediately downstream of the UGA selenocysteine codon. This specific structure is recognized by SelB, a specialized elongation factor which also binds the Thus, the binding of SelB to both the mRNA and the charged enables designation of the UGA selenocysteine codon and the direct presentation of the charged to the A site of the ribosome. Much less is known in eukaryotes about the selenocysteine incorporation mechanism. For example, the protein factors involved have not been identified yet. Remarkably, readthrough of the UGA selenocysteine codon has been shown to require the presence of SECIS (SElenoCysteine Insertion Sequence), an RNA stem-loop structure residing in the 3' untranslated region (3’UTR) of selenoprotein mRNAs (Berry et al., 1991). SECIS is essential for the recognition of UGA as a selenocysteine codon rather than a codon for termination of translation; its localization within the 3’UTR is different between different selenoprotein mRNAs. Rather surprisingly, it could even be found lying 5 kb downstream from the selenocysteine codon in the type 2 iodothyronine deiodinase (Buettner et al., 1998). Different proteins carrying binding specificities to the SECIS element were described (Shen et al., 1995; Hubert et al., 1996; Lesoon et al., 1997), but the demonstration that they actually participate in the decoding of the selenocysteine codon has not been brought yet. From the above description, it is obvious that the challenge rests in the discovery of the structural and functional relationships between the SECIS element and the selenocysteine codon. In particular, it is especially important to establish how the SECIS element can fulfill its role while residing far downstream from the codon to be designated. To address the issue, we i) proposed a model for the SECIS element (Walczak et al., 1996); ii) tested it by structure-function experiments (Walczak et al., 1998). Deduced from such investigations, it appeared that the detailed knowledge of structure-function constraints in the SECIS element can constitute an invaluable asset for unveiling new selenoprotein mRNAs in databases (see below).

2. STRUCTURE-FUNCTION STUDIES OF THE SECIS ELEMENT In an earlier work, we proposed an experimentally-derived secondary structure model for the SECIS element, generated by enzymatic and chemical structure probing of the SECIS RNA, as well as by molecular modeling (Walczak et al., 1996). Further, sequence comparisons with more than 30 different SECIS elements allowed us to refine the model, leading to the consensus secondary structure shown in Fig. 1. The stem-loop structure proposed by the consensus model consists in two helices I and II interrupted by an internal loop, an apical loop surmounting helix II. Among these

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structural features, one could observe that only helix II possesses a rather fixed, 14–15 bp length. That of helix I is variable, as is those of the internal and apical loops. The SECIS element is characterized by a low degree of sequence conservation, exhibiting invariant residues only in the internal and apical loops where an A and the sequence AAR were found, respectively. The AAR sequence does not show a strict positional conservation, the 5’A being either adjacent to, or separated by a few nucleotides from the top closing base pair of helix II. The most striking sequence/structure conservation occurs within helix II, where resides a quartet of non-Watson-Crick base pairs with the invariant G.A/A.G tandem occupying the central position. Clearly, based on these observations the SECIS element should be seen as an RNA stem-loop in which the global secondary structure is conserved, rather than the nucleotide sequence. The functional relevance of the structural features identified was subsequently tested, providing simultaneously a means for validating the merits of the model (Walczak et al., 1998). Single point mutations were introduced into the SECIS element by sitedirected mutagenesis at defined structural motifs or sequences. The resulting SECIS mutants were introduced separately into the 3’UTR of the cDNA of the glutathione peroxidase (GPx) in place of the residing wild-type SECIS element. After transfection into COS-7 cells of the GPx cDNAs carrying the SECIS variants, crude cell extracts were prepared. Since GPx contains a selenocysteine in the active center, the effects of the SECIS mutations will be readily reported by monitoring the residual GPx activity. In such a manner, we could verify the predictions of the model. With regard to helix II, removal

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of only one base pair diminished the GPx activity to 75%. Deletion of two base pairs, at different locations in the helix, led to abrogation of the enzymatic activity. The next structural feature investigated was the internal loop. Given the lack of sequence and length conservations, we asked whether an internal loop is strictly required in order for the SECIS element to function. Site-directed mutagenesis was performed on both sides of the loop. It changed the sequences so as to introduce Watson-Crick base pairs closing the loop and forming a contiguous helix I–II. With such a mutation, the SECIS element was profoundly debilitated since no residual GPx activity could be obtained. This experiment established a nice correlation between the model prediction and the functional requirement for an internal loop. Much to our surprise, replacement of the invariant A, within the internal loop, by G, C or U did not lead to very severe effects. When changed to G or U, the residual GPx activity dropped to 60–70%. Only when a C replaced the A, did the GPx activity drop significantly to 34% of the wt activity. Two possibilities can be invoked to interpret these results†: either the assay was not sensitive enough to detect such changes or the cohort of SECIS sequences then available in databases was too restricted, thus excluding putative SECIS not found so far and that would carry bases other than A. We next focussed our attention to the non-Watson-Crick quartet and first asked whether it could be changed to four adjacent Watson-Crick base pairs. Different substitutions, changing the sequence of either the 5', the 3' or both partners of the quartet to form four consecutive Watson-Crick base pairs, led to complete abrogation of the GPx activity. From this, we concluded that four non-Watson-Crick base pairs are strictly required for the function of the SECIS element. Rearrangements within the G.A/A.G tandem, such as A.G/G.A that was predicted by the structural model not to match the geometrical constraints of an RNA helix, convincingly demonstrated the strict requirement for a G.A/A.G tandem. In such a way, we could propose the non-Watson-Crick quartet as being a functional motif of the SECIS element (Walczak et al., 1998). These results raised the question as to whether the diminished, or abolished, GPx activities arose from the inabilities of the corresponding disabled SECIS elements to fulfill their roles. In other words, were they still capable to promote readthrough of the UGA selenocysteine codon. Using an appropriate approach we could effectively show that, in those cases where the SECIS elements were crippled, the lack of GPx activity correlated with translation of a truncated polypeptide ending at the UGA selenocysteine codon read as a stop codon. Collectively, these experiments verified the predictions of the SECIS structural model and especially pinpointed the crucial role of the non-Watson-Crick quartet (Walczak et al., 1998).

3. IDENTIFICATION OF NEW SELENOPROTEIN MRNAS USING THE SECIS ELEMENT AS A MOLECLAR TAG Radiolabeling of rats with suggested that there should exist more seleniumcontaining proteins than those described in the Introduction and that would not have been identified yet (Behne et al., 1996). As a matter of fact, it is very likely that identification of these, as yet undiscovered, selenoproteins would be very informative to better understand the pleiotropic role of selenium. Information concerning these proteins is certainly available in the hundreds of thousands of DNA sequences stockpiled in nucleic acids databases, as a result of the various sequencing projects that are underway. Solving

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the riddle, however, amounted to get access to this information. Since we were not in possession of the least, even partial, peptide sequence, the classical computer search programs could not be used to fetch them. The key issue, then, consisted in elaborating a ploy for deciphering the hidden information. Since every selenoprotein mRNA contains a SECIS element, the discovery of a novel SECIS element should reflect its belonging to an as yet unidentified selenoprotein mRNA. Given the constraints maintaining the conservation of the SECIS structure, it appeared to us that this element could be fit for acting as a molecular tag to be sought in databases. The information in the databases lying in one dimension only—the sequence itself—we needed an intermediary that would directly convert the sequence itself to SECIS RNA secondary structures. To perform this step, we took advantage of RNAMOT, an algorithm capable of detecting RNA secondary structures by reading out sequences from a nucleic acid database. A descriptor for the SECIS element was deduced from the structural studies described above and used by RNAMOT. This computer program searched, in databases, nucleotide sequences that were able to adopt a secondary structure similar to that proposed by the descriptor. The search conducted on different nucleic acids databases led to the identification of a large number of candidates that were further screened, either computationally or manually. The abilities of the selected sequences to act as bona fide SECIS elements were tested in vivo with the glutathione peroxidase reporter system described above which asked whether the SECIS winners could functionally replace the residing SECIS of the GPx mRNA. From these experiments, it turned out that seven of the candidates could lead to selenocysteine incorporation into GPx. The new SECIS elements are being used as a molecular handle to uncover the corresponding open reading frames (ORF) and hence novel selenoproteins. The work we have undertaken deserves attention in several respects and illuminates the mining treasures that lurk in databases. Needless to say, the main hurdle to overcome was the need for a tool permitting to unearth the relevant information. Bioinformatics, with the use of several smart programs, constituted precisely the means to attain the goal. Lastly, it is worthstressing that the detailed knowledge of the consensus structure of one RNA element provided the bedrock for disclosing new selenoproteins and gaining insight into their function.

ACKNOWLEDGMENTS This work was supported by grants from the Association pour la Recherche sur le Cancer, the Centre de Recherche Volvic sur les Oligo-Eléments (CRVOE) and the Ligue Nationale contre le Cancer. R.W. was awarded a fellowship from the CRVOE.

REFERENCES Behne, D., Kyriakopoeulos, A., Weiss, N.C., Kalckloesch, M., Westphal, C., and Gessner, H. 1996, Newly found selenium-containing proteins in the tissues of the rat. Biol Trace Elem Res, 55, 99–110. Berry, M.J., Banu, L., Chen, YY, Mandel, S.J., Kieffer, J.D., Harney, J.W., and Larsen, P.R. 1991, Recognition of UGA as a selenocysteine codon in type I deiodinase requires sequences in the 3' untranslated region. Nature, 353, 273–276. Buettner, C., Harney, J.W., and Larsen, P.R. 1998, The 3'-untranslated region of human type 2 iodothyronine deiodinase mRNA contains a functional selenocysteine insertion sequence element. J Biol Chem, 273,

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Burk, R.F. and Hill, K.E. 1999, Orphan selenoproteins [In Process Citation], Bioessays, 21, 231–237. Gladyshev, V.N., Jeang, K.T., Wootton, J.C., and Hatfield, D.L. 1998, A new human selenium-containing protein. Purification, characterization, and cDNA sequence. J Biol Chem, 273, 8910–8915. Guimaraes, M.J., Peterson, D., Vicari, A., Cocks, B.C., Copeland, N.G., Gilbert, D.J., Jenkins, N.A., Ferrick, D.A., Kastelein, R.A., Bazan, J.F., and Zlotnik, A. 1996, Identification of a novel selD homolog from eukaryotes, bacteria, and archaea: is there an autoregulatory mechanism in selenocysteine metabolism? Proc Natl Acad Sci U S A, 93, 15086–15091. Hubert, N., Walczak, R., Carbon, P., and Krol, A. 1996, A protein binds the selenocysteine insertion element in the 3'-UTR of mammalian selenoprotein mRNAs. Nucleic Acids Res, 24, 464–469. Hüttenhofer, A. and Böck, A. 1998, RNA structures involved in selenoprotein synthesis, in RNA Structure and Function, (R. Symons and M. Grunberg-Manago, eds), pp. 603–639, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (NY, USA). Lesoon, A., Mehta, A., Singh, R., Chisolm, G.M., and Driscoll, D.M. 1997, An RNA-binding protein recognizes a mammalian selenocysteine insertion sequence element required for cotranslational incorporation of selenocysteine. Mol Cell Biol, 17, 1977–1985. Shen, Q., Leonard, J.L., and Newburger, P.E. 1995, RNA-binding proteins that specifically recognize the selenocysteine insertion sequence of human cellular glutathione peroxidase mRNA, J Biol Chem, 270, 30448–30452. Stadtman, T.C. 1996, Selenocysteine. Annu Rev Biochem, 65, 83–100. Walczak, R., Carbon, P., and Krol, A. 1998, An essential non-Watson-Crick base pair motif in 3'UTR to mediate selenoprotein translation. RNA, 4, 74–84. Walczak, R., Westhof, E., Carbon, P., and Krol, A. 1996, A novel RNA structural motif in the selenocysteine insertion element of eukaryotic selenoprotein mRNAs, RNA, 2, 367–379.

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PROLONGED STIMULATION OF RATS WITH ADRENOCORTICOTROPHIC HORMONE (ACTH) ALTERS CONCENTRATION OF SELENIUM IN THE KIDNEY Reversibility and Possible Mechanism

B. Cheng, T. S. Srikumar, F. M. Al-Awadi, J. Thakkar, and F. Sequeira Biochemistry Department Kuwait University Faculty of Medicine POBox 24923, Safat Code 13110 Kuwait

1. INTRODUCTION In humans, certain physiologic conditions such as depression, anxiety, prolonged physical stress, and severe illness, are frequently associated with elevated concentrations of ACTH and glucocorticoids in the circulation. Moreover, chronic stimulation of humans and rats with ACTH causes the development of hypertension in both species (Li et al., 1992). The mechanistic relationships between these physiopathologic conditions and prolonged actions of ACTH remain largely unknown. Selenium is a key element which exerts anti-oxidative effects. One of the best described defense components in tissues is selenium-containing glutathione peroxidase. This enzyme exists in most, if not all, tissues (Mirochnitchenko et al., 1995) and in plasma (Schiavon et al., 1994). Circulatory glutathione peroxidase has been shown to be released primarily from the kidney (Avissar et al., 1994). Our initial effort was therefore directed to investigate whether prolonged stimulation of ACTH would affect selenium concentration in the kidney of rat.

2. EXPERIMENTAL Wistar male rats (mean 150 g) were injected subcutaneously with ACTH (Synatchen Depsito, Ciba-Geigy, Switzerland), or dexamethasone (Sigma, UK) per Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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100 g animal body weight, daily, for four consecutive days. Animals injected with saline or olive oil served as controls. To minimize possible acute effects, animals were killed 24 hours after the last injection of ACTH or the control substances. Some stimulated rats were kept for four additional days without any further treatment, and then killed (postACTH stimulation). During the entire period of treatment, rats were allowed free access to food and water. Kidney homogenates were centrifuged at 150,000 g for 60 minutes to prepare supernatants. The resulting supernatants were subjected to chromatographic analyses using a Sephacryl S-200 Superfine column (2.6 × 80 cm), equilibrated in 10 mM Tris-HCI (pH 7.8). Sixty fractions (10 ml each) were collected at an elution rate of 1.6 ml per minute. Selenium was determined using a graphite-furnace atomic absorption spectrophotometric technique with (tissues), or without (plasma and chromatographic fractions), prior acid-digestion. Lyophilized human reference serum (Seronorm) and bovine liver 1,577 extract served as reference materials. Glutathione peroxidase activity was measured in plasma and kidney supernatants using a commercial kit (Randox Laboratories Ltd., UK). The variation of assays with standards was < 2.3%. The Studentís t-tests were computed with the Glantzí Primer Biostatistics program.

3. RESULTS Prolonged stimulation of rats with ACTH resulted in a 20–50% decrease of selenium concentration in the kidney (P < 0.02). In contrast, plasma concentration of selenium increased in the stimulated animals by 15–20% (P < 0.01). The changes of selenium concentrations in plasma and kidney appeared to be persistent for four days post ACTHstimulation (P < 0.01 and 0.05, respectively). These ACTH-induced changes were duplicated by the treatment of rats with dexamethasone. Although the renal concentration of selenium was reduced by ACTH, the tissue glutathione peroxidase activity remained unchanged. In contrast, the plasma glutathione peroxidase activity of stimulated animals increased about 100% (P < 0.01), which was in parallel with an increased concentration of selenium in the tissue. Chromatographic analyses of renal supernatants revealed an alteration in the elution profile of selenium, but not that of proteins. The two major selenium peaks were shifted from the fraction numbers of 13–23 (unstimulated supernatant) to 8–12 (stimulated supernatant).

4. CONCLUSION Since the plasma concentration of selenium in stimulated rats was elevated, the diminution of selenium in the kidney does not seem to be secondary to a deficiency of the trace element in the circulation. In addition, the hormonal effects on both plasma and kidney selenium concentrations are likely to be mediated by adrenal glucocorticoids, because treatment of rats with dexamethasone caused similar effects. Since plasma glutathione peroxidase is primarily derived from renal proximal tubes (Avissar et al., 1994), it is conceivable that during long term stimulation of ACTH, kidney could produce and release more glutathione peroxidase into circulation by consumption of selenium in the tissue. This could provide an explanation for the findings that the enzyme activity in the kidney of stimulated rats remained unaltered, while the tissue concentration of selenium diminished.

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At present we speculate that the increases in both selenium concentration and glutathione peroxidase activity in plasma are needed to exert protective effects against undesired components in the circulation during chronic stress. In this context, the plasma selenium levels and glutathione peroxidase activity both decrease in humans with renal disorders (Schiavon et al., 1994; Richard et al., 1991). Since the change of selenium concentration in the kidney was not reversed for at least four days post ACTH-stimulation, and was accompanied by an altered distribution of selenium in the supernatant chromatogram, our data suggest that prolonged ACTHstimulation would have profound effects on the normal selenium metabolism in the kidney. (The authors wish to record their appreciation to Dr. J. Craik for his critical reading of the manuscript.)

REFERENCES Avissar, N., Ornt, D.B., Yagil, Y., Horowitz, S., Watkins, R.H., Kerl, E.A., Takahashi, K., Palmer, I.S., and Cohen, H.J., 1994, Human kidney proximal tubules are the main source of plasma glutathione peroxidase. Am. J. Physiol. 266:C367–C375. Li, M., Birchall, I., Kincaid-Smith, P.S., and Whiteworth, J.A., 1992, Role of the renal medulla in adrenocorticotrophin-induced hypertension in rats. J. Hyperten. 10:1129–1136. Mirochnitchenko, O., Palnitkar, U., Philbert, M., and Inouye, M., 1995, Thermosensitive phenotype of transgenic mice overproducing human glutathione peroxidase. Natl, Acad. Sci. USA. 92:8120–8124. Richard, M.J., Arnaud, J., Jurkovitz, C., Hachache, T., Meftahi, H., Laporte, F., Foret, M., Favier, A., and Cordonnier, D., 1991, Trace elements and lipid peroxidation abnormalities in patients with chronic renal failure. Nephron 57:10–15. Schiavon, R., Guidi, G.C., Biasioli, S., De Fanti, E., and Targa, L., 1994, Plasma glutathione peroxidase activity as an index of renal function. Eur. J. Clin. Chem. Clin. Biochem. 32:795–765.

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DIETARY SELENIUM INTAKE AND ANTIOXIDANT DEFENSES IN TISSUES OF PERIPUBERAL RATS Maria Cristina Santos1, Jean Nève2, Maria Leonor Pavão3, and Ana Maria Viegas-Crespo4 1

Centro de Estudos de Bioquímica e Fisiologia da Universidade de Lisboa Inst. Investigação Científica Bento da Rocha Cabral e Dept. de Química e Bioquímica Fac. Ciências da Universidade de Lisboa 1749-016 Lisboa, Portugal 2 Institute de Pharmacie de l’Université Libre de Bruxelles Campus Plaine, Bruxelles Belgique 3 CIRN, Universidade dos Açores R. Mãe de Deus, 9500-Ponta Delgada Portugal 4 Centro de Biologia Ambiental e Dept. Zoologia e Antropologia Fac. Ciências da Universidade de Lisboa 1749-016 Lisboa, Portugal

1. INTRODUCTION Free radicals are highly reactive chemical species that can oxidize and damage essential biological molecules. Their formation is a result of endogenous metabolism or of xenobiotics bio-transformation, but under normal physiological conditions cells are protected against oxidative challenge by enzymatic and non enzymatic antioxidants (Sies and Groot, 1992; Yu, 1994). Selenium (Se) is a trace element which essentiality for animals and humans is now well established. The element performs its functions mainly through selenoproteins and several glutathione peroxidases that degrade hydroperoxides using glutathione (GSH) as a reducer, are selenoenzymes playing an important role as antioxidant defenses (Ursini, Maiorino and Gregolin, 1985; Flohé, 1989). However, selenium is also a toxic agent with a narrow range of suitable levels. Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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The main objective of this study was to assess the physiological development of some antioxidant parameters in liver and testes of rats during the puberty until the adult hood and to know if a low selenium supplementation in the diet can have some effect on those parameters, especially on those related with glutathione metabolism.

2. ANIMALS AND METHODS 2.1. Animals and Diets This study was carried out with about eighty male Wistar rats. Twenty one days old animals (weaning rats) were fed a commercial standard diet containing 0.1 ppm Se or this diet supplemented with 0.5 ppm Se as sodium selenite in the drinking water. Five age groups were selected for study: 4, 6, 8, 10 and 16 weeks of age.

2.2. Methods Plasma selenium concentrations were measured by electrothermal atomic absorption according to Nève, Chamart and Molle (1987). Se-glutathione peroxidase (Se-GSHPx) was assayed with hydrogen peroxide as substrate according to Lawrence and Burk (1976). Glutathione transferase (GSH S-Tr) activity was measured using the method of Habig, Pabst and Jakoby (1974) with 1-chloro-2,4-dinitrobenzene (CDNB) as substrate. The activity of glutathione reductase (GSSG Rd), the enzyme that regenerates GSH was measured using the method developed by Pinto and Bartley (1969). Glutathione contents were assessed by the 5-5'-dithiobis-(2-nitrobenzoic acid) (DTNB)—GSSG reductase recycling assay, as described by Anderson (1985), in tissue samples pulverised under liquid nitrogen.

3. RESULTS Means of plasma selenium levels increased during the puberty in both normal and supplemented rats (Table 1), but the differences between the dietary groups at identical ages were not statistically significant. As it was expected, in the liver the Se-GSHPx activity increased during puberty and from about 8 weeks of age it stayed at levels concordant with adulthood

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(Table 2). When the diet was enriched with Se, a significantly higher activity in liver of young adults (16 weeks of age) was observed. The activity of GSHS-Tr increased in liver until the age of 6 weeks and after that it stayed unchanged (Table 2). The dietary selenium supplementation readily led to higher hepatic GSHS-Tr activity. In testes, the Se-GSHPx activity also increased during the puberty, but any change with the diet enriched with selenium was not observed (Table 3). On the other hand the activity of GSHS-Tr raised in testes only after maturity (8 weeks of age). Once more, the selenium supplementation in the diet had no effect on this testicular parameter. GSSG reductase activity and glutathione contents were mesured in the adult age groups. The diet with higher selenium level led to an increased content of total glutathione in liver, but the level of the oxidised form as well as the activity of its reductase were unchanged (Table 4). Any difference in the testicular parameters could not be detected.

4. DISCUSSION In Wistar rats, the development of Se-GSHPx and GSH S-Tr activities from weaning to the adult age is different in the two tissues. On the other hand, the hepatic activities are always higher than those measured in the testes. In the liver, the findings related to the first enzyme are similar to those observed by others (Pinto and Bartley, 1969), but the GSH S-Tr activity has an evolution which is different from some data in

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the literature (Peltola, Huhtaniemi and Ahotupa, 1992; Jung and Henke, 1996). When animals are fed the selenium supplemented diet, the liver shows to be sensitive to small changes of dietary selenium contents as it has been found elsewhere (Crespo, Pinto and Neve, 1993). In the testes, the developmental profile of Se-GSHPx is also according to that observed by Behne, Duk and Elger (1986) and it is very different from the GSH S-Tr profile. As it was expected, in these conditions the testes are not affected by selenium supplementation, due to efficient testicular homeostatic mechanisms (Behne, 1989). This study suggests that selenium in diet at moderated levels, that are not much higher than the recommended intake of the element for these animals, has influence on some steps of hepatic glutathione metabolism, increasing the activity of some enzymes related to GSH and also the level of this thiol, probably as a result of an adaptive response.

ACKNOWLEDGMENTS This study was performed with a finantial support of the “Fundação para a Ciência e Tecnologia” (F.C.T.)

REFERENCES Anderson, M.E., 1986, Tissue glutathione, in: CRC Handbook of Methods for Oxygen Radical Reseach (R.A. Greenwald, ed.) 2a ed., CRC Press Inc., Florida. Behne, D., 1989, Selenium homeostasis, in: Selenium in Medicine and Biology (J. Néve and A. Favier, eds.) pp. 83–91, Water De Gruyter, New-York. Behne, D., Duk, M., and Elger, W., 1986, Selenium content and glutathione peroxidase activity in the testis of the maturing rat. J. Nutr. 116:1442–1447. Crespo, A.M., Pinto, R.E., and Neve, J., 1993, Plasma and Liver Selenium Levels in the Rat During Supplementation with 0.5, 2, 6, and 15ppm Selenium in Drinking Water. Biol Trace Elem. Res. 38:139–147. Flohé, L., 1989, The selenoprotein glutathione peroxidase, in: Glutathione: Chemical, Biochemical and Medical Aspects (D. Dolphin, R. Poulson, and O. Avramovic, eds.) part A, pp. 644–731, John Wiley & Sons. New-York. Jung, K. and Henke, W., 1996, Developmental changes of antioxidant enzymes in kidney and liver from rats. Free Rad. Biol. & Med. 20:613–617. Lawrence, R.A. and Burk, R.F., 1976, Glutathione peroxidase activity in selenium deficient rat liver. Biochem. Biophys. Res. Commun. 71:952–958.

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Nève, J., Chamart, S., and Molle, L., 1987, Optimization of a direct procedure for determination of selenium in plasma and erytrocytes using Zeeman-eflect atomic absorption spectroscopy, in: Trace Elem. Anal. Chem. in Medic, and Biol. (P. Bratter and P, Schramel, eds.) vol. 4, pp. 349–358, Walter de Gruyter. Berlin, New York. Peltola, V., Huhtaniemi, I., and Ahotupa, M., 1992, Antioxidant enzyme activity in the maturing testes. J. Andrology 13:450–455. Pinto, R.E. and Bartley, W., 1969, The effect of age and Sex on glutathione reductase and glutathione peroxidase activities and on aerobic glutathione oxidation in rat liver homogenates. Biochem. J. 112:109–115. Sies, H. and Groot, H., 1992, Role of reactive oxygen species in cell toxicity. Toxicology Letters 64/65:547–551. Ursini, F., Maiorino, M., and Gregolin, C, 1985, The selenoenzyme phospholipid hydroperoxide glutathione peroxidase. Biochimica et Biophysica Acta 839:62–70. Yu, B.P., 1994, Cellular defenses against damage from reactive oxygen species. Physiol. Rev. 74:1390–162.

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SELENOPROTEIN P IN PLASMA IN RELATION TO CANCER MORBIDITY IN MIDDLE-AGED SWEDISH MEN Marie Persson-Moschos1, Lars Stavenow2, Björn Åkesson1, and Folke Lindgärde2 1

Division of Biomedical Nutrition Center for Chemistry and Chemical Engineering Lund University S-221 00 Lund, Sweden 2 Department of Vascular and Renal Diseases Malmö University Hospital S-205 02 Malmö Sweden

1. INTRODUCTION In several surveys the relation between selenium status and future risk of cancer has been studied. In most case-control studies, cancer cases had lower prediagnostic serum selenium levels than controls (Comstock et al., 1992; Knekt et al., 1990). Studies in animals have shown that high doses of selenium can protect against experimental cancer, and recently the interest in the preventive role of selenium supplementation in humans was stimulated by results from two intervention studies (Blot et al., 1993; Clark et al., 1996). In epidemiologic studies usually selenium concentration and in some cases glutathione peroxidase activity in plasma were used as indices on selenium status. The application of recently discovered selenoproteins as markers of selenium status has not yet been exploited, although this would be expected to give information about specific selenium functions. In plasma selenoprotein P, glutathione peroxidase and protein-bound selenomethionine are major fractions and selenoprotein P accounts for at least 40% of the plasma selenium (Åkesson et al., 1994). Recently an immunoassay for measurement of this protein was developed (Persson-Moschos, 1999), and using this assay the relation between selenoprotein P and cancer morbidity was investigated in middle-aged men from a well characterized population study. Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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2. MATERIAL AND METHODS The design was a case-control study nested in a cohort. During 1974 to 1982 12,500 men living in Malmö were invited to a health-screening. The subjects were born 1926 to 1937 (46 to 48 years old at screening), and from the screening and until the end of 1988, 400 cancer cases of the subjects were identified. For each case, two living controls of the same age who had not acquired any clinical cancer by the end of 1988 (i.e. 800 men) were selected from subjects screened on the same day. Totally 906 plasma samples were available for analysis of selenoprotein P (302 cases and 604 controls). The cancer cases were divided into four groups: respiratory tract (ICD7: 160–163, (n = 69)); digestive tract (ICD7: 140–158 (n = 115)); urinary tract (ICD7: 177–181 (n = 57) and remaining (ICD7: nr 164, 170, 190–209) (n = 61). Selenoprotein P levels were measured with a radioimmunoassay (Persson-Moschos et al., 1995), and the concentration of selenoprotein P was expressed in arbitrary units.

3. RESULTS Cases and controls were very similar with respect to body mass index, plasma cholesterol and blood pressure. As expected, the proportion of smokers was higher among cancer cases (60%) than among controls (48%) (p = 0.001). Cases in the respiratory tract (74%) and urinary tract (70%) cancer groups had a significantly higher proportion of smokers compared to the other cancer groups. The selenoprotein P levels in cancer cases and controls were 1.20 (1.16, 1.24) a.u. (mean (95% CI)) and 1.23 (1.21, 1.25) a.u., respectively, which was not significantly different. However, in cases divided into subgroups according to cancer site, selenoprotein P levels were significantly lower than the respective controls for respiratory tract cancer (1.20 (1.24, 1.26 ) and 1.30 (1.25, 1.35) a.u., respectively). In the digestive tract cancer group (1.15 (1.08, 1.22) and 1.21 (1.17, 1.25) a.u., respectively) the same non-significant trend was observed. In cases plus controls (n = 906) divided into smokers and non-smokers the selenoprotein P level in smokers was significantly lower than that in non-smokers (1.20 (1.17, 1.23) and 1.24 (1.21, 1.27), a.u., respectively). The association of relative risk for getting cancer with selenoprotein P concentration was also estimated from quintiles of selenoprotein P level. For increasing quintiles the ORs adjusted for smoking were 5.2 (1.2, 23.4) (mean (95% CI)), 2.3 (0.7, 8.2), 2.9 (1.2, 6.8), 2.0 (0.9, 4.4) and 1.0, respectively (p for trend = 0.01). Moreover, the ORs adjusted for smoking in tertiles of selenoprotein P level were calculated for cases in respiratory tract, digestive tract, urinary tract and remaining cancer groups. They were 6.0, 3.4, 0.2, and 0.6 respectively, in the lowest tertile compared to the cases in the highest. For respiratory and digestive tract cancer groups, the p values for trend were 0.004 and 0 002, respectively. Several biochemical and other variables were measured at screening. In cases plus controls selenoprotein P had low but significant (p < 0.05) correlations to plasma concentrations of albumin, IgG, fasting blood glucose, and body mass index. Inverse correlations to selenoprotein P were found for plasma levels of and transferase (r = –0.12; p = 0.02 and r = –0.10; p = 0.004, respectively). The correlation coefficients were similar in cases and controls. Correlations of selenoprotein P to plasma albumin, body mass index and negative correlations to transferase, carbonmonoxide-haemoglobin were significant in smokers but not in

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non-smokers. In cases plus controls, smokers had significantly higher plasma levels of transferase, orosomucoid, cholesterol and triglyceride compared with non-smokers.

4. DISCUSSION This seems to be the first study on the association between cancer risk and selenoprotein P, but previously similar studies have been made using plasma selenium as biomarker of selenium status. The selenoprotein P level of all cases was non-significantly lower than in all controls, but in respiratory tract cancer cases it was significantly lower than in the respective controls (7.7%). For previous prediagnostic studies we calculated that the cases had on average 7.2% lower plasma selenium than the controls for both respiratory and digestive tract cancer. Moreover, in this study, there was a significant trend of increasing risk of overall cancer with decreasing quintiles of selenoprotein P levels. The risks for respiratory cancer and digestive tract cancer were six-fold and three-fold, respectively, in the lowest tertiles compared to the highest tertiles of selenoprotein P concentration. In other studies plasma selenium was significantly correlated to selenoprotein P levels at low to moderate plasma selenium concentrations, but at higher plasma selenium concentrations the correlation was weaker (Persson-Moschos, 1999). Although the selenium concentration in plasma and other tissues is mainly determined by selenium intake, dietary forms of selenium can have different effects. The plasma levels of selenoprotein P seems to be equally affected by supplementation of major dietary forms of selenium (Persson-Moschos et al., 1998), whereas the plasma selenium concentration increased more after intake of selenomethionine than of other forms. Several factors other than selenium intake may affect biomarkers of selenium status. In this study, smokers had significantly lower levels of selenoprotein P than non-smokers. In other studies lower values of plasma selenium, whole blood selenium, erythrocyte selenium, and toenail selenium have been observed in smokers. One possible explanation to the lower selenoprotein P level in smokers is that smoking might contribute to chronic low grade inflammation due to the irritating effect on the respiratory tract and vascular endothelial cells. The positive correlation between selenoprotein P and albumin and negative correlation to suggest that selenoprotein P levels are decreased by inflammatory activity. Moreover, smoking may increase oxidative stress since cigarette smoke is a rich source of reactive nitrogen species such as nitric oxide, which together with superoxide can produce peroxynitrite. Recently it was demonstrated that selenoprotein P can scavenge peroxynitrite (Arteel et al., 1998). The natural presence of cadmium in tobacco smoke might also contribute to a lower selenium status in smokers. Recently, blood cadmium was found to be negatively associated both with selenium in blood and selenium and selenoprotein P in plasma (Osman et al., 1998). Experimental studies have shown that addition of high levels of selenium in the diet has a carcinostatic effect in animals treated with carcinogenic chemicals. It has been demonstrated that selenium compounds may induce cell death by several mechanisms and increase the activities of xenobiotic-metabolizing enzymes in vivo (Harrison et al., 1998). It is also possible that selenium in the form of glutathione peroxidases and selenoprotein P may prevent mutations by acting as free radical scavengers (Arteel et al., 1998). Definite evidence of a protective effect of selenium in human investigations has not yet been presented, however, there has been an increasing interest in the cancer preventive

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action of selenium supplementation since recent intervention studies have indicated beneficial effects (Blot et al., 1993; Clark et al., 1996). Additional studies are necessary to corroborate these findings. It is concluded that low selenoprotein P levels in middle-aged men may be associated with an increased risk of cancer in respiratory tract and digestive tract. Moreover, selenoprotein P was significantly lower in smokers compared to non-smokers, but the factors contributing to this effect are unclear.

REFERENCES Åkesson, B., Bellew, T., and Burk, R.F., 1994, Purification of selenoprotein P from human plasma, Biochim. Biophys. Acta 1204:243–249. Arteel, G.E., Mostert, V., Oubrahim, H., Briviba, K., Abel, J., and Sies, H., 1998, Protection by selenoprotein P in human plasma against peroxynitrite-mediated oxidation and nitration, Biol. Chem. 379:1201–1205. Blot, W.J., Li, J.-Y., Taylor, P.R., Guo, W., Dawsey, S., Wang, G.-Q., Yang, C.S., Zheng, S.-F., Gail, M., Li, G.Y., Yu, Y., Liu, B.-Q., Tangrea, J., Sun, Y., Liu, F., Fraumeni Jr, J.F., Zhang, Y-H., and Li, B., 1993, Nutrition intervention trials in Linxian, China: supplementation with specific vitamin/mineral combinations, cancer incidence, and disease-specific mortality in the general population, J. Natl. Cancer Inst. 15:1483–1492. Clark, L.C., Combs, G.F., Turnbull, B.W., Slate, E.H., Chalker, D.K., Chow, J., Davis, L.S., Glover, R.A., Graham, G.F., Gross, E.G., Krongrad, A., Lesher Jr, J.L., Park, K., Sanders Jr, B.B., Smith, C.L., and Taylor, J.R., 1996, Effects of selenium supplementation for cancer prevention in patients with carcinoma of the skin, JAMA 276:1957–1963. Comstock, G.W., Bush, T.L., and Helzlsouer, K., 1992, Serum retinol, beta-carotene, vitamin E and selenium as related to subsequent cancer of specific sites, Am. J. Epidem. 135:115–121. Harrison, P.R., Lanfear, J., Wu, L., Fleming, J., McGarry, L., and Blower, L., 1997, Chemopreventive and growth inhibitory effects of selenium, Biomed. Environ. Sci. 10:235–245. Knekt, P., Aromaa, A., Maatela, J., Alfthan, G., Aaran, R.-K., Hakama, M., Hakulinene, T., Peto, R., and Teppo, L., 1990, Serum selenium and subsequent risk of cancer among Finnish men and women, J. Natl. Cancer Inst. 82:864–868. Osman, K., Schütz, A., Åkesson, B., Maciag, A., and Vahter, M., 1998, Interactions between essential and toxic elements in lead exposed children in Katowice, Poland, Clin. Biochem. 31:657–665. Persson-Moschos, M. Selenoprotein P. Nutritional and clinical aspects. Doctoral thesis, University of Lund, Lund, 1999. Persson-Moschos, M., Huang, W., Lindeberg, S., Srikumar, T.S., and Åkesson, B., 1995, Selenoprotein P in serum as a biochemical marker of selenium status, Analyst 120:833–836. Persson-Moschos, M., Alfthan, G., and Åkesson, B., 1998, Plasma selenoprotein P levels of healthy males in different selenium status after oral supplementation with different forms of selenium, Eur. J. Clin. Nutr. 52:363–367.

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EFFECT OF SELENIUM SUPPLEMENTATION ON PLASMA SELENOPROTEIN P OF NEW ZEALANDERS 1

1

Christine D. Thomson , Anna J. Duffield , and Kristina E. Hill

2

1

Department of Human Nutrition University of Otago PO Box 56, Dunedin New Zealand 2 Department of Medicine Division of Gastroenterology Vanderbilt University Medical Center Nashville Tennessee, USA

1. INTRODUCTION Selenium exists in the human body in a number of selenoproteins, the one most studied being glutathione peroxidase (GSHPx), the activities of which in plasma and erythrocytes have been used to assess selenium status. Recent research in human subjects suggests that another functional selenoprotein, selenoprotein P is also a suitable indicator of selenium status (Marchaluk et al., 1994; Hill et al., 1996) and may in fact be superior to GSHPx because it constitutes a greater proportion of plasma selenium (Huang and Åkesson, 1993). As part of a larger study to determine the selenium requirements for maximisation of GSHPx (Duffield et al., in press), we investigated the effect of supplementation with varying sized doses of selenium as selenomethionine on plasma selenoprotein P in New Zealand residents with low selenium status.

2. METHODS Fifty-two New Zealand adults (17 males, 35 females) aged 19–59 years with whole blood selenium concentrations less than (100 ng/ml) were randomly assigned to one of five groups. Subjects received in a double-blind intervention a supplement containing a placebo or 10, 20, 30, or ; selenium as L-selenomethionine (Westar Nutrition Inc, 1239 Victoria Street, Costa Mesa CA 92626, USA) daily for 20 Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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weeks. Blood samples were collected into vacutainer tubes containing EDTA at baseline and at weeks 2, 4, 8, 12 and 20 for analysis of selenium, selenoprotein P and GSHPx. Plasma selenium was analysed by flow injection hydride generation atomic absorption spectrometry (Tiran et al., 1993) after digestion with nitric and perchloric acid. Selenoprotein P was assayed by a radioimmunoassay using human selenoprotein P (Hill et al., 1996). GSHPx activity was assayed in plasma using an automated modification of the coupled assay of Paglia and Valentine (1967). The SAS procedures PROC GLM (SAS Institute Inc, Cary, NC, USA 1996) was used to examine differences between the treatments at each time point. These were adjusted for the initial value and for gender by including a factor in the regression analysis.

3. RESULTS Selenium and selenoprotein P concentrations and GSHPx activities increased in all treatment groups with supplementation, rapidly during the first 2 weeks and then more slowly, reaching a plateau in some groups. Selenoprotein P concentrations are illustrated in Fig. 1. Percent change in selenoprotein P levels (25, 45, 31, 52, 88% of baseline values at week 20 for the 0, 10, 20, 30, selenium groups, respectively) were greater than those for plasma GSHPx (13, 17, 19, 21 and 30%) and for plasma selenium (4, 14, 23, 26 and 41%). When results were expressed as differences between each supplemental group and the control group at each week adjusted for gender and baseline measures, there was a dose response in the levels of selenoprotein P reached. Correlation coefficients for relationships among plasma selenium, selenoprotein P and GSHPx were high for the placebo group (Table 1). Correlations fell dramatically in the other groups for the GSHPx versus selenium relationship. For selenoprotein P, correlations with plasma selenium were greater than for GSHPx over the range of intakes.

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Statistical analysis of our results indicate that selenoprotein P was maximised at plasma selenium concentrations of around which was a little lower than the concentration needed for full expression of plasma GSHPx This suggests therefore that the selenium intake for maximisation of selenoprotein P is probably also similar to but a little lower than that for maximisation of GSHPx.

4. DISCUSSION Daily supplementation of low doses of selenomethionine produced higher concentrations or activities of all measures of selenium status. However plasma selenoprotein P reflected selenium intake more rapidly, showed the greatest increase relative to baseline values, reached a plateau at lower supplemental intakes than other measures and correlated more strongly than GSHPx with plasma selenium over the range of selenium intakes studied. Thus, in combination with other plasma and whole blood indices, plasma selenoprotein P concentration would be a very useful means of assessing selenium status. A response of selenoprotein P concentrations to selenium supplementation may reflect marginal selenium status of New Zealanders. However whether maximal levels of selenoprotein P are desirable for optimal health requires further investigation once the function of this protein has been established.

REFERENCES Duffield, A.J., Thomson, C.D., Hill, K.E., and Williams, S., An estimation of selenium requirements for New Zealanders, Am. J. Clin. Nutr. In press. Hill, K.E., Xia, Y., Åkesson, B., Boeglin, M.E., and Burk, R.F., 1996, Selenoprotein P concentration in plasma is an index of selenium status in selenium-deficient and selenium-supplemented Chinese subjects, J. Nutr. 126:138–145. Huang, W. and Åkesson, B., 1993, Radioimmunoassay of glutathione peroxidase in human serum, Clin. Chim. Acta 219:139–148. Marchaluk, E., Persson-Moschos, M., and Thorling, E.B., 1994, Variation in selenoprotein P concentration in serum from different European regions, Eur. J. Clin. Nutr. 49:42–48. Paglia, D.C. and Valentine, W.N., 1967, Studies on quantitative and qualitative characterization of erythrocyte glutathione peroxidase, J. Lab. Clin. Med. 70:158–169. Tiran, B., Tiran, A., Rossipal, E., and Lorenz, O., 1993, Simple decomposition procedure for determination of selenium in whole blood, serum and urine by hydride generation atomic absorption spectroscopy, J. Trace Elem. Electrolytes Health Dis. 7:211–216.

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TRACKING THE METABOLISM AND EXCRETION OF SELENIUM IN REAL TIME IN HUMANS 1

1

1

2

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G. Bellisola , M. Colombatti , G. Fracasso , F. Pasti , M. Valdes , 2 and A. Torboli 1

University of Verona, Institute of Immunology and Infectious Diseases Policlinico Borgo Roma Strada le Grazie, I-37134 Verona, ITALY 2 Ital Structures, Via Monte Misone 11/d I-38066 Riva del Garda, Italy

1. INTRODUCTION AND AIMS Selenium (Se) plays crucial functions in mammals and a poor selenium status is considered a risk factor for disease in humans (Daniels, 1996). The presence of the unusual amino acid Selenocysteine (SeCys) in proteins confers functional specificity to selenoproteins (Stadtman, 1996). On the contrary, no functional advantage has been attributed to the random incorporation of Selenomethionine (SeMet) instead of methionine in proteins (Deagen, Beilstein and Wanger, 1991). Nevertheless, SeMet is also utilized for the synthesis of SeCys in the liver by the transsulfuration pathway (Esaki et al., 1991). Plasma Se levels reflect the intakes of this element and selenoprotein synthesis is in relation with Se bioavailability (dosage and chemical form) in cell and tissues (Behne et al., 1991); therefore individual Se status can be evaluated with either quantitative and functional measurements of Se and selenoprotein activity in body fluids and cells (Nève, 1995). Almost total plasma Se distributes in three plasma proteins (Harrison, Littlejohn and Fell, 1996): Selenoprotein P (SeP), extracellular Glutathione Peroxidase (eGSHPx) and albumin. SeP is a SeCys-rich glycoprotein accounting for the most Se in the plasma (Burk and Hill, 1994) and selenite rapidly stimulates its synthesis in the liver (Burk et al., 1991). The different affinity for Heparin is useful to separate Se in SeP (high affinity) from Se in albumin and in eGSHPx (low affinity) (Akesson, Bellew and Burk, 1994). Present work describes the short-time variations of Se in plasma and urine after the assumption of a single oral dose of Se in healthy humans. Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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2. MATERIALS AND METHODS Blood plasma (EDTA) and urine samples were collected in 5 healthy males before and each hour for 6 hours after the ingestion of selenium as sodium selenite. Affinity chromatography on Heparin Sepharose (HS) was applied in order to distinguish Se in SeP from Se in eGSHPx and albumin (Mostert, Lombeck and Abel, 1998). Plasma samples (3 mL) were incubated for 1 h with 1 mL HS gel previously equilibrated with 0.05 M Tris buffer, pH 7.4 and containing 0.1 M ammonium acetate. The gel was then separated from the plasma supernatant by centrifugation (5 min at 1,500 g) and, after washing, SeP was eluted from HS with 2 mL of the same buffer containing 2 M ammonium acetate. Total Reflection X-Ray Fluorescence (TXRF) technique was applied to measure Se in samples (blood plasma, urine, supernatant and eluate, respectively) after the addition of Ga as an internal standard.

3. RESULTS AND DISCUSSION Table 1 shows the time-courses of Se in plasma and urine samples. Maximal plasma Se concentration is observed in plasma samples 3 h after the ingestion of selenite, thus suggesting the rapid absorption of this selenocompound. Se slightly decreases in the plasma from the h while Se excretion in urine progressively increases. Opposite trends for Se in the plasma and in the urine from the h indicate that the kidneys are involved also in the short-time regulation of Se homeostasis. Table 2 shows the time-courses of Se in eluate and supernatant samples separated by Heparin-affinity chromatography. Higher Se concentrations are always measured in

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eluate than in supernatant samples, thus suggesting that a major contribution to total plasma Se is due to the element present in the SeP molecule with respect to the element in connection with albumin and eGSHPx molecules. Moreover, different trends for Se in eluates and in supernatants also suggest the preferential incorporation of this element in SeP molecule. In conclusion, the time-course of Se can be arranged for the evaluation of both intestinal absorption of Se and the rate of selenoprotein synthesis. From this point of view, the quantitative measurement of Selenium in human fluids can assume the significance of a functional test.

REFERENCES Akesson, B., Bellew, T., and Burk, R.F., 1994, Purification of selenoprotein P from human plasma, Biochem. Biophys. Acta 1204:243–249. Behne, D., Kyriakopoulos, A., Scheid, S., and Gessner, H., 1991, Effects of chemical form and dosage on the incorporation of selenium into tissue proteins in rats, J. Nutr. 121:806–814. Burk, R.F. and Hill, K.E., 1994, Selenoprotein P. A selenium-rich extracellular glycoprotein, J. Nutr. 124:1891–1897. Burk, R.F., Hill, K.E., Read, R., and Bellew, T., 1991, Response of rat selenoprotein P to selenium administration and fate of its selenium, Am. J. Physiol. 261:E26–30. Daniels, L.A., 1996, Selenium metabolism and bioavailability, Biol. Trace Elem. Res. 54:185–199. Deagen, J.T., Beilstein, M.A., and Whanger, P.D., 1991, Chemical forms of selenium in selenium containing proteins from human plasma, J. Inorg. Biochem. 41:261–268. Esaki, N., Nakamura, T., Tanaka, H., Suzuki, T., Morino, Y., and Soda, K., 1981, Enzymatic synthesis of selenocysteine in rat liver, Biochemistry 20:4492–4500. Harrison, I., Littlejohn, D., and Fell, G.S., 1996, Distribution of Selenium in human blood plasma and serum, Analyst. 121:189–194. Mostert, V., Lombeck, I., and Abel, J., 1998, A novel method for the purification of Selenoprotein P from human plasma. Arch. Biochem. Biophys. 357:326–330. Nève, J., 1995, Human selenium supplementation as assessed by changes in blood selenium concentration and glutathione peroxidase activity, J. Trace Elem. Med. Biol. 5:65–73. Stadtman, T.C., 1996, Selenocysteine, Annu. Rev. Biochem. 65:83–100.

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CHANGES IN INDICES OF SELENIUM STATUS IN MEN ON LOW, MEDIUM, AND HIGH INTAKES 1

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T. Fox , C. Atherton , S. Fairweather-Tait , J. Dainty , J. Lewis , 2 2 M. Baxter , and H. Crews 1

Institute of Food Research Norwich NR4 7UA, UK 2 MAFF CSL Food Science Laboratory Norwich NR4 7UQ, UK

1. INTRODUCTION Selenium is essential element for human health (Reilly, 1996). There is evidence that the activity of GSHPx in the body is affected by selenium supply (Burke and Hill, 1993). Erythrocyte, plasma and platelet are recognised as reliable tissue for GSHPx activity measurements but direct measurement of selenium in whole blood and plasma is the most common method of assessing selenium status (Diplock, 1993). Stable isotopes of selenium have been used in pharmacokinetic studies to measure the size and turnover of body selenium pool for the assessment of selenium status (Janghorbani et al., 1990). In this present study we compared various measurements of selenium status in volunteers who had been given a controlled diet of a low, medium and a high selenium content for six weeks.

2. METHODS AND MATERIALS

2.1. Subjects Twelve healthy male volunteers aged between 23 and 57 years (mean weight 74.6kg) were recruited from advertisements in the local press and radio and screened for normal blood parameters by the local hospital.

2.2. Intervention The volunteers took part in three dietary intervention periods of six weeks each in which the selenium intakes totalled and respectively. Each Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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six week period was separated by a washout period of 5 months during which time the volunteers returned to their habitual diet. A standard diet consisting of a seven day rolling menu which contained an average selenium concentration of was used for all three interventions and was supplemented with yeast to increase selenium intake to the required level.

2.3. Blood Samples Blood was collected for plasma selenium concentration, erythrocyte, plasma and platelet GSHPx assays prior to each dietary intervention and one week after. Plasma, erythrocyte and platelet GSHPx activity was assayed using a Randox glutathione peroxidase analysis kit based on the method of Paglia and Valentine (1967) (Randox Laboratories UK) on a Cobas autoanalyzer.

2.4. Exchangeable Body Pools At the beginning of the sixth week, after an overnight fast, each of the volunteers were given stable isotope enriched selenious acid by intra-venous infusion into the radial vein and blood samples taken via an indwelling cannula at half-hourly intervals for 4 hours and thereafter at hourly intervals for the next 4 hours. Blood samples were collected by venepuncture at 24, 48, 72 and 168 hours. Urine was also collected as individual samples on the day of cannulation and thereafter as 24 hour samples for the next 4 days. The size of the exchangeable selenite pool of selenium in each volunteer was calculated from the kinetics of the disappearance of the labelled selenite infused bolus in the plasma using SAAM software based on a two compartment model (SAAM Institute Inc. Seattle, USA). Plasma selenium concentration and isotope enrichment were analysed by ICP-MS using an VG Plasmaquad PQ2 Turbo plus (Perkin Elmer). GSHPx assays in plasma, erythrocytes and platelets were compared with plasma selenium concentration and size of exchangeable selenium pools.

3. RESULTS 3.1. Selenium Status Plasma selenium concentration fell after the low diet (p < 0.001) and increased after the high diet (p < 0.001) (Table 1). There were no changes in GSHPx activity in erythrocyte and plasma samples but there was a significant increase in platelets following the high selenium diet (p < 0.01) and a significant decrease following the medium diet (p < 0.05) but no change after the low diet (Table 2).

3.2. Exchangeable Pools The size of exchangeable selenium pools differed significantly with an increase in pool size after the high diet and a decrease in size after the low diet. The plasma selenium concentration was significantly correlated with size of exchangeable selenium pool (R = 0.833 p < 0.001) (Table 1).

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4. DISCUSSION The WHO recommended daily allowances (RDA) are 40 and and the 1989 US National Research Council RDA for selenium is and for adult men and women, respectively. The US RDA is readily satisfied by typical diets in countries with relatively selenium rich soils and hence a moderate to high selenium content in plants, such as the US, but for some parts of the world, notably New Zealand, Scandinavia and parts of China the low selenium content of the soil does not provide sufficient selenium to meet these figures (Levander, 1991). In this study we fed diets containing to reflect low selenium areas and to reflect intakes in Europe and that would be achieved when taking selenium supplements. Neither erythrocytes nor plasma GSHPx activity changed in response to the different dietary intakes in selenium. However, there were significant differences in the plasma selenium concentration, the size of the exchangeable body selenium pools and platelet GSHPx activity. Platelets are a reliable tissue for estimating selenium status via GSHPx activity and their use avoids the problems associated with the presence of haemoglobin and glutathione-S-transferase and the response to selenium supplementation is faster than in plasma (Levander et al., 1983). However, the method has been criticised in that platelet GSHPx may reflect recent selenium intake rather than long term status (Whanger et al.,

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1988). This may be true where inorganic supplements are used since the selenium is readily available for selenocysteine synthesis but is also available for excretion. In the present study, organic supplements were used and in this form the selenium would be indistinguishable from its sulphur containing analogues and would be incorporated into general non-specific protein synthesis where it would not be available for selenium dependent enzyme synthesis until the proteins were catabalised. The fact that platelet GSHPx activity was the only tissue enzyme to change following the high selenium diet suggests that plasma and erythrocyte GSHPx levels were already saturated or that synthesis of platelet GSHPx took precedence over the other tissues at the expense of erythrocyte and plasma GSHPx. It has been shown that GSHPx activity has a significant correlation with whole blood and plasma selenium levels in populations with intakes of selenium below current USA intakes, but that the activity is saturated in populations with blood concentrations above (Whanger et al., 1988; Diplock, 1993). There were changes in platelet GSHPx activity after the medium diet which suggests that the organic selenium supplied to make up the total selenium intake was not available for selenocysteine synthesis and hence GSHPx enzyme synthesis resulting in a decrease in platelet activity during this period. For cytosolic GSHPx (cGSHPx) it is known that selenium supply in the body controls the levels of both cGSHPx protein and cGSHPx activity (Takahashi et al., 1986). Based on these findings the measurement of cGSHPx has been used to assess selenium status. However, in our study erythrocyte and plasma GSHPx did not reflect a change in dietary intake and selenium status over the six weeks intervention period when changes in the exchangeable selenium body pools were measured. The finding that the size of exchangeable selenium pools were highly correlated with plasma selenium concentration is consistent with the findings of other workers (Ducros et al., 1997) where the size of exchangeable pools in elderly institutionalised women correlated well with plasma selenium levels (R = 0.66, P < 0.01). The plasma selenium concentration is also consistent with other studies, e.g. in New Zealand subjects (Thompson et al., 1993), in Belgian subjects (Reilly, 1996) and in Finnish subjects (Levander et al., 1988). These findings suggest that plasma selenium concentration is a good indicator of selenium status given that low intakes of selenium in New Zealand support our findings of on the low diet whereas our findings of in the high diet is higher than those found in Belgium which reflect average selenium intakes of

5. CONCLUSION Erythrocyte and plasma GSHPx were not sensitive to large changes in selenium intake over a six week period but might change over a longer time. Conversely, plasma selenium concentration and platelet GSHPx activity were more sensitive indicators of a change in selenium status. Exchangeable pool size estimates did change as a result of changes in selenium intake and correlated with changes in plasma selenium concentrations.

ACKNOWLEDGMENTS This work was supported by the Ministry of Agriculture, Fisheries and Food, the Biotechnology and Biological Sciences Research Council.

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REFERENCES Burke R.F. and Hill K.E., 1993, Regulation of selenoproteins, Ann Rev Nutr; 13:65–81. Diplock A.T., 1993, Indexes of selenium status in human populations, Am J Clin Nutr; 57:256S–258S. Ducros V., Faure P., Ferry M., Couzy F., Biajoux I., and Favier A., 1997, The size of the exchangeable pools of selenium in elderly women and their relation to institutionalization, Br J Nutr, 78:379–396. Janghorbani M., Martin R.F., Kasper L.J., Sun X.F., and Young V.R., 1990, The selenite-exchangeable metabolic pool in humans: A new concept for the assessment of selenium status, Am J Clin Nutr; 51:670–677. Levander O.A., De Loach D.P., Morris V.C.X., and Moser P.B., 1983, Platelet glutathione peroxidase activity as an index of selenium status in rats, J Nutr; 113:55–63. Levander O.A., 1991, Scientific rationale for the 1989 Recommended Dietary Allowances, J Am Diet Assoc; 91:1572–1576. Reilly C., 1996, Selenium in food and health. Blackie Academic and Professional, London. Takahashi K. and Cohen H.J., 1986, Selenium-dependant glutathione peroxidase protein and activity: immunological investigations on cellular and plasma enzymes. Blood; 68:640–645. Thomson C.D., Robinson M.F., Butler J.A., and Whanger P.D., 1993, Long term supplementation with selenate and selenomethionine on selenium and glutathione peroxidase in blood components of New Zealand women. Br J Nutr; 69:577–588. Whanger P.D., Beilstein M.A., Thomson C.D., Robinson M.F., and Howe M., 1988, Blood selenium and glutatione peroxidase activity of populations in New Zealand, Oregon and South Dakota. FASEB J; 2:2296–3002.

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TRUE ABSORPTION, EXCRETION, AND TISSUE RETENTION OF SELENIUM AT WIDELY

VARYING SELENIUM SUPPLY TO RATS

W. Windisch and M. Kirchgessner Institute of Nutrition Sciences University of Technology Munich-Weihenstephan D-85350 Freising, Germany

1. INTRODUCTION Due to widely varying dietary Se contents the organism is forced to counterbalance changes in Se intake by regulating the Se fluxes from the diet through the body. For other essential trace elements this homeostatic regulation is known to work by adaptations of true absorption, endogenous faecal excretion and renal excretion (Kirchgessner, 1993). In the case of Se, the knowledge on the mode of homeostatic action is still fragmentary because there was no appropriate method available to differentiate true absorption from endogenous faecal excretion. In a recent study however, the isotope-dilution technique was established for selenium (Gabler et al., 1997). Therefore, the present study was designed to measure the mode of homeostatic adaptations to dietary Se ranging from deficient to excessive supplies and its interaction to tissue Se metabolism.

2. MATERIAL AND METHODS 32 male SD rats with an initial body weight of 50 g were fed ad libitum a semisynthetic diet poor in Se (40 ng native Se per g). After 18 days the animals were divided into 10 groups and received diets adjusted to total Se contents of 40, 70, 100, 150, 200, 300, 450, 600, 1,000 and 3,000 ng/g by adding NaSelenite. The diets were offered restrictively for 3 weeks (14.1 g/d during the last week). One week after the change in Se supply the animals were injected intramuscularly with Se-75. Subsequently, food intake was recorded and daily faecal and urinary excretions were collected quantitatively. At the end of the experiment, the animals were killed and dissected into blood, organs, samples of muscle (m. quadriceps) and bone (femur), hair and other tissue fractions (Kirchgessner et al., Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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1997). Endogenous faecal excretion and true absorption of Se was quantified by the isotope-dilution technique (Gabler et al., 1997).

3. RESULTS The apparent and true absorption of dietary Se remained constant over the whole range of Se supply and averaged 88% (apparent) and 96% (true) of Se intake (Table 1). There was also no change in endogenous faecal excretion rate (7% of Se intake). Up to dietary levels of 100ng/g the urinary Se excretions ranged between 10 to 14% of intake. At 150ng/g it increased abruptly to 32%, rose degressively to a maximum of 68% at 1,000ng/g and then decreased to 63% at highest Se intake. At Se contents between 40 and 100ng/g the Se retention averaged 78% and dropped abruptly to 58% at 150ng/g. Further increments of dietary Se contents reduced the Se retention continuously to a minimum of 22% of Se intake at 1,000 ng/g and increased it again to 26% at 3,000 ng/g. The Se concentrations of tissues increased with dietary Se levels rising from 40 to 150ng/g and from 600 to 3,000ng/g while within the middle range of Se supply the Se concentrations of tissues remained rather constant (Table 2; see also Kirchgessner et al., 1997). The strongest reaction of tissue Se concentration was found in the liver with a 15fold rise between the lowest and highest level of Se supply. In most tissues the differences ranged in the magnitude of factor 4 (see also Kirchgessner et al., 1997). Only in brain, testes and hair the Se concentrations remained constant except for an increase at the highest dietary Se level. The GSH-Px activity in blood plasma rose degressively from 2,540 U/l at lowest Se supply to a maximum of about 5,700U/l which was reached at dietary Se levels of 600 ng/g. The increase in GSH-Px activity was continuous and there was no indication of an abrupt change in reaction pattern on rising levels of dietary Se. The Se-75 activities found in the whole body of the animals at the end of the experiment decreased from 88% to 7% of injected Se-75 activity as dietary Se supply rose. The difference, between final whole-body activity and the injected Se-75 was recovered completely within the cumulative faecal and renal excretions of Se-75.

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4. DISCUSSION Neither true absorption rates nor endogenous faecal excretion of Se revealed any quantitatively relevant reaction on the wide change in Se supply. Obviously, there is no homeostatic adjustment of Se fluxes between the digestive tract and the ‘internal body’ with respect to the Se status. In total, the absorption capacity of dietary Se (selenite) was very high (96% of intake) which confirms other recent studies (Gabler et al., 1997; Windisch et al., 1998). The low urinary Se excretion rates at dietary Se levels below 150 ng/g indicate the attempt of the organism to counteract Se deficiency by minimising Se losses. However, at 150 ng/g the urinary Se excretion increased abruptly which is to be interpreted as the transition from a deficient to a sufficient Se supply and the onset of a homeostatically regulated elimination of excessively absorbed dietary Se. This break in urinary Se excretion pattern provides a direct estimate of Se requirement. The injected Se-75 was recovered completely in tissues and excrements irrespective of the level of Se supply. Therefore, gaseous exhalations of Se were not of quantitative importance. The urinary excretion revealed to be the only quantitatively relevant means of Se homeostasis. Consequently, the homeostatic regulation takes place after the dietary Se has already been absorbed. However, the excretory potential of the kidneys is limited. This seemed to be already the case at dietary Se concentrations of 1,000 ng/g, because at this Se supply the urinary Se excretion (expressed as % of Se intake) turned from increasing to decreasing rates. Thus, rising amounts of absorbed Se were no more eliminated via kidney and accumulated in tissues. The changes in tissue Se concentrations due to the rise in Se supply were most pronounced in the liver. This organ seemed to act as Se storage which was mobilised in Se deficiency and filled with substantial quantities of Se at excessive Se intake. In contrast to liver, the brain and testes revealed to be subject to a privileged homeostasis within the body since their Se concentrations remained constant from deficient to high dietary Se supply. Also in the hair, the Se concentration remained constant except for a severe increase at highest Se supply. Presumably, the excess in dietary Se intake induced the pronounced metabolic formation of seleno amino acids which were incorporation into the hair (see also Windisch et al., 1998).

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The GSH-Px activity and the concentration of Se in blood plasma are widely used as parameter to diagnose the Se status in terms of a deficient or sufficient dietary Se supply. However, an important precondition for this purpose is a distinct change in reaction pattern when Se intake passes from deficient to sufficient supply. But neither GSHPx activity nor Se concentration in blood plasma showed such a distinct break. Thus, their use as a parameter to diagnose the Se status seems to be limited. In conclusion, the homeostatic control of Se metabolism is based on the urinary excretion. The compensatory capacity of Se homeostasis ranges between dietary Se contents of about 150ng/g (minimum requirement) and 600ng/g (threshold to an unphysiological excess).

REFERENCES Gabler, S., Kirchgessner, M., and Windisch, W., 1997: Isotope-dilution technique for determination of endogenous faecal excretion and true absorption of selenium in Se-75 labeled rats. J. Anim. Physiol. a. Anim. Nutr. 78:10–19. Kirchgessner, M., 1993: Homeostasis and homeorhesis in trace element metabolism. In: Trace Elements in Man and Animals—TEMA 8. (Eds. Anke, M., Meissner, D., and Mills, C.F.) Verlag Media Touristik, Gersdorf, Germany. Kirchgessner, M., Gabler, S., and Windisch, W., 1997: Homeostatic adjustments of selenium metabolism and tissue selenium to widely varying selenium supply in Se-75 labeled rats. J. Anim. Physiol. a. Anim. Nutr. 78:20–30. Windisch, W., Gabler, S., and Kirchgessner, M., 1997: Effect of selenite, seleno cysteine and seleno methionine on the selenium metabolism on Se-75 labeled rats. J. Anim. Physiol. a. Anim. Nutr. 78:67–74.

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THE ORAL INTAKE OF ANTIOXIDANT NUTRIENT ASSOCIATED WITH SELENIUM DOES PROTECT SKIN AGAINST ACTINIC EXPOSURE

Jean Pierre Césarini and S. Demanneville L.R.T.P.H.—I.N.S.E.R.M.—Fondation A. de Rothschild 75019 Paris, France

Exposures of the human skin to solar ultraviolet radiations (UVR) induce several acute effects and cumulative damages. Among the acute effects, the actinic erythema is the consequence of a cascade of events starting with the DNA direct absorption of ultraviolet radiations (mainly UVB) or the DNA indirect damages through oxygen reactive species produced by the absorption of UVA by cellular chromophores (Pathak and Stratton, 1968). Either directly or indirectly, several constituents of the cells in the epidermis may be directly damaged, leading to reduction or suppression of some functions, i.e.: cytoplasmic membranes, membrane receptors, enzymes, or, as an adaptation to the aggression, they may be engaged in cell division or in the production of protecting material like melanins. As a result of cumulative damages throughout a life time, skin ageing (helio-dermatoses) and skin cancers will develop (Darr and Fridovich, 1994). It has been recognised for a long time that free radicals are pathologic agents which result from the UVR absorption with deleterious consequences. In the epidermis, keratinocytes, melanocytes and Langerhans cell have built several lines of defence to neutralise the production of free radicals by the respiratory bust or by the UVR of solar exposures. Products of lipid peroxidation, melanin radicals and antioxidant depletion have been observed in UV-irradiated skin. Following skin UV irradiation, superoxidedismutase, catalase and glutathione peroxidase activities decline as well as the levels of vitamins C and E. Supplementation of the skin with trace elements or vitamins, either supplied topically or systemically, is supposed to bring a better resistance or to restore

Address all correspondence to Dr. Jean-Pierre Césarini, L.R.T.P.H.—I.N.S.E.R.M.—Fondation A. de Rothschild, 25, rue Manin, 75019 Paris, France. Telephone: +33-1-48036948; fax: +33-1-48036510; email: [email protected] Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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an adequate line of defense. Indeed, in experimental conditions, in vitro or in animal studies, antioxidant nutrients were constantly demonstrated efficient against the oxidative stress. Among several experiments, realised in vitro or in laboratory animals, we have selected some convincing experiments. Vitamin A promotes the repair of the dermal damage in UVB -irradiated hairless mouse (Bryce et al., 1988). In hairless mice, topical and systemic vitamin E are able to reduce the UV-induced lipid peroxidation and to restore the thymidine incorporation reduced by UV radiations (Record et al., 1991). Skin tumours, induced by ultraviolet radiations in Skh: HR-1 hairless mice, were significantly reduced in a dose-related manner when the animals were maintained on a diet containing increased levels of selenium (incorporated in yeast). It was found, by the monitoring of several epidermal antioxidant enzymes, that the selenium deficiency decreased the glutathione peroxidase and resulted in increase of superoxide-dismutase and catalase in response to the UV aggression (Pence et al., 1994). In a series of experiments, the percutaneous applications of selenium-rich water on mice skin, in a UVR induced carcinogenesis protocol, reduced the incidence of skin tumours (Cadi et al., 1991). On human cutaneous fibroblasts in culture, the supplementation of the medium in selenium-rich water, resulted in a significant resistance to the UVR aggression (Moysan et al., 1995). In human, a diet supplemented in ascorbic acid and in was found efficient to protect against sunburn reaction (Eberlein-Köning et al., 1998), antioxidant nutrients increase the UV-induced pigmentation (Postaire et al., 1997). In a double blind versus placebo three weeks supplementation of volunteers with selenium and a vitamin complex (vitamins E and A), we have been able to demonstrate a reduction in UV-induced cell damages (reduction in erythema) and a protection against UV-genotoxic lesions i.e. reduction of sunburn cells (La Ruche and Césarini, 1991). The aims of the present study was to demonstrate the efficacy of the daily intake during 28 consecutive days of a complex of vitamins (vit. A: 1.000U.I., vit. C: 60mg, vit.E: 15 U.I.) associated with selenium in the form of organic salt: metabolised by saccharomyces cerevisiae yeast (150mg) in 30 healthy volunteers against the UV aggression represented by a solar simulator delivering sub and supra erythemal doses. Three parameters were studied: actinic erythema threshold (MED), pigmentary modification, both evaluated by chromametry, and dosage of lipoperoxides in the stratum corneum obtained by non invasive skin surface biopsies (fluorimetric method): before and after irradiation, before and after intake of the complex.

Effect on Actinic Erythema For all the six study doses ranging from 0.8 MED to 2.44 MED, the redness was significantly reduced (p < 0.001). Among the 30 volunteers, 14 presented at least a 25% increase in the threshold corresponding to MED.

Effect on Surface Lipoperoxides On non irradiated skin, the spontaneous level of lipoperoxides was reduced by an average of 12% (tendency p = 0.06). After irradiation, for the doses between 0.8 MED and 1.25 MED, the level of cutaneous lipoperoxides was multiplied by fivefold, the reduction of this increase being in average of 50%. It has been noticed that 35% of the subjects were not responders, while 23% had a reduction of more than 70%.

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We concluded that association of selenium with vitamin complex, taken orally for 28 days, at a dosage within the recommended daily intake, was able to improve the skin defence against free radical generated by UV exposures (Césarini et al., 1998). In a more recent experience, a topical preparation containing the same complex was applied on the skin of volunteers either during a few days (the last application being 24 hours before irradiation), immediately (15 minutes) before irradiation, or after irradiation. The threshold for the MED was elevated by at least 25% in average, only when the gel was applied a few minutes before the irradiation. As a conclusion, we believe that antioxidant complex at a recommended daily intake regimen, or applied topically, may reduce significantly the toxicity of solar radiation corresponding to a normal daily exposure. Potent sunscreens (SPF > 15) are mandatory to filter out the aggressive radiations corresponding to several hours of exposure. However, the consequences of minimal daily exposures, known to be responsible of the regular insult of the skin and chronic effects, like skin ageing, skin cancers and immunosuppression, can be efficiently fight by the daily intake of micro nutrients.

ACKNOWLEDGMENT This work was made possible by a grant from Laboratoires Richelet, 15, rue la Pérouse, 75116 Paris, France.

REFERENCES Bryce, G.F., Bogdan, B.S., and Brown, C.C., 1988, Retinoic acids promote the repair of the dermal damage and the effacement of wrinkles in the UVB—irradiated hairless mouse, J. Invest. Dermatol. 91:175–180. Cadi, R., Beani, J.C., Belanger, S., Richard, M.J., Richard, A., Favier, A., and Amblard, P., 1991, Effet protecteur de l’application percutanée d’eau thermale Roche-Posay vis-à-vis de la peroxydation lipidique et de la carcinogénése cutanée induites par les UVB, Nouv. Dermatol. 10:266–272. Césarini, J.P., Girard, P., Garcia, E., and Demanneville, S., 1998, La prise orale d’un supplément nutritionnel antioxydant réduit les conséquences de 1’agression actinique, Nouv. Dermatol. 17:305–308. Darr, D. and Fridovich, I., 1994, Free radicals in cutaneous biology, J. Invest. Dermatol. 102:671–674. Eberlein-König, B., Placzek, M., and Przybilla, B., 1998, Protective effect against sunburn of combined systemic ascorbic acid (vitamin C) and (vitamin E), J. Am. Acad. Dermatol. 38:45–48. La Ruche, G. and Césarini, J.P., 1991, Protective effect of oral selenium plus copper associated with vitamin complex on sunburn cell formation in human skin, Photodermatol. Photoimmunol. Photomed. 8:232–235. Moysan, A., Morlière, P., Marquis, I., Richard, A., and Dubertret, L., 1995, Effects of selenium on UVAinduced lipid peroxidation in cultured human skin fibroblasts, Skin Pharmacology 8:135–148. Pathak, M.A. and Stratton, K., 1968, Free radicals in human skin before and after exposure to light, Arch. Biochem. Biophys. 123:468–476. Pence, B.C., Delver, E., and Dunn, D.M., 1994, Effects of dietary selenium on UVB-induced skin carcinogenesis and epidermal antioxidant status, J. Invest. Dermatol. 102:759–761. Postaire, E., Jungmannn, H., Bejot, M., Heinrich, U., and Tronnier, H., 1997, Evidence for antioxidant nutrients-induced pigmentation in skin: results of a clinical trial, Biochemistry and Molecular Biology International 42:1023–1033. Record, I.R., Dreosti, I.E., Konstantinopoulos, M., and Buckley, R.A., 1991, The influence of topical and systemic vitamin E on ultraviolet light-induced skin damage in hairless mice, Nutrition and Cancer 16:219–225.

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BIOAVAILABILITY OF SELENIUM FROM UNTREATED AND PROCESSED SELENOMETHIONINE-ENRICHED FILLETS OF ATLANTIC SALMON (SALMOSALAR)

R. Oernsrud and M. Lorentzen Institute of Nutrition, Directorate of Fisheries P.O. Box 185. N-5002, Bergen Norway

Wild Atlantic salmon has a higher concentration of selenium (Se) in muscle than farmed Atlantic salmon. To inscrease Se levels in farmed fish, fish feed can be supplemented with selenomethionine. Se bioavailability and the effect of processing on these salmon fillets were examined in the present study. The bioavailability of Se from untreated and processed salmon (“gravlaks”) was assessed in Se-deficient albino rats (Mol: Wist). A low-Se Torula yeast feed was supplemented with 0, 50, 100, 150 or as sodium selenite or as Se from untreated or processed salmon, and fed to weanling rats for 30 days. Bioavailability of Se was assessed by metabolic balance, tissue Se levels (femur, muscle, liver and plasma) and activity of Se-dependent glutathione peroxidase (EC 1.11.1.9) in plasma, and calculated by the slope-ratio method using sodium selenite as a standard. All response parameters showed a higher bioavailability of Se from the selenomethionine-enriched salmon fillets than selenite. Differences in Se absorption, retention and Se levels in rat muscle from untreated and processed salmon suggested that processing alters the bioavailability of Se. The results showed that the bioavailability of Se from selenomethionine-enriched salmon fillets was higher than selenite, and that processing of the fillets altered the utilisation of Se.

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STABILITY OF GLUTATHIONE PEROXIDASE ACTIVITY DURING STORAGE AND HEAT TREATMENT OF WHEY H. Lindmark Månsson1, J. Chen2, and B. Åkesson2 1

Swedish Dairy Association Lund, and 2 Division of Biomedical Nutrition Chemical Center, Lund University

Lund, Sweden

Antioxidant enzymes may have important roles for the oxidative stability of foods. Recently it has been discovered that the family of the selenium-containing glutathione peroxidases (GSHPx) contain at least four members in mammalian tissue but their role in foods is not well known. To some extent this is due to a lack of suitable assays since activity measurements only cannot distinguish between all enzymes and only few specific immunochemical methods are available. In this study a modified version of the coupled enzymatic assay for GSHPx using glutathione reductase was applied to whey. Moreover the stability of GSHPx activity was followed at storage and heat treatments simulating those used in the food industry. Whey was prepared by treatment of milk with lactic acid and then stored frozen. GSHPx activity was assayed using tert.butylhydroperoxide and glutathione as substrates. As blanks enzyme-free incubations or incubations containing mercaptosuccinate (4 mmol/l) were used. In acid whey (pH 4.5) stored at +8° and at room temperature (approx. 22°) GSHPx was more stable than in whey adjusted to pH 6.7. After 9 days in the cold 89% of the starting activity remained at pH 4.5 and 65% at pH 6.7. After storage for 9 days at room temperature 49% of the activity remained at pH 4.5 and 35% at pH 6.7. On the contrary when whey with different pH was heated the stability of GSHPx was higher at pH 6.7 than at pH 4.5. After heat treatment at 63° for 30 min which simulates pasteurization, 79% of GSHPx activity remained at pH 6.7 but only 9% at pH 4.5. As expected heating at higher temperatures resulted in more rapid loss of GSHPx activity. After heating at 72° for 5 min 75% of GSHPx activity remained at pH 6.7 and 5% at pH 4.5. After heating at 80° for 5min most of the activity was lost but after 2min 81% of GSHPx activity remained in whey at pH 6.7 and 44% in whey maintained at pH 4.5. These results indicate that GSHPx in whey is stable to several treatments used in the dairy industry suggesting that is has importance for food stability and quality. 892

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PURIFICATION AND ENZYME-LINKED IMMUNOASSAY OF BOVINE EXTRACELLULAR GLUTATHIONE PEROXIDASE H. Lindmark Månsson1,2 and B. Åkesson3 1

Department of Applied Nutrition and Food Chemistry Lund University, S-221 00 Lund Sweden 2 Swedish Dairy Association S-223 70 Lund, Sweden 3 Division of Biomedical Nutrition Chemical Center, Lund University S-221 00 Lund, Sweden

Milk lipids easily undergo auto-oxidation which may lead to development of offflavour. The mechanisms involved include a complex interplay of pro- and antioxidants. e. g. different enzymes. Among antioxidant enzymes, superoxide dismutase and catalase have been demonstrated in milk. Another enzyme family with potential antioxidant functions are the selenium-containing glutathione peroxidases. Glutathione peroxidase activity occurs in bovine milk and in milk from other species but its importance is not well known. In this study extracellular glutathione peroxidase (eGSHPx) was purified from bovine plasma and an immunological method for quantitation of the protein in whey was developed. eGSHPx was isolated from bovine plasma according to the method of Madipatti and Marnett1 modified by Huang and Åkesson.2 It included ammonium sulphate fractionation and column chromatography using phenyl-Sepharose CL-4B, DEAESephadex A-50 and Sephadex G-200. The degree of the purification was approx. 20,000-fold, and the preparation gave rise to only one band at SDS-PAGE. Antibodies to the purified eGSHPx was raised in a rabbit. An enzyme-linked immunosorbent assay (ELISA) was developed. The ELISA-metod was used to quantify eGSHPx in bovine milk and whey samples. 893

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REFERENCES Maddipati, K.R. and Marnett, L.J. Characterization of the major hydroperoxide-reducing activity of human plasma. J. Biol. Chem. 1987;262:17298–17403. Huang, W. and Åkesson, B. Radioimmunoassay of glutathione peroxidase in human plasma. Clin. Chim. Acta, 1993;219:139–148.

281

SELENIUM DISTRIBUTION IN LIVER OF HENS FED WITH SUNFLOWER OIL

I. Falnoga, V. Stibilj, D. Mazej, and M. Tušek Znidaric Department of Environmental Sciences Jozef Stefan. Institute, Ljubljana Slovenia

Peroxidation of lipids or oxygen free radical generation in general is physiological process important for cell metabolism, division and differentiation and also for biosyntesis of hormons and prostaglandins. Free radicals generated through these processes are effectively scavened by antioxidant defence system. A part of these system are selenoproteins like cellular glutathionperoxidase (GPx) and regarding some literature data also metallothioneins (MTs). Cellular GPx is an important antioxidant and MTs comprise a family of low molecular weight metal-binding proteins that are reported to function in the detoxification of heavy metals, in Cu and Zn homeostasis, in the scavening of free radicals, in the acute phase response and maybe also in the regulation of energy balance (1). So in wider less specific view it could be said that the metabolism of lipids is tightly connected with selenium and metallothioneins. Regarding these data we were interested in selenium distribution in livers of laying hens fed with feed containing 5% extra sunflower oil where the total amount of crude fat in feed was higher than the recommended value. The purpose of this study was to determine the total selenium concentrations in water soluble protein fractions after size exclusion chromatography (Sephadex G-75) of liver supernatants; to compare the selenium, Zn and Cu distribution in supernatants from lyophilized and wet liver samples; and to verify the presence of Zn,Cu MT—like proteins and its posible association with selenium. The procedure for determination of selenium concentrations by HG-AFS (atomic fluorescence spectrometry coupled with the hydride-generation technique) in column fractions was developed in our laboratory. It is relatively simple and rapid with a very low detection limit of 0.2 ng/g. After size-exclusion chromatography (Sephadex G-75) of both supernatants, from wet and lyophilised liver samples, no essential difference was found regarding the position of the Se, Zn and Cu peaks. But a difference was observed in the peak heights, particularly in the range near the void volume and a smaller one in the range of MT-like 895

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proteins (position near cytochrome c). In both cases it was found the presence of selenium associated with Zn,Cu MT—like proteins.

REFERENCES Beattie J.H., Wood A.M., Newman A.M., Bremner I., Choo K.H.A., Michalska A.E., Duncan J.S., and Trayhurn P. 1998, Obesity and hyperleptinemia in metallothionein (-I and -II) null mice Proc. Natl. Acad. Sci. 95, 358–363. Takatera K., Osaki N., Yamaguchi H., and Watanabe T., 1994, HPLC/ICP Mass Spectrometric Study of Selenium Incorporation into Cyanobacterial Metallothionein Induced under Heavy-Metal Stress. Analytical Science. 10, 567–572.

282

SELENIUM AND LIPOPROTEINS Preliminary in Vivo and in Vitro Observations in Humans

V. Ducros1,2, F. Laporte1,2, N. Belin2, A. David2, A-M. Roussel1, and A. Favier1,2 1

Laboratoire de Biologie du Stress Oxydant UFR des Sciences Pharmaceutiques et Biologiques Domaine de La Merci F-38700 La Tronche, France 2 Fédération des Laboratoires de Biochimie CHU de Grenoble, BP 217 F-38043 Grenoble cedex 9

Different hypotheses have been proposed to account for the role of selenium (Se) in the prevention of cardiovascular diseases: Se is present at the active site of the enzyme glutathione peroxidase and thus may protect endothelial cells and lipoproteins from lipid peroxidation. Selenoperoxidases can also regulate the production of pro-aggregant eicosanoids from arachidonic acid. In addition Se may play a role in the metabolism of lipoproteins since Se deficiency has been shown to induce hypercholesterolemia in animals. The present work was undertaken to examine whether Se is present in plasma lipoproteins and to investigate the role of Se in the protection of lipoproteins against oxidative damages. Lipoproteins were isolated by density gradient ultracentrifugation of EDTA plasma, HDL was further purified by ultracentrifugal flotation in KBr. Se content in isolated lipoproteins was determined by gas chromatography-mass spectrometry using the isotopic dilution technique. The kinetics of conjugated diene formation during copperinduced LDL oxidation was determined by monitoring the change in absorbtion at 234 nm. Our results demonstrate that human plasma lipoproteins (LDL + HDL) contain about 4% of total plasma Se, probably in the form of selenomethionine occuring as a random substitute for methionine in the apolipoproteins. We have evaluated the efficiency of different Se compounds to inhibit LDL oxidation: selenate and selenite failed to delay 897

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the Cu-induced formation of conjugated dienes while both selenomethionine and selenocystine at micromolar concentrations were more potent antioxidants than the corresponding sulfur aminoacids. Our results suggest that Selenoaminoacids may exert their protective effect either by chelating the copper ion or by a peroxidase-like activity.

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TRANSFORMING GROWTH SELENOPROTEIN P EXPRESSION IN CULTURED HUMAN LIVER CELLS

INHIBITS

V. Mostert1, I. Dreher2, J. Köhrle2, and J. Abel1 1

Medizinisches Institut für Umwelthygiene an der Heinrich-Heine-Universität Department of Toxicology Auf’m Hennekamp 50 40225 Düsseldorf, FRG 2 Klinische Forschergruppe Medizinische Poliklinik Universität Würzburg Röntgenring 11 97070 Würzburg, FRG

Selenoprotein P (SeP) is a plasma protein which contains up to ten selenocysteine residues and accounts for about 50% of total selenium in human plasma. Its biological function is still unknown, but an antioxidative role appears to be probable. Data that demonstrate a protective role of SeP against oxidation and nitration reactions mediated by the potent endotoxin peroxynitrite implicate an involvement of SeP in inflammatory processes. This is also suggested by the finding of the SeP promoter being negatively regulated by inflammatory cytokines such as and In this study, the effect of on the expression of SeP in the human liver cell line HepG2 was investigated. Western analysis revealed a dose-dependent reduction of SeP content in cell supernatant with an of 31 pM Treatment with 100pM for 48 h led to a decrease to 21 ± 9% of controls. RT-PCR analysis of SeP-mRNA expression demonstrated an inhibition of SeP transcription to 40 ± 2% of control levels after 24 h treatment with 100pM Experiments with inhibitors of transcription and translation revealed the necessity of mRNA and/or protein-synthesis for to exhibit its inhibitory effect. The mRNA expression of other selenoproteins investigated in this study, GPX3 and GPX4, remained unaffected by treatment. 899

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A luciferase expression vector under control of the human SeP promoter was negatively regulated by treatment in a dose-dependent fashion indicating a transcriptional regulation of the SeP gene by We suggest that downregulates SeP synthesis by liver cells during the late phase of inflammation after previous upregulation by a yet-unknown pro-inflammatory mediator during acute phase.

284

EXPERIMENTALLY INDUCED SELENIUM AND VITAMIN E DEFICIENCY IN GROWING RABBITS

J. Pallauf, A. Müller, and Erika Most Institute of Animal Nutrition and Nutrition Physiology Justus-Liebig-University Senckenbergstraße 5, D-35390 GIESSEN Germany

Little information is available on the importance of selenium for rabbits with adequate vitamin E supply. Therefore 4 × 5 New Zealand White weanling rabbits with an initial weight of 610 ± 62 g received diets based on torula yeast for 12 weeks. The Se content of the basal diet was below the detection limit of 0.03mg/kg. The concentration was only 1.5mg/kg diet. The diets for groups II and III were supplemented with 150 mg/kg and 0.40 mg/kg Se as sodium selenite respectively. To obtain an initial status 5 additional animals were sacrificed at the beginning of the experiment. Blood was taken every two weeks from the vena auricularis. Activity of glutathione peroxidase (GPx) was measured according to a modified method of LAWRENCE and BURK (1976). For the determination of Se dependent plasma-GPx was used as substrate. Total activity of liver cytosolic-GPx was estimated with t-butyl-hvdroperoxide. After 5 weeks plasma-GPx activity as compared to the initial status group (45.2 ± 4.53mU/mg protein) was decreased in group I (19.1 ± 7.08), remained unchanged stable in group II (46.3 ±11.2) and increased in the Se sufficient groups III (62.4 ± 23.9) and IV (106 ± 19.9). After 12 weeks a plateau of enzyme activity was reached in groups III (100 ± 27.6) and IV (110 ± 19.4) while in groups I (5.54 ± 1.09) and II (16.4 ± 8.97) extremely low values were obtained. At the end of the experiment plasma Se concentrations were significantly lower in the Se deficient groups I (38.3 ± 6.24ng/ml) and II (42.6 ± 9.77) than in groups III (149 ± 33.4) and IV (126 ± 6.45). The concentration in plasma was significantly higher in the vitamin E supplemented groups II (9.76 and IV (9.33 ± 3.36) as compared to groups I (1.06 ± 0.41) and III (1.34 ± 0.24). Compared to the initial status group (529 ± 108mU/mg protein) selenium depletion caused a significant decrease of liver c-GPx activity in groups I (66.6 ± 24.9) and II (127 ± 79.9) whereas increased activities were found for the supplemented groups III (655 ± 197) and IV (819 ± 80.8). Liver Se concentration was drastically lower in groups I (1.60 ± 0.35ng/mg protein) and II (1.79 ± 0.44) than in the Se supplemented groups III (15.9 901

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±4.13) and IV (16.1 ± 1.58). The concentration in the liver was much higher in the supplemented groups II (32.2 ± 10.2mg/kg fresh matter) and IV (34.6 ± 9.13) as compared to the vitamin E deficient groups I (1.25 ± 0.15) and III (2.34 ± 0.97). The TBA-RS indicated the highest rate of lipid peroxidation in the unsupplemented group I (2,399 ± 413nmol/mg protein). Se treatment (group III) resulted in significantly lower values (191 ± 70) for lipid peroxides. Vitamin E treatment (group II, 31.4 ± 9.87) and especially combined supplementation of Se and vitamin E (group IV, 19.8 ± 6.5) indicated an effective protection against lipid peroxidation. In summary Se considerably modulated the activity of GPx in the peroxide metabolism of rabbits. However vitamin E is needed to establish the enzyme activity of Gpx and to avoid lipid peroxides.

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IDENTIFICATION OF 5 NOVEL SELENOPROTEINS BASED ON RNA STRUCTURAL TAGS

A. Lescure, D. Gautheret*, P. Carbon, and A. Krol *EP91 CNRS “Information Génétique et Structurale” Marseille, France UPR 9002 du CNRS, IBMC 15 rue René Descartes 67084 Strasbourg France

Selenoproteins have been shown to play an important role in essential cellular processes and the prevention of cancer. However, the physiological role of selenium is not fully understood. In order to better address this question, we have undertaken the identification of new selenoproteins by an original approach. Selenocysteine incorporation into selenoproteins arises from readthrough of an in frame UGA codon. In eukaryotes, this mechanism requires the presence of the SECIS element, a specific RNA structure residing in the 3’UTR of selenoprotein mRNAs. Based on experimental data, a consensus secondary structure for the SECIS element has been proposed in our laboratory (Walczak et al., 1996; see also communication by Dr Alain Krol). Using a program enabling detection of RNA secondary structures, computational searches of genomic or EST databases led to the discovery of 52 potential SECIS elements capable of adopting the consensus secondary structure. Among these, 33 corresponded to already characterized selenoprotein mRNAs. The remaining unknown SECIS elements were tested for their abilities to promote insertion of selenocysteine in vivo. Five novel functional SECIS elements were identified in this manner. The corresponding cDNAs were subsequently sequenced and, as expected, the new SECIS elements are indeed localized in the 3’UTRs of the mRNAs. The coding sequences have been identified and all contain at least one in frame UGA codon. Potential functions for these selenoproteins, based on protein domain homologies and cellular localization, will be presented. To our knowledge, it is the first time that a strategy based on a search with a consensus RNA secondary structure has been taken to uncover the existence of new proteins. This allowed us to identify cDNAs coding for five novel selenoproteins of as yet unknown function. 903

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TAT PROTEIN OF HIV1 DECREASES SELENOGLUTATHIONE PEROXIDASE AND INCREASES UV-A CYTOTOXICITY INDEPENDENTLY OF SELENIUM SUPPLEMENTATION

Richard M. J., Guiraud P., and Favier A. Laboratoire de Biologie du Stress Oxydant (LBSO) Université Joseph Fourier UFR de Medecine Pharmacie 38700 La Tronche, France

Tat protein of HIV1 has been reported to influence cellular phenotype by affecting the expression of cellular genes. These modifications include a decrease of manganese superoxide dismutase expression and may lead to oxidative stress by different events including increased generation of reactive oxygen species (ROS), and glutathione depletion. In this work we looked at the role of Tat protein on intracellular pro-/antioxidant balance using HeLa cells stably transfected with a tat expression vector. We focused on selenium dependant glutathione peroxidase (GSH-Px). We showed that Tat protein affects intracellular trace element levels and metalloenzymes involved in peroxides metabolism. Se, Zn and Mn were not modified by the expression of Tat whereas intracellular Cu and Fe levels were significantly decreased in HeLa-tat cells. Interestingly, Se-GSH-Px activity was dramatically decreased in tat transfected cells compared with the wild-type HeLa cells (p < 0.001). This alteration is not related to cellular deficiency in selenium. In addition the redox status related to glutathione (GSH) was altered. The ratio GSSG/GSH is significantly increased (p < 0.016) in transfected cells compared with parental cells whereas GSH reductase is significantly increased in transfected cells. In order to understand Se metabolism in HeLa-tat cells we studied the uptake, distribution and cytotoxicity of sodium selenite. incorporation and release were similar in HeLa-tat and wild cells. Nethertheless Se cytotoxicity was significantly different, HeLa parenteral cells being more sensitive to selenite than HeLa-tat cells 904

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The same qualitative pattern was obtained when analysing the total intracellular selenoproteins by SDS-PAGE electrophoresis and autoradiography after a 24 h labelling. However, differences were observed between HeLa wild and HeLa-tat in intensity of the spots. Se was added to the culture medium, intracellular GSH-Px activity increased but was maintained to low values comparing parenteral cells. GSH-Px reach a plateau at Se in HeLa-tat cells whereas a good correlation between Se and GSH-Px is obtained until in wild cells. Tat amplifies oxidative lethal damages induced by UVA irradiation. When HeLa cells were UVA irradiated the lethal dose 50 (LD50) in wild cells (90J-cm2) was significantly different from that obtained for HeLa-tat cell (65 J-cm2). The culture of the cells in a selenium supplemented medium did not lead to a modification of UV cytotoxicity neither in HeLa-wild nor in tat cells although GSH-Px activity increased. In this work we demontrated for the first time that Tat protein modifies cellular selenium metabolism and GPX activity. In addition, tat transfected cells were more susceptible to UV irradiation even in selenium supplemented cells.

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SERUM SELENIUM CONCENTRATIONS AND THE ACUTE PHASE RESPONSE

T. M. T. Sheehan and Aleha Khatun Regional Laboratory for Toxicology City Hospital NHS Teaching Trust Dudley Road, Birmingham B18 7QH U.K.

Serum selenium concentrations in patients undergoing elective surgery have been shown to fall as part of the Acute Phase Response.1 The contribution of this phenomenon to the low selenium values encountered in patients on parenteral and other nutritional therapies has yet to be determined. A preliminary investigation into this question has been made using specimens (n = 476, from 448 adults patients) submitted to this laboratory, over a three-month period, for the measurement of essential trace elements. Selenium was assayed using graphite furnace atomic absorption spectroscopy (Local adult reference range: Specimens were investigated for evidence of Acute Phase Response by turbidimetric assay of C-reactive protein (CRP; normal values <6mg/L). 107 specimens (22%) yielded results that were normal for both CRP and selenium . 70 specimens (15%) had normal CRP values, but depleted selenium The majority of specimens (247: 52%) had raised CRP (median = 62mg/L) and depressed selenium A fourth group of 52 specimens (11%) with raised CRP values (media = 24mg/L) was within the normal range for selenium Comparison between the two ‘normal’ selenium groups apart, differences between the median selenium values were all significant at 0.05% (MannWhitney Test). Within-group correlation between the two analytes was limited, the best exhibited by the positive trend within the normal selenium/raised CRP group. Correlation within the low selenium/raised CRP group was poorer and inverse. Overall, it was apparent that the proportion of specimens with raised CRP values increased as the selenium concentration range fell. Thus, for all specimens (n = 159) with normal selenium the proportion with raised CRP was 33%; the equivalent finding for those (n = 319) with depleted selenium was 78%. In the specimen population studied it is evident that selenium concentration are influenced by the Acute Phase Response. However, the apparent increase in selenium with 906

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CRP in one specimen group illustrates the complexity of investigationg this category of patient; in this instance the extent of selenium supplementation will need to be determined. Such considerations may make the interpretation of isolated results problematic even when the degree of Acute Phase Response is known. REFERENCE 1. Nichol C., Herdman J., Sattar N. et al. Changes in concentrations of plasma selenium and selenoproteins after minor elective surgery: further evidence for a negative acute phase response? Clin. Chem. 1988, 44:1764–1766.

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288

NEUROCHEMICAL ALTERATIONS FOLLOWING PERINATAL COPPER DEFICIENCY IN RODENTS

Joseph R. Prohaska Department of Biochemistry & Molecular Biology School of Medicine University of Minnesota-Duluth Duluth, Minnesota 55812

1. INTRODUCTION Essentiality of copper for mammals was established in the 1920s at the University of Wisconsin. Less than a decade later the essentiality of copper for brain was documented by characterizing enzootic ataxia in lambs, a condition resulting from ewes grazing on pastures low in copper (Bennetts and Chapman, 1937). Another striking confirmation of copper’s role in brain development was work on copper-deficient guinea pigs (Evenson et al., 1968). Cerebellar development was grossly impaired and hypomyelination was evident. Underscoring copperís importance for human brain was the discovery of the link between abnormal copper metabolism and the X-linked neurodegenerative disorder, Menkes’ disease by David Danks and colleagues (Danks et al., 1972). Abundant research attempting to link the lack of brain copper and its associated neuropathology exists. This hypothesis-based research follows the sequence: 1) inadequate copper intake in the diet; 2) leads to a lower than normal concentration in brain copper; 3) which leads to an impairment in the activity of a copper-dependent enzyme; 4) which causes a metabolic pathway to be altered; 5) resulting in an altered phenotype. An example might be useful. The lack of pigmentation which accompanies copper deficiency is due to limitation in the activity of tyrosinase, a copper-dependent enzyme which converts tyrosine to L-DOPA enroute to melanin formation. Several cuproenzymes are candidate proteins for brain hypothesis testing (Table 1).

2. MODELS Several approaches have been employed to study the neurochemical changes/ functions dependent on copper. Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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2.1. Genetic The most successful genetic models are those using mutant mice. Hunt and coworkers discovered that the coat color mottled mutants had a defect in norepinephrine synthesis (Hunt and Johnson, 1972) and later discovered this was connected with a defect in copper transport (Hunt, 1974). Mouse mutations at the mottled locus and those of Menkes’ disease are homologous. Both the human and mouse genetic mutants demonstrate the essentiality of the copper-ATPase-efflux transporter for normal brain metabolism. There were a few bumps along the research path involving two other murine mutations: crinkled and quaking. Neither is convincing that copper is involved (Prohaska, 1987). Non-lethal mutations of two human cuproenzymes are known. Humans lacking ceruloplasmin (CPL) have a CNS neuropathy which is caused by iron overload rather than copper deficiency (Morita et al., 1995). Humans lacking a functional dopamine(-monooxygenase (DBM) exhibit alterations of the autonomic nervous system (Gary and Robertson, 1994). Thus no single cuproenzyme deletion matches the observed phenotype of dietary copper deficiency. Contemporary research using transgenic mice holds promise for hypothesis testing and studying the neuropathology of copper deficiency. Knockout mice lacking DBM must be rescued with norepinephrine (NE) during embryonic development for survival, strongly supporting a key role for NE in development (Thomas et al., 1995). CuZn-superoxide dismutase (SOD) knockout mice have compromised fertility but display no increase susceptibility to hyperbaric oxygen (Ho et al., 1998). Brain tissue was not elevated except to confirm lack of SOD.

2.2. Dietary The principal experimental paradigm used to study brain and copper deficiency has been semi-purified diets. The principal animal model has been the rat. Limited studies have been done on mice, and few on guinea pigs, and miniature swine. Details of earlier studies are reviewed by O’Dell and Prohaska (1983). Two main factors influence the neurological impact of copper deficiency (the dietary copper concentration and the timing of the copper deficiency. If we assume that about 5mgCu/kg diet are adequate to support reproduction and development than a diet of 0.4mgCu/kg and 1 mgCu/kg would be classified as “deficient” Rats fed the lower level prior to breeding produce not viable offspring whereas the higher level supports normal reproduction (see Prohaska, 1997). When mouse dams were fed a diet containing 0.5 mg

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Cu/kg beginning at various times relative to parturition dramatic differences in the offspring were observed including neurochemical changes in copper and catecholamines (Prohaska and Bailey, 1993a). These points need to be considered when generalizing about the effects of perinatal copper deficiency on brain. However, as Hunt and Idso (1995) demonstrated even a marginal copper deficiency (1.8mg Cu/kg) during gestation and lactation was sufficient to alter morphology of the hippocampus. Thus, both timing and degree of copper deficiency have an impact on the development of the nervous system.

3. NEUROCHEMICAL CHARACTERISTICS 3.1. Copper Adult rat brain contains less than 50 nmol Cu/g and a weanling rat about 30 nmol Cu/g. Restriction of dietary copper during gestation and lactation lowers brain copper content of offspring to about one-fifth the normal level in most regions except the hypothalamus where the reduction is less (Prohaska and Bailey, 1994). Similar patterns were observed following perinatal copper deficiency in mice except for the “sparing” effect of the hypothalamus (Prohaska and Bailey, 1993b). Tracer studies with 67Cu indicated that copper-deficient mice incorporated more newly arriving copper into SOD compared to MT or neurocuprein (Prohaska, 1983). Restoration of copper to diets of mice (Prohaska and Bailey, 1993b) and rats (Prohaska and Bailey, 1995a) demonstrated a slow recovery of brain copper. In fact even after one-year of repletion brain copper in the cortex was still 20% lower than control concentrations (Prohaska, 1997).

3.2. Cuproenzymes The activity of a number of brain cuproenzymes change following perinatal copper deficiency. Activity of CCO—Cytochrome c Oxidase—in both rat and mice offspring is markedly lower (Prohaska, 1981a, 1991). Regional assessment of CCO indicated a 50–85% decrease in activity in deficient compared to sufficient offspring for mice and rats (Prohaska and Bailey, 1993b and 1995b). Following repletion CCO activity was restored slowly and was still low in repleted group compared to controls after four months (Prohaska and Bailey, 1995a). Activity of SOD is lower in copper-deficient rat brain (Prohaska and Wells, 1974). The decrease in SOD activity is usually less than CCO for most organs including brain (Phillips et al., 1986; Prohaska, 1991). There are modest regional changes in the decrease in brain SOD in both mice and rats (Prohaska and Bailey, 1993b and 1995b). SOD activity is restored faster than CCO in both mice (Prohaska and Bailey, 1993b and 1995a). DBM measured in whole brain homogenates of mice (Prohaska and Smith, 1982) or regionally in rats (Prohaska and Bailey, 1995b) indicates a robust increase in enzyme activity. However, in vivo evidence of lower DBM is supported by catecholamine data (see below). Normalization of DBM activity in rat brain is accomplished rapidly, within a month (Prohaska and Bailey, 1995a and 1995b). Regional rat brain PAM—Peptidyl glycine (-amidating monooxygenase—activity is lower following perinatal copper deficiency provided copper is not added to the assay

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(Prohaska and Bailey, 1995b; Prohaska et al., 1995). Midbrain PAM activity is not lower in brains of copper-deficient rats that began depletion postweanling (Prohaska et al., 1995). PAM was reported to be higher in anterior pituitary and submandibular glands of postnatal copper deficient rats when copper was added to the assay (Mains et al., 1985). In most brain regions PAM activity was restored to control levels following onemonth of copper repletion (Prohaska and Bailey, 1995b). No one has yet published measurements of brain CPL—ceruloplasmin—(ferroxidase) activities. Nor has CPL in brain been assessed following copper deficiency. Humans with genetic loss of CPL have elevated brain iron. Brain iron concentration is not altered by dietary copper deficiency (Prohaska and Wells, 1975).

3.3. Metabolites Several metabolites have been quantified to test the hypothesis the lower enzyme activity actually has a biological impact. To evaluate CCO brain levels of adenine nucleotides were measured. Neither in rats (Prohaska and Wells, 1975) nor in mice (Rusinko and Prohaska, 1985) was there evidence of altered nucleotides or energy charge. However, in both cases there was a several-fold elevation in brain lactate suggesting some perturbance in energy metabolism. It is not clear whether CCO activity is limiting in brain mitochondria following copper deficiency. To elevate altered SOD activity endogenous peroxides and the ability to generate peroxides was evaluated in rat brain (Prohaska and Wells, 1975). Samples from deficient and control brains were equivalent. Furthermore, analysis of brain fatty acids indicates no loss of polyunsaturated fatty acids from brain tissue of copper-deficient rats (Prohaska, 1981b; Sun and O’Dell, 1992). The composition of brain lipids in copper-deficient rats is consistent with the observed hypomyelination (DiPaolo, Kanfer, and Newberne, 1974). Ascorbate pools in brain might be altered if excess oxygen radicals were produced. In mice we found a significant drop in brain ascorbate that was reversed by copper treatment (Prohaska and Cox, 1983); however, in rat brain copper deficiency had no apparent effect on ascorbate concentration (Kubat and Prohaska, 1996). There is little compelling evidence that the deficit in brain SOD activity is responsible for the delayed development and neuronal pathology observed. To evaluate DBM activity brain norepinephrine (NE) and dopamine (DA) have been evaluated, the product and substrate, respectively, of DBM. Brain NE concentration is lower following perinatal copper deficiency (Prohaska and Wells, 1974). Brain DA levels vary regionally following copper deficiency. Whole brain DA levels are usually not greatly altered (Prohaska and Smith, 1982; O’Dell and Prohaska, 1983). However, regional DA analysis supports a lower DBM activity hypothesis in that elevated DA is observed in both mice and rats in those regions enriched in noradrenergic neurons (Prohaska and Bailey, 1993b and 1994). In the corpus striatum, a region enriched in dopaminergic neurons, there is an irreversible lowering of DA in brains of copperdeficient male rats (Feller and O’Dell, 1980; Prohaska and Bailey, 1994). Carlton and Kelley 1969 demonstrated loss of neuronal cells in the corpus striatum of copper-deficient rats. The decrease in brain NE in copper deficiency is likely not due to enhanced NE turnover (Gross and Prohaska, 1990). Following postnatal copper deficiency neither brain NE or DA were altered again underscoring the key role of timing of the deficiency (Prohaska et al., 1990). Perhaps the altered DBM activity and subsequent disturbance in catecholamine pools is responsible for the altered development of brain and the abnormal behavior of copper-deficient rats (Carlton and Kelley, 1969; DiPaolo et al.,

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1974). Only one study has evaluated brain PAM by metabolite analysis. PAM converts certain peptides with a C-terminal glycine into an amide. Levels of glycine-extended (MSH-related molecules were not elevated in brains of rats after postnatal copper deficiency (Mains et al., 1985).

4. REVERSIBILITY It was discussed above how most enzyme changes seem reversible within a few months. NE rises and DA falls to control ratios within a month indicating DBM reversibility (Prohaska and Bailey, 1995a). Yet, even after months of copper repletion of former copper-deficient female and male offspring exhibit altered acoustic startle responses (Prohaska and Hoffman, 1996). Thus perinatal copper deficiency permanently alters behavior of rats with phenotypically and biochemically normal characteristics. Recall that brain copper does not reach control levels even after one year of repletion. Hopkins and Failla (1995) reported lower (11–15%) brain copper in 6-month old female and male offspring of dams fed 2.8mgCu/kg. Essentially no other copper-dependent variable was altered in these rats. Thus, sufficient copper is required during perinatal development to ensure that brain receives normal copper accretion. Perhaps full cognitive capacity cannot be reached unless the brain receives adequate copper during development.

ACKNOWLEDGMENT Financial support for the recent neurochemical work was provided by USDA NRI Competitive Grants Programs.

REFERENCES Bennetts, H.W. and Chapman, F.E., 1937, Copper deficiency in sheep in Western Australia: A preliminary account of the etiology of enzootic ataxia of lambs and an anemia of ewes, Aust. Vet. J. 13:138–149. Carlton, W.W. and Kelly, W.A., 1969, Neural lesions of the offspring of female rats fed a copper-deficient diet, J. Nutr. 97:42–52. Danks, D.M., Campbell, P.E., Stevens, B.J., Mayne, V., and Cartwright, E., 1972, Menkes’ kinky hair syndrome, Pediatrics 50:188–201. DiPaolo, R.V., Kanfer, J.N., and Newberne, P.M., 1974, Copper deficiency and the central nervous system. Myelination in the rat: Morphological and biochemical studies, J. Neuropathol. Exper. Neurol. 33:226–236. Everson, G.J., Shrader, R.E., and Wang, T.-I., 1968, Chemical and morphological changes in the brains of copper-deficient guinea pigs, J. Nutr. 96:115–125. Feller, D.J. and O’Dell, B.L., 1980, Dopamine and norepinephrine in discrete areas of the copper-deficient rat brain, J. Neurochem. 34:1259–1263. Gary, T. and Robertson, D., 1994, Lessons learned from dopamine (-hydroxylase deficiency in humans, News in Physiological. 9:35–39. Gross, A.M. and Prohaska, J.R., 1990, Copper-deficient mice have higher cardiac norepinephrine turnover, J. Nutr. 120:88–96. Ho, Y.-S., Gargano, M., Cao, J., Bronson, R.T., Heimler, I., and Hutz, R.J., 1998, Reduced fertility in female mice lacking copper-zinc superoxide dismutase, J. Biol. Chem. 273:7765–7769. Hopkins, R.G. and Failla, M.L., 1995, Chronic intake of a marginally low copper diet impairs in vitro activities of lymphocytes and neutrophils from male rats despite minimal impact on conventional indicators of copper status, J. Nutr. 125:2658–2668.

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Hunt, C.D. and Idso, J.P., 1995, Moderate copper deprivation during gestation and lactation affects dentate gyrus and hippocampal maturation in immature male rats, J. Nutr. 125:2700–2710. Hunt, D.M and Johnson, D.R., 1972, Aromatic amino acid metabolism in brindled (Mobr) and viable-brindled (Movbr), two alleles at the mottled locus in the mouse, Biochem. Genet. 6:31–40. Hunt, D.M., 1974, Primary defect in copper transport underlies mottled mutants in the mouse, Nature 249:852–854. Kubat, W.D. and Prohaska, J.R., 1996, Copper status and ascorbic acid concentrations in rats, Nutr. Res. 16:237–243. Mains, R.E., Myers, A.C., and Eipper, B.A., 1985, Hormonal, drug, and dietary factors affecting peptidyl glycine (-amidating monooxygenase activity in various tissues of the adult male rat, Endocrinology 116:2505–2515. Morita, H., Ikeda, S., Yamamoto, K., Morita, S., Yoshida, K., Nomoto, S., and Kato and Yanagisawa, N., 1995, Hereditary ceruloplasmin deficiency with hemosiderosis: A clinicopathological study of a Japanese family, Ann. Neurol. 37:646–656. O’Dell, B.L. and Prohaska, JR., 1983, Biochemical aspects of copper deficiency in the nervous system, in: Neurobiology of the Trace Elements, Volume 1 (I.E. Dreosti, and R.M. Smith, eds.), Humana Press, Clifton, pp. 41–81. Phillips, M., Camakaris, J., and Danks, D.M., 1986, Comparisons of copper deficiency states in the murine mutants blotchy and brindled, Biochem. J. 238:177–183. Prohaska, J.R., 1981, Comparison between dietary and genetic copper deficiency in mice: Copper-dependent anemia, Nutr. Res. 1:159–167. Prohaska, J.R., 1981b, Changes in brain enzymes accompanying deficiencies of the trace elements, copper, selenium, or zinc, in: Trace Element Metabolism in Man and Animals (TEMA-4), (J. McC. Howell, J.M. Gawthorne, and C.L. White, eds.), pp. 275–282, Australian Academy of Science, Canberra. Prohaska, J.R., 1983, Comparison of copper metabolism between brindled mice and dietary copper-deficient mice using 67Cu,J. Nutr. 113:1212–1220. Prohaska, J.R., 1987, Function, of trace elements in brain metabolism, Physiol. Rev. 67:858–901. Prohaska, J.R., 1991, Changes in Cu, Zn-superoxide dismutase, cytochrome c oxidase, glutathione peroxidase and glutathione transferase activities in copper-deficient mice and rats, J. Nutr. 121:355–363. Prohaska, J.R., 1997, Persistent neurochemical and behavioral abnormalities in adult rats following recovery from perinatal copper deficiency, in: Trace Elements in Man and Animals-9: Proceedings of the Ninth International Symposium on Trace Elements in Man and Animals, (P.W.F. Fischer, M.R. LíAbb”, K.A. Cockell, and R.S. Gibson, eds.), pp. 208–212, NRC Research Press, Ottawa. Prohaska, J.R. and Bailey, W.R., 1993a, Copper deficiency during neonatal development alters mouse brain catecholamine levels, Nutr. Res. 13:331–338. Prohaska, J.R. and Bailey, W.R., 1993b, Persistent regional changes in brain copper, cuproenzymes and catecholamines following perinatal copper deficiency in mice, J. Nutr. 123:1226–1234. Prohaska, J.R. and Bailey, W.R., 1994, Regional specificity in alterations of rat brain copper and catecholamines following perinatal copper deficiency, J. Neurochem. 63:1551–1557. Prohaska, J.R. and Bailey, W.R., 1995a, Persistent neurochemical changes following perinatal copper deficiency in rats, J. Nutr. Biochem. 6:275–280. Prohaska, J.R. and Bailey, W.R., 1995b, Alterations of rat brain peptidylglycine (-amidating monooxygenase and other cuproenzyme activities following perinatal copper deficiency, Proc. Soc. Exp. Biol. Med, 210:107–116. Prohaska, J.R. and Cox, D.A., 1983, Decreased brain ascorbate levels in copper-deficient mice and in brindled mice, J. Nutr. 113:2623–2629. Prohaska, J.R. and Hoffman, R.G., 1996, Auditory startle response is diminished in rats after recovery from perinatal copper deficiency, J. Nutr. 126:618–627. Prohaska, J.R. and Smith, T.L., 1982, Effect of dietary or genetic copper deficiency on brain catecholamines, trace metals and enzymes in mice and rats, J. Nutr. 112:1706–1717. Prohaska, J.R. and Wells, W.W., 1974, Copper deficiency in the developing rat brain: A possible model for Menkes’ steely-hair disease, J. Neurochem. 23:91–98. Prohaska, J.R. and Wells, W.W., 1975, Copper deficiency in the developing rat brain: Evidence for abnormal mitochondria, J. Neurochem. 25:221–228. Prohaska, J.R., Bailey, W.R., Gross, A.M., and Korte, J.J., 1990, Effect of dietary copper deficiency on the distribution of dopamine and norepinephrine in mice and rats, J. Nutr. Biochem. 1:149–154. Prohaska, J.R., Bailey, W.R., and Lear, P.M., 1995, Copper deficiency alters rat peptidylglycine (-amidating monooxygenase activity, J. Nutr. 125:1447–1454.

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Rusinko, N. and Prohaska, J.R., 1985, Adenine nucleotide and lactate levels in organs from copper-deficient mice and brindled mice, J. Nutr. 115:936–943. Sun, S.H.-H. and O’Dell, B.L., 1992b, Low copper status of rats affects polyunsaturated fatty acid composition of brain phospholipids, unrelated to neuropathology, J. Nutr. 122:65–73. Thomas, S.A., Matsumoto, A.M., and Palmiter, R.D., ••, Noradrenaline is essential for mouse fetal development, Nature 374:643–646.

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MOLECULAR ANALYSIS OF WILSON DISEASE GENE IN FRENCH PATIENTS 1

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I. Ceballos-Picot , A. Nicole , B. Aral , C. Franvel , C. Soulié , 1 3 3 2 H. Lassal , F. Woimant , M. Haguenau , and P. Chappuis 1

Laboratoire de Biochimie B and CNRS UMR8602 Hôpital Necker—Enfants Malades 149 rue de Sèvres 75743-Paris Cedex 15 2 Laboratoire de Biochimie et Biologie Moléculaire Hôpital Lariboisière, 2 rue Ambroise Paré 75475 Paris Cedex 10 3 Service de Neurologie Hôpital Lariboisière 2 rue Ambroise Paré 75475 Paris Cedex 10

Wilson disease (WD) is an autosomal recessive disorder of copper transport resulting from toxic copper accumulation in liver and brain. Its clinical expression includes hepatic and/or neurological manifestations. The disease has a worldwide prevalence of 1 in 35,000 people, and a corresponding carrier frequency of 1 in 90. The WD gene which maps to chromosome 13 encodes for ATP7B, a copper-transporting P-type ATPase (Thomas et al., 1995).

1. THE DISEASE In WD, uptake of copper by intestine and liver is normal; however patients have a marked impairment in biliary copper excretion and are also unable to synthesize holoceruloplasmin suggesting that ATPase is a key factor in these processes. Impairment of biliary excretion leads to copper accumulation which is toxic for liver. As the disease progresses, increasing liver damage leads to the release of large amounts of copper which accumulates in peripheral organs such as brain, kidneys and cornea. Deposition of copper in the limbus of the cornea gives rise to the dull-yellow Kayser-Fleisher (KF) rings. The initial clinical presentation is highly variable. Main signs are chronic or fulminent liver disease, progressive neurological disorder with tremor, dysarthria and Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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dystonia, psychiatric illness, hematologic and, less often, renal disturbances. Approximately 25% of the patients have an initial clinical presentation that involves two or more organ systems. Patients generally become symptomatic between 6 and 45 years of age. KF rings are frequently absent in children or patients with exclusively hepatic manifestations (Roberts and Cox, 1999).

2. BIOCHEMICAL DIAGNOSIS A low serum ceruloplasmin level has for long been considered as a diagnostic test for WD. However serum ceruloplasmin may also be decreased in patient presenting an hepatic insufficiency, an intestinal malabsorption or a malnutrition. Consequently both serum ceruloplasmin and 24 hour copper urine excretion should be used as a screening procedure only. An increased serum free-copper concentration has sometimes be considered as a valuable diagnostic tool. In patients who present primarily with hepatic dysfunction a liver biopsy for quantitative copper determination should be recommended to establish the diagnosis of WD if either KF rings are absent, or if ceruloplasmin levels are normal, as it may occur in up to 15% of cases. However the hallmarks of the disease such as KF rings, biochemical evidences of low plasma Cu and high hepatic Cu overload are not always present or may be misleading and molecular diagnosis provides now an additional reliable tool for identifying WD patients and heterozygous carriers. As this diagnosis can be preclinicaly settled any invasive hepatic biopsy could be avoided and copper-chelating treatment early instaured in order to prevent the disease progression.

3. MOLECULAR DIAGNOSIS OF WILSON DISEASE The exon-intron structure of the WD gene allows now to realize a molecular diagnosis. Polymorphic microsatellite markers flanking the gene were used for presymptomatic diagnosis and carrier detection inside a family. PCR-SSCP (Single-Strand Conformation Polymorphism) analysis was also used to screen for regions of the gene containing a mutation which was later identified by sequencing.

3.1. Indirect Genotypic Diagnosis The microsatellite haplotypes were constructed in 35 French unrelated families with WD. Four polymorphic markers flanking the WD locus (D13S294, D13S295, D13S296, D13S301), mostly dinucleotide repeats, were amplified by PCR for microsatellites haplotyping (Chappuis et al., 1998). Within each family these markers were combined to determine haplotypes on WD and on normal chromosomes. The use of this procedure in the diagnosis of WD in a family requires DNA sample from at least one parent and an affected patient for which the diagnosis has already been firmly established.

3.2. Spectrum of Mutations in ATP7B Gene in French Patients and Genotype-Phenotype Relations We realized mutation screening in 21 exons of the gene in 49 french unrelated WD patients by using PCR-SSCP followed by a direct sequence analysis of the shifted

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fragments. Two Turkish, one Tunisian and one Chinese patients were also included in our study. The majority of patients had a neurological presentation of the disease. We identified the mutation of 65% of the WD chromosomes analyzed. Forty-four per cent of mutations were found to be clustered to exons 14, 8 and 7 (21% of the mutations in exon 14, 16% in exon 8 and 7% in exon 7). This localization which corresponds to the membrane spanning segments and to the phosphorylation domain of ATPaseB supports the importance of these structures for the ATP-dependent copper transport function of Wilson protein (Fig. 1). The major mutations in exon 7 were novel splice-site mutations at the intron-exon boundary (Fig. 2). These mutations are expected to result in exon skipping. A Met665Ile mutation, already described, is expected to disrupt the structure of the transmembrane domain 1 (Tm1). In exon 8, we found six different mutations. Three of these are new mutations. The first one is a large deletion of 16 bases which is expected to alter the reading frame of the gene product. The two others represent non conservative aminoacid changes which are expected to disrupt the structure of Tm2 and Tm4, respectively.

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The three other missense mutations were already described. In particular Arg778Gly was found in a family of turkish origin and Arg778Leu was found in a Chinese family. This last mutation is a common mutation found in asian patients. In exon 14 we described 4 different mutations including the most common one, His1069Gln. Val1039Phe is a novel mutation, located within the phosphorylation domain of the protein. His1069Gln in exon 14 accounts for 18% of total mutations. The investigation of this mutation in diverse populations, including those of central Europe, North America, Great Britain, and several Mediterranean countries underlines its prevalence in the disease. After screening of the mutated exon by PCR-SSCP and identification of the mutation by sequencing, we conclude that many mutations correspond to a typical SSCP profile in specific condition of DNA strand renaturation. A representative example is shown in Fig. 3 in which the band in the middle shows the mobility shift of exon 14 corresponding to His1069Gln mutation which is absent in control. The missense mutation Val1039Phe leads to an SSCP profile different from His1069Gln. The index patient shows SSCP profiles typical for each specific mutation. Two homozygous patients for His1069Gln had a mild, late hepatic/neurologic presentation and presented subnormal ceruloplasmin levels, in accordance with previous published results (Houwen et al., 1995) but in disagreement with other papers (Ha-Hao et al., 1998). Several mutations have been associated with different haplotypes so that it is difficult to conclude from our study that haplotypes may be useful indicators of specific mutations. The only exception is His1069Gln which was shown to be linked to one haplotype with an heterogeneity at the level of one marker (D13S294). In many other papers, this mutation is also associated with an identical haplotype indicating that it is likely the most

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common molecular defect of the WD gene and suggesting that it arose from a single and very ancient mutational event. Molecular genetic investigations confirm that WD shows considerable genetic heterogeneity: 30 different mutations in ATP7B gene, have been found in our French population (16 novel mutations). Nearly half of them (44%) were clustered in exon 14, 8 and 7 with a prevalence of for His1069Gln mutation located in exon 14. The variability of symptoms we observed in individuals carrying the same mutation implies the involvement of additional genetic and/or environmental factors in the modulation of phenotypic response. Functional analysis of novel mutated gene after transfection may give insights into the basis of the disease variability.

REFERENCES Chappuis P., Aral, B., and Ceballos-Picot, I., 1998, Copper related diseases, in: Metal Ions in Biology and Medicine, Volume 5 (P. Collery, P. Brätter, V. de Negretti de Brätter, L. Khassanova, and J.C. Etienne, eds), pp 729–736, John Libbey, London. Ha-Hao D., Hefter H., Stremmel W., Castañeda-Guillot C., Hernandez A.H., Cox D.W., and Auburger G., 1998, Hisl069Gln and six novel Wilson disease mutations: analysis of relevance for early diagnosis and phenotype. Eur. J. Hum.Genet. 6:616–623. Houwen R.H.J., Juyn J., Hoogenraad T.U., Ploos V.A., and Berger R., 1995, H714Q mutation in Wilson disease is associated with late, neurological presentation. J. Med Genet. 32:480–482. Roberts E.A. and Cox D.X., 1999, Wilson disease, Baillieres Clin. Gastroenterol. 12(2):237–256. Thomas G.R., Forbes J.R., Roberts E.A., Walshe J.M., and Cox D.X., 1995, The Wilson disease gene: spectrum of mutations and their consequences, Nature Genet. 9:210–217.

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DEFINING OPTIMAL COPPER STATUS IN HUMANS Concepts and Problems

J. J. Strain Northern Ireland Centre for Diet and Health (NICHE) University of Ulster Coleraine, BT52 1SA Northern Ireland, UK

1. INTRODUCTION This review indicates some putative functional indices that can be used to assess copper status in humans. The problems associated with the use of such indices are then explored by reference to their response to supplementation and depletion studies.

2. FUNCTIONAL INDICES OF COPPER STATUS IN HUMANS Overt copper deficiency in humans is rare and has been largely confined to premature infants, malnourished infants and children, patients receiving total parental nutrition and in those receiving special diets or unmodified cow milk. The most frequent signs of copper deficiency in humans are anaemia, neutropaenia and bone abnormalities while less frequent signs are hypopigmentation, impaired growth, increased incidence of infections, abnormalities of glucose and cholesterol metabolism and electrocardiograms (Uauy et al., 1998). Various attempts have been made to relate these symptoms to alterations in copper metalloenzymes and non-copper enzymes that may be copper responsive and to identify the role of copper as an antioxidant and in carbohydrate metabolism, immune function, bone health and cardiovascular mechanisms (Strain, 1994). If suboptimal copper status is a problem in humans populations, then it is probable that subtle changes in these molecular, biochemical and physiological systems might eventually lead to ill health (Klevay, 1998). It also follows that a search for a valid functional index of Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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sub-optimal copper status in humans might be well placed in a thorough exploration of such systems. A valid functional index of copper status in humans must respond sensitively, specifically and predictably to changes in the concentration supply of dietary copper or copper stores, be accessible for measurement and measurable, and impact directly on health. The validation of a functional index for copper status, therefore, presents major difficulties, not least the design of studies to establish cause and effect relationships not only between copper status and the functional index but also between copper status and ill health. Establishment of the latter would require long-term studies with, probably, insurmountable problems but these might be circumvented by the use of surrogate measures of ill health. Table 1 gives some of the functional indices (classified as molecular, biochemical and physiological) that might be used to define sub-optimal copper status in humans. The most commonly used putative measures of copper status, viz plasma, white cell and platelet copper, cannot be regarded as functional indices and have intractable problems in interpretation (Johnston, 1999). The inclusion of candidate functional indices in Table 1 does not imply validity and the problems associated with their use are outlined in the following section.

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3. RESPONSE OF PUTATIVE FUNCTIONAL INDICES OF COPPER STATUS TO COPPER SUPPLEMENTION AND DEPLETION STUDIES IN HUMANS 3.1. Copper Supplementation Studies Copper supplementation studies in humans have resulted in few changes in any putative index of sub-or supra-optimum copper status. For example, Pratt, Omdahl and Sorenson (1985) found no change in the concentration of copper in the serum, urine or hair nor in haematocrit, serum lipids and liver and kidney function tests in seven adult subjects after 10 mg of copper per day as copper gluconate for 12 weeks in a doubleblinded placebo-controlled trial. Similarly, Salmenpera, Siimes, Nanto and Perheentupa (1989) demonstrated that concentrations of plasma copper and caeruloplasmin in healthy full-term infants were resistant to dietary supplementation (from 3.5 to 12 months) of 9.1 (mol copper/l in liquid cow-milk-based formulas compared with two similar groups (n = 16) of infants fed either a liquid cow-milk based formula containing 1.3 (mol copper/l or exclusively breast-fed. Although Medeiros, Milton, Brunett and Stacy (1991) found that supplementation with either 2 or 3 mg copper per day had no effect on serum copper and caeruloplasmin protein, erythrocyte superoxide dismutase (SOD) and haematocrit, in two 6-week placebo-controlled double-blind studies comprising a total of 27 young adult subjects, low density lipoprotein (LDL) and total cholesterol, haemoglobin and caeruloplasmin oxidase activity increased in the copper supplemented subjects compared with controls. These increases with respect to cholesterol fractions were not supported by a subsequent copper supplementation study (2 mg/day for 4 weeks in a copper/placebo double-blinded crossover design) in 20 adult men with moderately high plasma cholesterol (Jones, DiSilvestro et al., 1997). The latter workers also found that copper supplementation had no effect on lag times for very low density lipoprotein (VLDL) and LDL oxidation in vitro, and on the activities of three copper metalloenzymes, erythrocyte SOD, plasma caeruloplasmin and diamine oxidase (DAO). Copper supplementation, however, significantly increased SOD and DAO activities as well as lipoprotein oxidation lag times in the 10 subjects in the lower half of a median split based on precopper values. These results are important in that they suggest that copper supplementation of healthy volunteers may result in observable changes in various molecular, biochemical or physiological indices if there was a sensitive method of prior screening to distinguish between responders and non-responders in any population. Findings from my own laboratory indicate that copper supplementation of 22 (11 men, 11 women) healthy adult volunteers in three separate double-blinded, placebo-controlled cross-overs, each of 6 week duration, using 3 mg copper/day as sulphate or glycine chelate or 6 mg copper/day as glycine chelate resulted in a lack of response to supplementation of many parameters including erythrocyte and leucocyte SOD activities, leucocyte cytochrome c oxidase (CCO) activity and (except at 6 mg copper/day) platelet CCO activity (Strain et al., 1998), LDL susceptibility to oxidation (Bonham et al., 1998) and measurers of DNA damage and haemostasis (Faughan et al., 1998). A lack of response of some of these physiological parameters would indicate that supra-optimal copper status may not have been reached over the 6 week periods even at the high (6 mg/day) dose of copper. One putative functional index of copper status, however, was markedly and consistently affected by copper supplementation in each cross-over. This index was serum DAO activity which was higher in women and was significantly increased after copper supplementation in both men and women (Strain et al., 1998).

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In a companion study using a similar protocol as part of the EC-funded FOODCUE project, collaborators from INRA at Clermont-Ferrand, France (Y, Rayssiguier and co-workers, unpublished data) found that DAO activity was also significantly increased in 26 older adults, compared with wash-out, in each of the three copper supplementation periods. Interestingly, resistance to haemolysis of erythrocytes was also increased after supplementation with 3 mg copper/day as copper sulphate and the 6 mg copper/day as glycine chelate in these subjects. The latter data contribute further to the idea that copper acts as an antioxidant in vivo (Mazur et al., 1998). The conclusions which can be drawn from the copper supplementation studies, therefore, are that very few candidate functional indices are affected by supplementation of the usual diet of healthy volunteers. There is some evidence that oxidative stress might be decreased after supplementation and that DAO activity alone of the molecular indices appears to be consistently affected by copper supplementation. The coppermetalloenzyme, DAO, is responsible for the oxidative deamination of biological diamines such as cadaverine, putrescene and histamine (Wolvekamp and DeBruin, 1994). It is possible, however, that DAO activity might not have sufficient specificity for a valid molecular functional index of copper as activity is raised in pregnancy, (Kusche et al., 1994) uremia and kidney dialysis patients (DiSilvestro et al., 1992) and after gastrointestinal damage (Wolvekamp and De Bruin, 1994) and is decreased in patients with Chrohnís disease and celiac disease (Bayless et al., 1981). Moreover, DAO activity may simply be a biomarker of dietary copper intake rather than status. Nevertheless, only plasma DAO activity and hepatic copper content were sufficiently sensitive to differentiate rats fed a copper adequate (6.70 mg copper/kg) diet from a copper marginal (1.73 mg copper/kg) diet for 57 days (J. J. Strain unpublished data). Erythrocyte, liver, and heart SOD activities, liver and heart CCO activities and plasma caeruloplasmin activity were lowered by feeding a copper deficient (0.52 mg copper/kg diet) diet but none of these was lowered feeding the copper marginal diet compared with the copper adequate diet.

3.2. Copper Depletion Studies In contrast to copper supplementation studies, copper depletion studies in humans have resulted in changes in a range of functional indices and these have been reviewed by Milne (1998). Researchers at the US Department of Agriculture Human Nutrition Research Centers have undertaken a number of highly controlled copper depletion and repletion studies in metabolic units. In one study, Milne and Nielsen (1996) fed 12 postmenopausal women 0.57 mg copper/day for 105 days followed by copper repletion with a 2.0 mg copper/day for 35 days and found that the low copper diet was associated with lower erythrocyte SOD, platelet CCO and erythrocyte glutathione peroxidase activities and higher clotting factor VIII. Some of these indices, however, did not return to baseline on copper repletion. Decreased plasma copper or caeruloplasmin was not observed during copper depletion on the low copper diet. Turnlund, Scott, Peiffer, Jang, Keyes, Keen and Sakanashi (1997), however did observe decreased plasma copper, caeruloplasmin protein and activity in 11 young men after a low copper (0.38 mg/day dietary period of 42 days which had been preceded by a 24 day period on a diet of 0.66 mg/day copper. Skin lysyl oxidase activity was also found to decrease on copper depletion and increase again on repletion in this study. In addition, feeding the lowest copper diet was associated with significant decrease in the proliferation of peripheral blood mononuclear cells cultured with phytohaemogglutinin, Concanavalin A and pokeweed and an increase in the percentage of circulating B cells (CD19+) in these men (Kelley et al., 1995). These

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immunological data support findings from marginal copper deficiency studies in experimental animals which have exhibited lowered resistance to infection, lowered concentration of neutrophils and T-lymphocytes and impaired neutrophil, monocyte and T-cell function (Hopkins and Failla, 1995) and in vitro cell work which has shown that decreased cellular copper produced decreases in interleukin—2 mRNA and changes in the structure and function of T-cells (reviewed by Percival, 1998). In another copper depletion study, healthy adult males (n = 11) were fed medium (1.6 mg copper/day), low (0.7 mg copper/day) and then high (6.0mg copper/day) over consecutive 8 week periods with a minimum of 4 week wash out periods (Baker et al.). Biomarkers of bone resorption viz urinary pyridinoline and deoxypyridinoline were significantly increased after the low dietary copper period. These data indicate that measures of bone health might be sensitive indices of copper status and support longterm copper supplementation studies which have shown decreased loss of vertebral bone mineral density in perimenopausal women taking copper compared with controls (EatonEvans et al., 1996) and, when copper was given with calcium and other trace minerals, decreased spinal bone loss in postmenopausal women compared with controls (Strause et al., 1994).

REFERENCES Baker, A., Harvey, L., Majask-Newman, G., Fairweather-Tait, S., Flynn, A., and Cashman, K., 1999, Effect of dietary copper intakes on biochemical markers of bone metabolism in healthy adult males, Eur. J. Clin. Nutr. 52:1–5. Bayless, T.M., Luk, G.D., Baylin, S.B., and Thomas, M.E., 1981, Decreased plasma diamine oxidase levels in Chrohnís disease and celiac disease, Gastrointerology 80:1106. Bonham, M., Turley, E., McKeown, A., OíConnor, J.M., Gilmore, W.S., Chopra, M., Eaton-Evans, M.J., McKelvey-Martin, V.J., and Strain, J.J., 1998, Copper supplementation and LDL susceptibility to oxidation: FOODCUE Project, Proc. Nutr. Soc. 57:157A. DiSilvestro, R.A., Jones, A.A., Smith, D., and Wildman, R., 1997, Plasma diamine oxidase activities in renal dialysis patients, a human with spontaneous copper deficiency and marginally copper deficient rats, Clin. Biochem. 30:559–563. Eaton-Evans, J., McIlrath, E.M., Jackson, W.E., McCartney, H., and Strain, J.J., 1996, Copper supplementation and the maintenance of bone mineral density in middle-aged women, J. Trace Elem. Exp. Med. 9:87-94. Faughan, M.S., Bonham, M., McKeown, A., OíConnor, J.M., Turley, E., Coulter, J., Gilmore, W.S., and Strain, J.J., 1998, The effects of copper sulphate supplementation on fibrinolytic factors in healthy subjects, Proc. Nutr. Soc. 57:25A. Hopkins, R.G. and Failla, M.L., 1995, Chronic intake of a marginally low copper diet impairs in vitro activities of lymphocytes and neutrophils from male rats despite minimal impact on conventional indicators of copper status, J. Nutr. 125:2658–2668. Johnston, M.A., 1999, Copper: Physiology, dietary sources and requirements, in Encyclopedia of Human Nutrition, Volume 1 (M.J. Sadler, J.J. Strain, and B. Cabellero, eds). pp 442–450, Academic Press, London. Jones, A.A., DiSilvestro, R.A., Coleman, M., and Wagner, T.L., 1997, Copper supplementation of adult men: Effects on blood copper enzyme activities and indicators of cardiovascular disease risk, Metabolism. 46:1380-1383. Kelley, D.S., Daudu, P.A., Taylor, P.C., Mackey, B.E., and Turnlund, J.R., 1995, Effects of low-copper diets on human immune response, Am. J. Clin. Nutr. 62:412–416. Klevary, L.M., 1998, Lack of a recommended dietary allowance for copper may be hazardous to your health, J. Am. Coll. Nutr. 17:322–326. Kusche, J., Trotha, U.V., Muhlberger, G., and Lorenz, W., 1974, The clinical-chemical application of the NADH test for the determination of diamine oxidase activity in human pregnancy, Agents Action 4/3:188189.

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Medeiros, D.M., Milton, A., Brunett, E., and Stacy, L., 1991, Copper supplementation effects on indicators of copper status and serum cholesterol in adult males, Biol. Trace. Elem. Res. 30:19–35. Milne, D.B., 1998, Copper intake and assessment of copper status, Am. J. Clin. Nut. 67(suppl):1041S–1045S. Milne, D.B. and Nielsen, F.H., 1996, Effects of a diet low in copper on copper-status indicators in postmenopausal women. Am. J. Clin. Nutr. 63:358–364. Percival, S.S., 1998, Copper and immunity, Am. J. Clin. Nutr. 67:1064S–1068S. Pratt, W.B., Omdahl, J.L., and Sorenson, J.R.J., 1985, Lack of effects of copper gluconate supplementation, Am. J. Clin. Nutr. 42:681–682. Salmenperä, L., Siimes, M.A., Nanto, V., and Perheentupa, J., 1989, copper supplementation: Failure to increase plasma copper and ceruloplasmin concentrations in healthy infants, Am. J. Clin. Nutr. 50:843–847. Strain, J.J., 1994, Never aspects of micronutrients in chronic disease: Copper, Proc. Nutr. Soc. 53:583–589. Strain, J.J., Kehoe, C.A., Turley, E., Bonham, M., OíConnor, J., McKeown, A., Faughnan, M.S., and Howard, A.N., 1998, The response of serum diamine oxidase activity to copper supplementation in humans, FASEB. J. 12:A199 (abstract). Strause, L., Saltman, P., Smith, K.T., Bracker, M., and Andon, M.B., 1994, Spinal bone loss in postmenopausal women supplemented with calcium and trace minerals, J. Nutr., 124:1060–1064. Turnlund, J.R., Scott, K.C., Peiffer, G.L., Jang, A.M., Keyes, W.R., Keen, C.L., and Sakanashi, T.M., 1997, Copper status of young men consuming a low-copper diet, Am. J. Clin. Nutr., 65:72–78. Uauy, R., Olivares, M., and Gonzalez, M., 1998, Essentially of copper in humans. Am. J. Clin. Nutr., 67(suppl):952S–959S. Wolvekamp, M.C. and De Briun, R.W., 1994, Diamine oxidase: An overview of historical biochemical and functional aspects, Dig. Dis., 12:2–14.

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DIETARY COPPER, VANADATE, LYSYL OXIDASE ACTIVITY, AND LYSINE TYROSYL QUINONE FORMATION

Robert B. Rucker, Changtai Cui, Eskouhie H. Tchaparian, Alyson E. Mitchell, Michael Clegg, Janet Y. Uriu-Hare, Brian Rucker, Tracy Stites, and Carl L. Keen Department of Nutrition University of California Davis, California 95616

1. INTRODUCTION Lysyl oxidase activity is critical for the assembly and crosslinking of extracellular matrix proteins, such as collagen and elastin. Lysyl oxidase can also serve as a vehicle for copper transport from extracellular matrix cells. Lysyl oxidase activity is sensitive to changes in dietary copper status and is influenced by metabolic and genetic perturbations in copper transport, specifically, mutations in the copper-transporting ATPase gene, PATPase-7A. Herein, the effects of nutritional copper deprivation, sodium vanadate toxicity, and Menkes’ disease are described with respect to copper accumulation in connective tissue cells and lysyl oxidase activation. A principle goal was to characterize features of lysyl oxidase activation. Dietary copper deficiency, sodium vanadate treatment, and observations using Menkes’ cells in culture are compared. Dietary copper deficiency causes a decrease in lysyl oxidase activity, but not in the amount of expressed lysyl oxidase protein (Rucker et al., 1996). Feeding sodium vanadate caused increased accumulation of copper in cells, but decreased accumulation of activated lysyl oxidase. The Menkes’ phenotype is also characterized by increased copper accumulation and decreased expression of active lysyloxidase (Kosonen et al., 1997). One explanation is that copper deficiency affects the post-translational formation of the active-site quinone cofactor of lysyl oxidase, lysine tyrosyl quinone (Wang et al., 1997). Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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2. MATERIAL AND METHODS Animals, Cells, and Diets Methodological approaches involved the use of several animal models and cells in culture. In studies of nutritional copper deficiency, male white leghorn chicks were used. Diets varying in copper content were prepared as described by Opsahl et al. (1982) using spray-dried skim milk and as a source of copper. The chicks were fed the semi-purified skim milk diet containing copper at 0, 1, 4, 8, or of diet. In studies with vanadate, weanling rats were used and fed Na metavanadate at 0, 10, 20, 40, or 80 of diet. For cells in culture normal human or Menkes’ fibroblasts were used as described by Kosonen et al. (1997). Sodium vanadate was also added to cultures of normal fibroblasts (0–20mmol/mL).

Enzyme Assays and mineral analyses Lysyl oxidase functional activity was assayed by modification of a fluorescent assay first described by Trackman et al. (1981). Ceruloplasmin activity and benzylamine oxidase activity in plasma were also determined (Schosinsky et al., 1974; McEwen, 1967). Copper and vanadium analyses were performed by graphite furnace atomic absorption spectrophotometry or by inductively coupled plasma atomic emission spectrometry (Jarrel Thermal Ash ICP analyzer).

Cell Cultures Normal human and Menkes’ fibroblasts were propagated in plastic culture flasks in complete Dulbecco’s Modified Eagle Medium (DMEM) containing 10% (v/v) fetal bovine serum, penicillin and streptomycin at a final concentration of 100 units/ml and respectively. When fibroblasts reached confluency, the medium was replaced with DMEM containing copper at a final concentration of and (when added) the vanadium levels were 0, 5, 10 or

Mass Spectral Analysis Mass spectral analysis was also used to characterize changes in mass of purified fractions of lysyl oxidase. Both electrospray ionization and matrix assisted laserdesorption ionization/time-of-flight/mass spectrometric (MALDI/TOF/MS) techniques were used. A novel aspect of these experiments was estimation of protein mass directly from the surface of chick tendon. Tissue slices of tendon were pulse-sonicated in sinnapinic acid, formed into sheer sheets, and mounted onto the MALDI/TOF/MS probe tip. A layer of sinnapinic acid was next crystallized under vacuum over the top of the mounted tissue.

3. RESULTS In chicks, weight and copper levels in liver and plasma tended to plateau (at 35 days) when the dietary copper intake was between of this was in contrast to a progressive increase in tendon copper in response to changing the dietary copper.

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Tendon lysyl oxidase activity increased in relationship to dietary copper concentration Since the levels of copper in chick tendon were positively correlated with dietary copper intake, i.e. an increase from ~1 to there was also a positive collation between tendon copper and tendon lysyl oxidase activity. Sodium vanadate was used since it is a broad-spectrum inhibitor of ATPase activity. Further, V toxicity results in developmental defects (Elfant et al., 1987), signs of which include extra cellular matrix (ECM) defects similar to those common to dietary Cu deficiency or intoxication with In response to V, in ECM-enriched tissues, e.g. tendon or skin, the Cu concentration increased progressively from 0.46 to tissue (values plateau at diet). Importantly, no change in liver Cu was observed. Liver Cu values were at all levels of V intake. The V concentration increased in response to dietary V in all tissues that were examined. Dietary V markedly inhibited skin and tendon lysyl oxidase activity (>50% at intakes of diet or more). In contrast plasma copper, plasma ceruloplasm, and benzyl amine oxidase, principally secreted from non-connective tissue cells, was not changed. The results in vivo were validated in studies in vitro using cultured fibroblast. As expected the Menkes’ cells elicited their usual phenotypic response (increased cellular M and decreased lysyl oxidase). As expected the Menkes’ cells elicited their usual phenotypic response (increased cellular Cu and decreased lysyl oxidase). The molecular masses of selected tendon proteins were also estimated using electrospray and MALDI/TOF/MS. The molecular weight of protein corresponding to lysyl oxidase in tendon from Cu-dencient birds was 28386 Da +/–86 and in tendon from copper-supplemented birds, 28639 Da +/– 122 (cf. Wu et al., 1992). These observations are consistent with an increase in protein mass that would occur from the formation of lysyl tyrosyl quinone, the active site cofactor, and the binding of 3–4 mol of Cu per mol of lysyl oxidase.

4. DISCUSSION We suggest that Cu deficiency cause post-translational modifications in tendon proteins corresponding to lysyl oxidase, e.g. a decrease in apparent mass. The decrease in mass is consistent with decreased copper binding and formation of lysyl tyrosyl quinone, particularly if copper is severely limiting. The data presented also suggest that the degree of inhibition of P-ATPases (upon exposure to V) may differ at least in an in vivo context. For example, copper accumulation in response to dietary vanadium is more pronounced in skin and tendon than in liver. In connective tissue, P-ATPase 7-A, the so-called “Menkes gene”, predominates, but in liver, P-ATPase 7-B, the “Wilson’s gene” appears to predominate (Kosonen et al., 1997 and papers cited). With respect to lysyl oxidase, an interpretation of the response to vanadium is inhibition of P-ATPase-7A, which in turn causes decreased copper delivery to lysyl oxidase. Lysyl oxidase activity is directly dependent on the amount of copper directed into post-golgi secretary vesicles (Kosonen et al., 1997). The cellular accumulation of copper in response to V suggests that it is diverted from pathways important to copper egress and lysyl oxidase secretion.

AKNOWLEDGMENTS Supported by PHS NIH Grants HD 26777, AM 25358 (CLK, JYU-A, RBR), an ARS-USDA grant from the Human Nutrition Research Initiative (RBR), and a grant

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from the Academy of Finland (TK). The authors wish to thank Drs. A. Daniel Jones, T.T. Yip and William Hutchens for advice and use of resources at the Facility for Advanced Instrumentation, University of California, Davis.

REFERENCES Elfant, M. and Keen, C.L. 1987 Vanadium toxicity in the adult and developing rat. Biol. Trace Elem. Res. 14:193-208. Kosonen, T., Uriu-Hare, J.Y., Clegg, M.S., Keen, C.L., and Rucker, R.B. 1997 Incorporation of copper into lysyl oxidase. Biochemical J. 327:283-289. Schosinsky, K.H., Lehmann, H.P., and Beeler, M.F. 1974 Measurement of ceruloplasmin from its oxidase activity in serum by use of o-dianisidine dihydrochloride. Clin. Chem. 20:1556–1563. McEwen, C.M. Jr. Human plasma monoamine oxidase. 1. Purification and identification. J. Biol. Chem. 240:2003-2010. Opsahl, W., Zeronian, H., Ellison, M., Lewis, D., Rucker, R.B., and Riggins, R.S. 1982 Role of copper in collagen cross-linking and its influence on selected mechanical properties of chick bone and tendon. J. Nutr. 112:708–716. Rucker, R.B., Romero-Chapman, N., Wong, T., Lee, J., Steinberg, F.M., McGee, C., Clegg, M.S., Reiser, K., Kosonen, T., Uriu-Hare, J.Y., Murphy, J., and Keen, C.L. 1996 Modulation of lysyl oxidase by dietary copper in rats. J Nutr. 126:51–60. Trackman, P.C., Zoski, C.G., and Kagan, H.M. 1981 Development of a peroxidase-coupled fluorometric assay for lysyl oxidase. Anal. Biochem. 113:336–342. Wang, S.X., More, M., Medzihradszky, K.F., Burlingame, A.L., Brown, D.E., Dooley, D.M., Smith, A.J., Kagan, H.M., and Klinman, J.P. 1996 A crosslinked cofactor in lysyl oxidase: redox function for amino acid side chains. Science 273:1078-1084.

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COPPER METABOLISM IN THE RODENT KIDNEYS Masaaki Kurasaki1, Masashi Okabe1, Mika Suzuki-Kurasaki1, Shigeru Saito2, Toshiyuki Hosokawa3, Osamu Yamanoshita1, and Takeshi Saito4 1

Department of Environmental Medicine and Informatics Graduate School of Environmental Earth Science Hokkaido University Sapporo, Japan 2 Department of Preventive Medicine St. Marianna University School of Medicine Kawasaki, Japan 3 Center for Research and Development in Higher Education Hokkaido University Sapporo, Japan 4 Department of Hygiene and Preventive Medicine Hokkaido University School of Medicine Sapporo, Japan

1. INTRODUCTION Cu is an essential trace element which requires a delicate cellular balance between a necessity and toxicity. Excess Cu is noxious in mammalian tissues. Cu accumulation in kidneys was observed in genetic disorders of Cu metabolism, such as Long-Evans Cinnamon (LEC) rats, a model for Wilson’s disease and Macular mice, a model for Menkes’ disease. It was believed that metallothionein (MT) was associated with Cu metabolism. MT is a low molecular weight protein with a high metal and sulfur content, and were induced by heavy metals and several other factors. The major function of MT is thought to be detoxification of heavy metals and homeostasis of essential trace metals (Kägi and Kojima, 1987). In this study, we revealed the histochemistry of Cu-MT in the kidneys of LEC rats and Macular mice. In addition, to understand the mechanism of Cu metabolism in the Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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tissue, we investigated the histochemical distribution of Cu-MT in the kidney of rat administered with Cu-MT.

2. MATERIALS AND METHODS 2.1. Animals Twenty one male wister rats, 6 male LEC rats, and 6 male Macular mice were used for the investigation. In the wister rats, Cu-MT was administered to the rats 3 times by intraperitoneally injections of 1.5 mg Cu-MT / rat. Each rat was sacrificed 24, 48 and 120 h after the final injection. All rodents were transcardinally perfused with 40 mM Tris-20 mM HC1 containing 152 mM NaCl. Their kidneys were quickly removed. All procedures were performed by the regulations as defined by National Institute of Health guide for the care and use of laboratory animals.

2.2. Experiments All histochemical procedures were carried out according to the method by Okabe, Nakayama, Kurasaki, Yamasaki et al. (1996). To prove the existence of MT, the immunoreactivity for MT was detected according to the method previously reported (Kurasaki, Okabe, Saito and Suzuki-Kurasaki, 1998). To examine the genomic expression of MT in the kidney, the distribution of mRNA encoding MT was observed by the method described previously (Suzuki-Kurasaki et al., 1997).

3. RESULTS AND DISCUSSION Although Cu-MT has been reported to be accumulated in the cortex (Evering, Haywood, Elmes, Jasani and Trafford, 1990; Schmid, Morgan, Öfner, Hittmair et al., 1993), it is noted that Cu-MT was intensely detected in the outer stripe of outer medulla in the kidney of LEC rats (Fig. 1A). In contrary, in Macular mice and rats administered

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with Cu-MT, MT was only observed in the cortex of the kidneys (Fig. 1B,C). The reason of the difference is still unclear. However, we have postulated that the difference depended on function and/or dysfunction of a Cu transport substance. Recently, two types of the gene encoding P-type cation-transporting ATPases (ATP7A and ATP7B) have been shown to be responsible for Cu-transport (Bull et al., 1993; Vulpe et al., 1993). ATP7A is a candidate gene for Menkes disease, and the other (ATP7B) is believed to be responsible for Wilson disease. In addition, we found that the Cu-MT injected to rats was transported to the cortex of kidneys (Fig. 1C). During the time course from 24hr to 120hr, although the level of the MT mRNA increased, the level of immuno-reactivity of MT was almost the same (Fig. 2). From the microscopic studies, Cu-MT was colocalized with acids phosphatase, an marker enzyme of lysosome in the PCT cells of kidney. From these results, it was thought that Cu bound to MT released in lysosome and became an inducer of de novo biosynthesis of MT in the same region. We propose a lysosomal cycle of Cu-MT in rat kidney as follows. Cu-MT was transported into PCT cells in the cortex and was taken up by the lysosomes. The released Cu ions from Cu-MT in the lysosome were reabsorbed by these cells and played as an inducer of MT mRNA in the same region, suggesting that Cu-MT is continually synthesized. Then the newly synthesized Cu-MT could be degraded in the lysosome. During the circulation of Cu ions, the metals in the PCT cells could cause renal injury in the rat. Cd-MT in cells causes renal damage due to free Cd ions released from Cd-MT by the lysosomal degradation (Nordberg and Nordberg, 1987; Dorian, Gattone II and Klaassen, 1992). The physiological significance of this result is that PCT cells are considered to be the primary site of the nephrotoxicity caused by heavy metals.

REFERENCES Bull, P.C., Thomas, G.R., Rommens, J.M., Forbes, J.R., and Cox, D.W., 1993, Nature Genet. 5:327–337. Dorian, C., Gattone II, V.H., and Klaassen, C.D., 1992, Toxicol. Appl. Pharmacol. 117:242–248. Evering, W.E., Haywood, S., Elmes, M.E., Jasani, B., and Trafford, J., 1990, J. Pathol. 160:305–312. Kägi, J.H.R. and Kojima, Y., 1987, Experientia suppl. 52:25–61. Kurasaki, M., Okabe, M., Saito, S., and Suzuki-Kurasaki, M., 1998, Am. J. Physiol. 274:F783–790. Nordberg, M. and Nordberg, G., 1987, Experientia suppl. 52:669–675. Okabe, M., Nakayama, K., Kurasaki, M., Yamasaki, F., Aoyagi, K., Yamanoshita, O., Sato, S., Okui, T, Ohyama, T., and Kasai, N., 1996, J. Histochem. Cytochem. 44:865–873. Schmid, K.W., Morgan, J.M., Öfner, D., Hittmair, A., Haywood, S., and Jasani, B., 1993, J. Histochem. Cytochem. 41:727–731. Suzuki-Kurasaki, M., Okabe, M., and Kurasaki, M., 1997, J. Histochem. Cytochem. 45:1493–1501. Vulpe, C., Levinson, B., Whitney, S., Packman, S., and Gitschier, J., 1993, Nature Genet. 3:7–13.

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EFFECT OF COPPER IN THE FOOD CHAIN ON HUMAN HEALTH (FOODCUE: FAIR CT95-0813) 2

1

1

1

E. Rock , J. J. Strain* , J. M. O’Connor , M. P. Bonham , 2 2 3 3 Y. Rayssiguier , A. Mazur , B. Sandström , S. Hodjberg-Bugel , 4 4 4 S. J. Fairweather-Tait , I. J. Harvey , G. Majsak-Newman , 5 5 5 5 6 A. Flynn , K. Cashman , A. Baker , F. Ginty , J. H. Beattie , 6 6 7 7 7 I. Bremner , M. D. Reid , G. Rotilio , M.-L. Scarino , Y. Sambuy , 7 7 7 S. Ferruzza , M. R. Ciriolo , and A. DeMartino 1

Northern Ireland Centre for Diet and Health (NICHE) University of Ulster Coleraine, Northern Ireland 2 Centre de Recherches en Nutrition Humaine Institut National de la Recherche Agronomique Clermont-Ferrand, France 3 Research Department of Human Nutrition Royal Veterinary and Agricultural University Copenhagen, Denmark 4 Institute of Food Research Norwich, England 5 Department of Nutrition University College Cork Ireland 6 Trace Elements and Gene Expression The Rowett Research Institute Aberdeen, Scotland 7 Department of Experimental Nutrition Instituto Nazionale Della Nutrizione Rome, Italy

1. INTRODUCTION Copper (Cu) has a range of important functional roles in the body, which appear to relate to the maintenance of immune function, bone health, arterial compliance, haemostasis and protection against oxidative and inflammatory damage. Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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Severe or clinically defined Cu deficiency in humans is rare but there are concerns that marginal or sub-optimal Cu status may, in the long term, precipitate a number of degenerative and inflammatory conditions including arthritis, cancer, osteoporosis and cardiovascular disease. The aims of the FOODCUE project were to provide information on the precise requirements for dietary Cu and to elucidate the balanced interactions that occur within the whole diet with respect to Cu. The specific scientific objectives of this research were: i) to provide data on the significance of increased dietary Cu (provided by Cu supplements) as a pro-oxidant or antioxidant in vivo in both free-living men and women; ii) to provide data on how formulated low, medium and high Cu diets fed to male and female volunteers in metabolic units can affect risk factors for cardiovascular disease, fructose/copper interactions and biomarkers of bone turnover; iii) to provide data on Cu bioavailability in humans using an oral challenge of stable isotope; iv) to provide in vitro data on the molecular mechanisms of Cu absorption and metabolism in the intestinal cell.

2. BIOAVAILABILITY STUDIES Characterisation of Cu Uptake from the Apical Membrane of Caco-2 Cells The apical uptake of copper was studied as a function of pH in the range pH 5.0 to pH 7.0. Uptake of Cu was shown to have a maximum at pH 6.0–6.5 and did not involve SH-groups in the apical proteins. When copper was presented to the apical membrane as Cu (I) ascorbate, its uptake was strongly inhibited as compared to Cu (II), given as Fructose however, did not affect Cu uptake. After 6 minutes of uptake from the apical membrane copper was bound intracellularly to low molecular weight ligands (possibly glutathione-GSH). With time (30 and 60 minutes) however, intracellular copper distribution shifted towards higher molecular weight ligands (possibly metallothioneinsMT). Cu, Zn Superoxide dismutase (SOD) was identified in Caco-2 cells by western blotting and its activity was not enhanced by copper treatment showing that, under the experimental conditions used, no apoSOD was present in the Caco-2 cells. To investigate the mechanisms of action of copper on the tight junctions, the localization of F-actin after copper treatment was investigated by staining the cells with fluorescent falloidin and visualising by fluorescence microscopy. but not resulted in a concentration—dependent decrease in F-actin staining in areas of the cell monolayer. To better characterize this effect, control and treated cells were scanned by confocal microscopy to localize F-actin within the cell. Copper treatment reduced F-actin staining at the level of the microvilli and of the peri-junctional actin ring. Ultrastructural analysis by transmission electron microscopy also showed that copper treatment resulted in disorganization of the microvilli and structural alterations to the tight junctional complexes.

Development of Alternative Techniques for Assessing Cu Bioavailability One healthy, non-smoking, female volunteer (aged 36) was recruited to take part in the study. On two occasions, the subject was given an oral dose of stable isotope, in order to measure the efficiency of absorption from a standard breakfast in the presence and absence of an inhibitor to copper absorption. Copper absorption was calculated by

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two methods; firstly as apparent absorption from the oral dose minus the excreted oral isotopic copper in faeces and secondly by the appearance of copper isotope on plasma albumin. The Dialysis-Chelex method was used for the extraction of albumin-bound copper with no cross-contamination between albumin and caeruloplasmin compartments. Analysis of the retentate after dialysis also demonstrated the utility of this method for measurement of caeruloplasmin-bound copper. The accuracy of the Dialysis-Chelex method was independently confirmed when the enrichment of total plasma copper, obtained 15–120 minutes after ingestion of a tracer by the volunteer, was found to compare favourably with the albumin copper data. At later time points, caeruloplasminbound tracer increased and was the predominant form in the days after faecal excretion of all unabsorbed tracer. Ingestion of inhibitors of copper absorption at the same time as the tracer reduced the level of albumin-bound tracer recovered within the initial 120 minutes, indicating that inhibition by ferric sulphate and ascorbic acid may be particularly effective in the stomach. Additionally, the method has considerable potential for the study of copper kinetics in the human liver and for the evaluation of copper status, because the turnover of hepatic copper is related to reserves of copper in the liver.

3. PUTATIVE INDICES OF BODY CU STATUS AND BIOLOGICAL EFFECTS OF CU SUPPLEMENTATION These parameters were determined from human supplementation trials and in vivo dietary manipulation studies in different research centers as following: Human dietary intervention trial carried out in the University of Ulster at Coleraine: the study design was a double blind, repeated cross-over with the supplementation and intervening placebo wash-out periods each lasting six weeks. Supplementation was carried out with 24 subjects (18–45 years) and involved doses of CuSO4 at levels of 3mgCu/d, Cu amino acid chelate at 3mgCu/d and 6mgCu/d, during the treatment periods. Human Study carried out in the Human Nutrition Research Center of ClermontFerrand: four groups of 7 people were randomly formed and included in an experimental design consisting on giving supplements as before but fully randomized The pills containing the supplements and placebos were given in a blinded way. Within this design, the data obtained during the supplementation period were compared to the mean values obtained after the three washout periods. All subjects were free-living and consumed self selected diets throughout the study. Human Study carried out in the Royal Veterinary & Agricultural University, Copenhagen: this study was performed as a double-blind, placebo-controlled supplementation study, divided into three six week periods and was a cross-over repeat design. The overall study was divided into two parts (A and B) with eight female subjects participating in each. In Part A weeks 1 and 6 were designed to be balance periods in which the 8 subjects were given a strictly controlled diet. For Part B week 6, only, was designed as the balance period and as in Part A the 8 subjects had a strictly controlled diet. The diets were designed to contain a constant constituent of nutrients with a Cu content of less than 1 mg/person/day. The diets were then supplemented with Cu as to give a basic intake of 1.5mgCu/d. Cu supplements in tablet form (Omg, 3mg and 6mg/d) were then given in a double-blind fashion daily from days 5–35 in Part A and days 1–35 in Part B for each of the 6 week supplementation periods.

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Human doetary intervention carried out in the Institute of Food Research (IFR), Norwich: the subjects lived in and ate all meals at the Human Nutrition Unit (HNU) for three periods of eight weeks with a minimum of 4 weeks between study periods. A diet low in copper (0.69 mg/d) was fed throughout the study in a 7-d rotating menu, each day consisting of three meals and snacks. The diet was adequate in all nutrients except copper and all dietary variables were within customary limits. Additional copper was added to the diets in the form of a copper supplement with each meal, such that the total daily intake was 1.6mg and 6.0mg during the first and third dietary periods respectively. The energy intakes of the subjects were adjusted as necessary in order to maintain body weight. This was achieved by the addition of either a commercially available dextrose drink or yoghurt.

Measurement of Putative Indices of Body Cu Status These included plasma Cu levels, superoxide dismutase (SOD) activity in red and white blood cells, cytochrome c oxidase activity in white blood cells, plasma diamine oxidase and plasma caeruloplasmin activity. These activities were all performed in the University of Ulster at Coleraine. Very significant increase of diamine oxidase activity was seen in both Coleraine and Clermont-Ferrand studies after Cu supplementation. Such data indicate that among the parameters tested, plasma diamine oxidase seems to a better marker for Cu status. However, further studies are necessary to determine the mechanisms underlying such an increase. For the other activities, no significant changes were observed in the Coleraine, Norwich and Clermont Ferrand studies. However, significant variations have been observed in young Danish women. Indeed, there was a statistically significant increase RBC-SOD activity on 6 mgCu/day compared to placebo. This is consistent with serum Cu concentrations (S-Cu) lower in placebo periods compared with 3 or 6 mg Cu/day. Interestingly, the plasminogen activator inhibitor 1 (PAI-1) activity was significantly lower after 4 weeks of an intake of 6 mgCu/day compared to placebo. Lower concentrations have been associated with a lower risk for cardiovascular diseases a low habitual dietary intake of copper in this population of young Danish females. As the intake of copper in the dietary intervention periods was 0.85 ± 0.08 mg/10MJ, these results indicate that Cu status was improved when given 3 mg Cu/day, but no further improvements were seen with 6 mg Cu/day. It should be noticed that for the highest Cu intake level (6 mg Cu/day), the generation of 5,6 epoxy cholesterol by peroxynitrite induced LDL oxidation was also increased.

The Biological Effects of Cu Intake They were evaluated through plasma lipid and lipoprotein changes, measurement of plasma fibrinogen and fibrinolytic activity, determination of transforming growth factor-beta, assessment of bone turnover and oxidative modifications including leukocyte DNA strand breakage (comet assay), susceptibility of red blood cells and lipoproteins to in-vitro-induced oxidizability. Among them, the only significant changes were observed on the erythrocyte oxidation and bone turnover in volunteers of Clermont-Ferrand and Norwich respectively.

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Vulnerability of Erythrocyte Membranes to an Applied in vitro Oxidative Stress Vulnerability of erythrocytes obtained from healthy elderly volunteers (50–70 years old) was assessed in vitro by AAPH-induced peroxidation. The data showed that supplementation with either 3 CuS or 6 Cu-AA increased the time for 50% of total lysis as compared with the values obtained after the washout period taken as control. Our results provided experimental evidence that supplementation with 3mg CuS and 6mg Cu-AA was able to protect the vulnerability of red blood cells against in vitro-induced peroxidation. Plasma antioxidants determination including vitamins (A and E) and carotenoids (lutein, lycopene and showed a significant correlation between the carotenes and half-time hemolysis.

Assessment of Bone Turnover In Norwich study, bone turnover was determined by the measurement of urinary pyrodinoline (Pyr) and deoxypyrodinoline (Dpyr) crosslinks and serum osteocalcin. Evaluation of the data has shown that dietary copper intake significantly affects bone turnover. Urinary creatinine (Cr) concentration was unaffected by dietary copper intake after six weeks equilibration at each intake level. However, there was a significant increase (p < 0.05) in urinary Pyr/Cr and urinary Dpyr/Cr excretion when subjects were on the low copper diet compared with the medium. Furthermore, there was, a significant decrease (P < 0.05) in urinary Pyr/Cr excretion and urinary Dpyr/Cr excretion when volunteers had equilibrated on the high copper diet compared to the medium. The ratio of Pyr to Dpyr in urine was unaffected by dietary copper intake. There were no significant differences in serum osteocalcin, a marker of bone formation, between the three dietary periods.

4. CONCLUSIONS Using Caco-2 human intestinal as in vitro model, the mechanisms of Cu uptake and transport have been investigated in great detail. Much valuable information has been collated on the tight junction permeability response of this cell line to different Cu complexes. The dialysis-chelex protocol for the extraction of albumin-bound Cu from plasma was developed and found to be both simple and accurate. This methodology may be a very suitable alternative to faecal monitoring of subjects for Cu bioavailability studies. Further studies are required to confirm the methodology but it has considerable potential for use in the study of the kinetics of Cu in the human liver and for evaluation of Cu status as hepatic Cu turnover is thought to be related to the liver Cu reserves. For all four centres, it was found that the mean dietary Cu intake was at the lower end of the recommended intakes. In the Danish study, dietary intake was estimated to be 0.85+/– 0.08mg/10MJ, indicating a low habitual dietary intake of Cu. Despite this, commonly used putative markers of Cu status are currently inadequate to assess Cu status. Of all the putative indices of Cu status analysed, the results showed that DAO was the only index which was consistently altered across all three Cu supplementations given. It is suggested that activity levels of this enzyme may be a suitable indicator of body Cu status.

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Cu has long been known to act as a pro-oxidant in vitro and indeed is often used to induce oxidation in the laboratory, although this is at concentrations which are million fold above physiological levels. Comet assay indicated that Cu supplementation at the doses described did not induce any in vitro alteration in DNA oxidative damage of mononuclear leukocytes. Cu supplementation did not result in any alteration in either Cu- or peroxynitrite-induced in vitro LDL oxidation. In one human trial, protective effect against in vitro-induced oxidation of red blood cells was seen. Moreover Cu supplementation had little physiologically relevant effect on traditional measures of inflammatory status, e.g. fibrinogen, CRP and white blood cell count and did not effect t-PA, PAI-1, factor V and factor VIII. To conclude, Cu supplementation, even at these high intakes (for 6 weeks) appears to have no effect on measures of inflammation lending further support to the idea that high dietary Cu does not act as a systemic pro-oxidant in vivo. These findings may alleviate concerns about increasing dietary Cu levels and provides further evidence against high dietary Cu intakes having a systemic pro-oxidant function in vivo.

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THE EFFECT OF COPPER SUPPLEMENTATION ON PUTATIVE INDICES OF BODY COPPER STATUS AND ON OXIDATIVE AND INFLAMMATORY MEASURES (FOODCUE PROJECT) J. M. O’Connor1, M. P. Bonham1, E. Turley1, C. Kehoe1, J. S. Coulter1,

M. S. Faughnan1, A. McKeown1, V. J. McKelvey-Martin1, E. Rock2, Y. Rayssiguier2 A. Mazur2, A. Flynn3, K. Cashman3, A. Baker3, and J. J. Strain*1 1

School of Biomedical Sciences University of Ulster Coleraine, Northern Ireland 2 Centre de Recherche en Nutrition Humaine Institut National de la Agronomique 3

Clermont-Ferrand, France Department of Nutrition University College Cork Ireland

Copper is routinely used to promote oxidation in vitro although this is at levels which million fold higher than physiological concentrations (Allen and Klevay, 1994). It has been demonstrated to be mutagenic and cause oxidative DNA damage and LDL oxidation in vitro. Although acute copper toxicity in humans is rare, there is concern that high body Cu may be associated with increased oxidative damage to healthy tissue. However, in contrast, other work indicates that Cu may act as an antioxidant in vivo and that adequate body Cu status may be required for the maintenance of antioxidant defences. In in vivo studies Cu deficiency has been linked to increased LDL susceptibility to oxidation and to an increased vulnerability of erythrocytes to oxidation. Severe or clinically defined Cu deficiency is rare in Western populations but it has been proposed

* correspondance Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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that marginal or sub-optimal Cu status may precipitate a number of degenerative or inflammatory conditions including cancer, arthritis, osteoporosis and cardiovascular disease (Strain, 1994). Human dietary Cu requirements are uncertain and there is currently no satisfactory method to adequately diagnose sub-optimal Cu status. The aim of this study was to provide data on how increased intake of Cu affected: the composition and physical characteristics of circulating levels of lipoproteins; susceptibility of LDL to in vitro oxidation; putative indices of body Cu status; inflammatory status; mononuclear leukocyte DNA damage and measures of bone turnover in healthy young free-living men and women. The study design was a double-blind repeated crossover trial with treatment and intervening placebo periods each of 6 weeks duration. The following supplementations were given orally in sequence: at a dose of 3 mg/d and Cu glycine chelates at doses of 3 mgCu/d and 6 mgCu/d. Blood and urine sample collection was at the end of each 6 week period. Mean Cu intake for females was 1.03 mg/d and for males was 1.48 mg/d. These data are the mean of data collected at the start and again at the end of the trial and were calculated from food photographs with diet history. Putative indices of body Cu status measured included red and white blood cell superoxide dismutase, white blood cell and platelet cytochrome c oxidase, diamine oxidase (DAO) and caeruloplasmin oxidase activities and caeruloplasmin protein levels. Of these measures, DAO was the only index which was consistently and significantly altered across all three supplementation periods (comparisons of means +/– SE for the each of the 3 supplementation periods: = 1.34 (+/–1.32) compared to placebo 1 = 0.30 (+/–0.48); 3mgCu glycine chelate = 1.63 (+/–1.34) compared to placebo 2 = 0.44 (+/–0.50) and 6mgCu glycine chelate = 2.00 (+/–2.02) compared to placebo 3 = 0.89 (+/–0.94). In all cases p < 0.01). LDL in vitro oxidizability was assessed by two methodologies. Cu induced in vitro oxidation was monitored by conjugated diene formation at 234 nm and the duration of lag time was interpreted to give a measure of the intrinsic resistance of the LDL to oxidation (Esterbauer, 1989). Peroxynitrite induced in vitro oxidation was assessed by measurement of lipoprotein relative electrophoretic mobility (REM) using gel electrophoresis (Patel et al., 1996). Neither methodology induced any alteration in LDL in vitro oxidizability. Similarly Cu supplementation resulted in no significant alteration in LDL lipoprotein a, apolipoprotein B, apolipoprotein A, triglycerides, free cholesterol, cholesterol esters and phospholipids nor in plasma total cholesterol. Mononuclear leukocyte (MNL) DNA damage was assessed by the comet assay. The Comet assay is a microelectrophoretic method for the quantitative measurement of DNA strand breaks and alkali labile sites (McKelvey-Martin et al., 1993). A measure of oxidative base damage in addition to DNA strand breaks was obtained by digesting the MNL DNA with endonuclease III before processing for the comet assay (Collins et al., 1997). Of the three supplementation periods supplementation with Cu did not induce any alteration in DNA damage. The effect of Cu supplementation on bone turnover was assessed by measurement of plasma osteocalcin as a marker of bone formation and urinary excretion of pyridinium scrosslinks as a measure of bone resorption (Robins and New, 1997 and Eaton-Evans et al., 1996). There was no alteration in measures observed during the three supplementation periods. Similarly, Cu supplementation at the three doses given had little physiologically relevant effect on traditional measures of inflammatory status including: fibrinogen, C

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reactive protein and white blood cell count and no effect on the related measures of transforming growth factor tissue plasminogen activator, plasminogen activator inhibitor 1, factor V and factor VIII. In conclusion, DAO, of the Cu indices measured, was the only index consistently and significantly altered and may be a suitable indicator of changes in body Cu status or Cu intakes with Cu supplementation. Cu supplementation, even at these high intakes, which were up to more than 7 times the normal daily Cu intake, did not appear to cause any significant alteration in DNA damage and did not result in any alteration in LDL oxidizability. Supplementation did not alter the characteristics of circulating lipoproteins nor effect traditional measures of inflammation and bone turnover. The data from this supplementation provide further evidence against high intakes of Cu contributing to oxidative stress in vivo. This work was funded by the European Commission (CT95-0813 FOODCUE) and the Ministry of Agriculture, Fisheries and Food (MAFF A181 (AN0511). The supplements were supplied by Thomson and Joseph Ltd., Norwich, England.

REFERENCES Allen, K.G.D. and Klevay, L.M., 1994, Copper an antioxidant nutrient for cardiovascular health. Curt Opinion in Lipid. 5:22–28. Collins, A.R., Dobson, ••, Dusinska, M., Kennedy, G., and Stevebrevetina, R., 1997, The Comet Assay: What can it really tell us? Mut. Res. 375:183–93. Eaton-Evans, J., McIlwrath, E.M., Jackson, W.E., McCartney, H., and Strain, J.J., 1996, Copper supplementation and the maintenance of bone mineral density in middle-aged women. J. Trace Elem. Exp. Med. 9:87–94. Esterbauer, H., Striegl, G., Puhl, H., and Rotheneder, M., 1989, Continuous monitoring of in vitro oxidation of low density lipoprotein. Free Rad. Res. Com. 6:67–75. McKelvey-Martin, V.J., Green, M.H.L., Schmezer, P., Pool-Zobel, DeMeo, M.P., and Collins, A., 1993, The Single Cell Gel Electrophoresis Assay (comet assay): A European Review, Mut. Res. 288:47-63. Patel, R.P., Diczfalusy, U., Dzeletovic, S., Wilson, M.T., and Darley-Usmar, V.M., 1996, Formation of oxysterols during oxidation of low density lipoprotein by peroxynitrite, myoglobin and copper. J. Lip. Res. 37:2361–2371. Robins, S.P. and New, S.A., 1997, Markers of bone turnover in relation to bone health. Procs. Nut. Soc. 56:903–914. Strain, J.J., 1994, Newer aspects of micronutrients in chronic disease: Copper Proc. Nut. Soc. 53:583–589.

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ROLE OF CERULOPLASMIN IN Fe EFFLUX FROM PLACENTA Identification of an Endogenous Cu Oxidase in Human Placental Cells (BeWo)

Ruth Danzeisen and Harry J. McArdle The Rowett Research Institute Greenburn Road, Bucksburn Aberdeen AB21 9SB

1. INTRODUCTION It has been known for many years that the metabolism of copper (Cu) and iron (Fe) are inter-related. In Cu deficient animals and in patients with aceruloplasminemia, Fe accumulates within specific tissues (Lahey et al., 1952; Harris et al., 1995). Ceruloplasmin (Cp), a plasma Cu protein with ferroxidase activity, stimulates Fe effux from perfused dog liver rapidly, while Cu-albumin has no effect (Osaki and Johnson, 1969). These data indicate a role for Cp in Fe release from cells. The hypothesis is that circulating Cp oxidises Fe (II) to Fe (III) for incorporation into transferrin (Tf) (Kaplan and O’Halloran, 1996). During pregnancy, Fe mobilisation is critical, but iron release from the placenta to the fetal circulation is not well understood. Cp is detectable in human fetal serum from early in gestation (Fryer et al., 1993), so we hypothesised, that Fe release is mediated by fetal Cp. To test this, we used a human placental cell line (BeWo). We have studied the role of exogenous Cp on Fe efflux from these cells, and we have investigated the possibility of an endogenous Cu oxidase in this cell line.

2. MATERIALS AND METHODS BeWo cells were grown under 100% humidity in Nutrient mixture Hams F12 Glutamax (10% fetal bovine serum, 2% Penicillin/Streptomycin). Confluent cells Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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were labelled with for 18 hours, release into serum-free medium (substituted with apo-Tf and Cp) was measured subsequently. BeWo cells were made Fe deficient by incubation with desferrioxamine or Cu deficient by incubation with diamsar for 24 hours. Cell differentiation was induced by addition of forskolin for 48 hours. Azide sensitive ferroxidase activity was measured as described previously. Cpimmunoreactivity was detected in BeWo cell membranes (brush border and non-brush border) and in BeWo cell homogenate (control and Fe deficient cells) using anti-Cp antibody and ECL.

3. RESULTS Extracellular had no effect on efflux from BeWo cells (Fig. 1). Similarly, increasing concentrations of Cp or changing labelling times with also did not stimulate release. This was the case in both differentiated and undifferentiated cells. We tested the possibility of an endogenous Cu oxidase in BeWo cells. There is azidesensitive ferroxidase activity in cell homogenate, equivalent to (mean ± SEM, n = 7). Treatment with diamsar decreased Cu oxidase activity activity, but not significantly, to (mean ± SEM, n = 3). Cu oxidase activity in BeWo cell homogenate is inhibited by an antibody to serum Cp (12.81 ± 4.18% inhibition (mean ± SEM, n = 5), antibody dilution of 1/50), but less than the effect on serum Cp (73.3% inhibition, antibody dilution of 1/50). Immunoreactivity to anti-Cp was detected in cell homogenate (two protein bands at approximately 140 and 100 kDa) and in the non-brush border fraction (one band at 100 kDa) (Fig. 2). Fe deficiency appears to increase the expression of the 140kDa band.

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4. DISCUSSION This paper tested the hypothesis that Cp is involved in Fe release from placental cells. Exogenous Cp appeared to have no effect on Fe efflux, instead we found an endogenous, membrane associated Cu oxidase activity in BeWo cells. These data contrast with that shown in liver (Osaki and Johnson, 1969), (Young et al., 1997), where exogenous Cp clearly has a role in iron release. However, an endogenous Cu oxidase has also been found in intestine (Vulpe et al., 1999) and a membrane-bound form has been identified in brain (Patel and David, 1997). It is possible, therefore, that different tissues, performing different functions, have to regulate iron efflux differently. Placenta and intestine must transfer large amounts of iron from one compartment to another, and as such may need to be able to regulate efflux independently. In support of this hypothesis, preliminary data suggests that Cu-oxidase activity and expression in BeWo cells may be increased in iron deficiency.

REFERENCES Fryer, A. A., Jones, P., Strange, R., Hume, R., and Bell, J.E. 1993, Plasma protein levels in normal human fetuses: 13 to 41 weeks’ gestation, Brit. J. Obstet. Gynaecol. 100:850–855. Harris, Z.L., Takahashi, Y., Miyajima, H., Serizawa, M., MacGillivray, R.T.A., and Gitlin, J.D. 1995, Aceruloplasminemia: Molecular characterization of this disorder of iron metabolism, Proc. Natl. Acad. Sci. USA. 92:2539–2543. Kaplan, J. and O’Halloran, T.V. 1996, Iron metabolism in eukaryotes: Mars and Venus at it again., Science. 271:1510–1512. Lahey, M.E., Gubler, C.J., Chase, M.S., Cartwright, G.E., and Wintrobe, M. 1952, Studies on copper metabolism II; Hematologic manifestations of Cu deficiency in swine, Blood. 7:1053–1074. Osaki, S. and Johnson, D.A. 1969, Mobilization of liver iron by ferroxidase (ceruloplasmin), J. Biol. Chem. 244:5757–5758. Patel, B.N. and David, S. 1997, A novel glycosylphosphatidylinositol-anchored form of ceruloplasmin is expressed by mammalian astrocytes, J. Biol. Chem. 272:20185–20190.

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Vulpe, C.D., Kuo, Y.M., Murphy, T.L., Cowley, L., Askwith, C., Libina, N., Gitschier, J., and Anderson, G.J. 1999, Hephaestin, a ceruloplasmin homologue implicated in intestinal iron transport, is defective in the sla mouse, Nat Genet. 2:195–199. Young, S.P., Fahmy, M., and Golding, S. 1997, Ceruloplasmin, transferrin and apotransferrin facilitate iron release from human liver cells, Febs Lett. 411:93–96.

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DIETARY MOLYBDENUM Effect on Copper Absorption, Excretion, and Status in Young Men

Judith R. Turnlund and William R. Keyes USDA/ARS, Western Human Nutrition Research Center Davis, California, USA

BACKGROUND There is a well established interaction between molybdenum and copper. The interaction has been demonstrated in ruminants, with sheep particularly susceptible to the effects (Mills and Davis, 1987). When molybdenum in low in soils, copper accumulates rapidly in sheep, and when molybdenum is high in soils signs of copper deficiency have been observed. There have been few observations of the interaction in humans. One study reported that molybdenum intake of over 0.5 mg/d was associated with increased urinary copper excretion and the authors suggested this could increase the risk of copper depletion (Deosthale and Gopalan, 1974). We studied copper metabolism in one group of subjects during molybdenum depletion and in another group fed increasing levels of molybdenum, to explore the effects of molybdenum intake on copper metabolism in humans.

EXPERIMENTAL DESIGN AND METHODS A study was conducted in 8 young men to determine the effect of the amount of dietary molybdenum and of molybdenum status on copper absorption, excretion, retention, and status. The 120-day study was conducted in a metabolic research unit. Volunteers were confined to the unit during the entire study. The men were divided into two groups of four men each. In one group, dietary molybdenum was (depletion) for 102 day, followed by 18 days with (repletion) (Turnlund et al., 1995). The other group was fed five levels of dietary molybdenum, 22, 72, 121, 467 and for 24 Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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days each (Turnlund, Keyes and Peiffer, 1995). The diet, a three-day rotating menu, was comprised of low-molybdenum foods and was supplemented with a liquid drink containing additional energy and the essential vitamins and minerals low in the lowmolybdenum food. The diet contained 1.62mg/d of copper throughout the study. Complete urine and stool collections were made throughout the study. Blood was collected for plasma copper at intervals selected during the study. The stable isotope was fed at the beginning, middle, and end of depletion and during repletion in the first group and at each molybdenum intake in the other to determine copper absorption. Serum copper were determined by flame atomic absorption spectrophotometry and urinary copper was determined by electrothermal atomic absorption spectrophotometry. Fecal copper and dietary copper were determine by isotope dilution. appearing in stools and added to the diet was determined by isotope dilution. Isotope ratios were measured using thermal ionization mass spectrometry (TIMS). Absorption was calculated based on the difference between the amount of fed and the amount appearing in the stools. ANOVA was used to compare the effects of molybdenum depletion and the level of dietary molybdenum on copper absorption and retention, urinary and serum copper, and to evaluate differences between subjects.

RESULTS The effects of molybdenum depletion and repletion on copper metabolism are summarized in Table 1. Copper absorption averaged 38.9 ± 1.5% during molybdenum depletion and 36.0 ± 2.6% during repletion. Copper retention averaged 0.048 ± 0.056mg/d during depletion and 0.002 ± 0.134mg/d during repletion. Serum copper averaged 14.4 during depletion and during repletion. Urinary copper averaged during depletion and during repletion. There were no differences in these parameters due to molybdenum depletion. The effects of five increasing levels of dietary molybdenum, ranging from 22 to are shown in Table 2. Average copper absorption at the five levels of dietary molybdenum ranged from 31.6% to 40.8% (SEM 2.5). Average copper retention ranged from +0.025 to–0.035mg/d (SEM 0.075). Average serum copper ranged from 13.0 to 14.0 (SEM 0.5). Average urinary copper ranged from (SEM 1.1). There were no differences due to the amount of dietary molybdenum. In both treatment groups, copper absorption, urinary copper and serum copper differed significantly between subjects.

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CONCLUSIONS The results suggest that diets very low in molybdenum do not alter copper metabolism in young men and that dietary molybdenum intake in amounts up to does not influence copper metabolism. In addition, copper status was maintained in both groups on a copper intake of 1.62mg/d for 120 days.

REFERENCES Deosthale Y.G. and Gopalan C. 1974, The effect of molybdenum levels in sorghum (Sorghum vulgare Pers.) on uric acid and copper excretion in man. Br J Nutr 31:351–355. Mills C.F. and Davis G.K. 1987, Molybdenum. In: Mertz W, ed. Trace Elements in Human and Animal Nutrition. 5th ed. San Diego: Academic Press, 429–463. Turnlund J.R., Keyes W.R., Peiffer G.L., and Chiang G. 1995, Molybdenum absorption, excretion, and retention studied with stable isotopes in young men during depletion and repletion. Am J Clin Nutr 61:1102–1109. Turnlund J.R., Keyes W.R., and Peiffer G.L. 1995, Molybdenum absorption, excretion, and retention studied with stable isotopes in young men at five intakes of dietary molybdenum. Am J Clin Nutr 62:790–796.

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IDENTITY AND REGULATION OF THE COPPER TRANSPORT PROTEIN, TRANSCUPREIN

N. Liu, L. Lo, T. Tran, L. Jones, and M. C. Linder California State University Fullerton, California 92834-6866 USA

Some time ago we identified a new protein in rat plasma, referred to as transcuprein, as involved in the initial distribution of incoming dietary copper to liver and kidney (K. C. Weiss et al., Am. J. Physiol. 249: E77–E88, 1985; Linder et al., Am. J. Clin. Nutr. 67: 965S–971S, 1998). We found this protein to have an even higher affinity for copper than albumin, and to exchange copper rapidly and directly with albumin under physiological conditions. In studies reported here, we have obtained amino acid sequence for, and identified, the major subunit of transcuprein and have begun to study its regulation by copper. Rat transcuprein was purified by a combination of techniques previously reported, including size exclusion chromatography on large and medium pore gels, and Cu(II) chelate affinity chromatography. Transcuprein was identified by its attached tracer, actual copper content, and apparent (native) molecular weight. As previously reported, it contained subunits of 200 and 69kDa in SDS-polyacrylmide gel electrophoresis. Amino acid sequence was obtained for peptides generated by in gel treatment of the subunits with modified trypsin. The 200k subunit sequences had 100% homology with rat alpha-1-inhibitor 3, a member of the macroglobulin family. The 69k subunit sequences were identical to those of rat albumin. Transcuprein (270k Da) thus appears to be composed of a complex of the macroglobulin, alpha-1-inhibitor 3, with albumin. To confirm a connection between this macroglobulin and copper metabolism, levels of alpha-1-inhibitor mRNA were measured by competitive PCR in livers of rats that were and were not copper deficient. Copper deficiency was induced by placing weanling pups on a starch-based copper deficient diet, with distilled water for drinking. Half of the animals had cupric sulfate added to the drinking water. Copper deficiency was monitored by measuring serum ceruloplasmin, as determined by its p-phenylene diamine oxidase activity. Deficient rats had about 60% of the ceruloplasmin oxidase activity of the copper-sufficient rats. A competitive PCR assay to quantitate alpha-1-inhibitor3 mRNA was developed using the MIMIC approach of Clontech and confirmed by 955

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Northern analysis. Copper deficient rats expressed about twice as much mRNA for alphal-inhibitor-3 in their livers as the copper-sufficient ones. We conclude that the initial transport of copper from the intestine to the liver and kidney that occurs in mammals involves its binding to members of the macroglobulin family, and that in the rat this specifically involves binding to a complex between alphal-inhibitor-3 and albumin. Liver expression of transcuprein/alpha-l-inhibitor-3 appears to be inversely regulated by copper status, which would be analogous to what occurs with transferrin expression in different states of iron sufficiency. Supported in part by NSF Grant DUE 9352396 and a minigrant from California State University.

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RESPONSE OF DIAMINE OXIDASE AND OTHER COPPER STATUS BIOMARKERS TO MODIFICATIONS IN DIETARY COPPER IN THE RAT

C. Feillet-Coudray, A. Mazur, C. Coudray, E. Rock, and Y. Rayssiguier Centre de Recherches en Nutrition Humaine Unité Maladies Métaboliques et Micronutriments INRA, Theix, St Genès Champanelle France

Copper (Cu) is an essential trace element, which plays an important role in various cellular functions. Several studies have shown that frequently Cu requirements may not be covered by diet Cu. However, there is a lack of suitable biochemical markers that are able to track Cu status. Classically, protein Cu-dependent activities, like SOD or ceruloplasmin, are used as biomarkers. Recently diamine oxidase (DAO), a Cu-dependent enzyme, has been proposed as a valuable biomarker of Cu status in humans and in animals. Furthermore, the initial results of a multicenter study (European program FOODCUE) have demonstrated that increasing levels of dietary Cu in healthy subjects results in increased levels of plasma DAO activity but does not affect other biomarkers of Cu status. The purpose of this work was to assess the sensitivity of DAO to modifications in Cu intake in comparison with other biomarkers of Cu status. Towards that aim, 3 groups of 8 male Wistar rats were fed for 3 weeks with either a control diet (6 ppm Cu), a Cu-supplemented diet (30 ppm Cu) and a Cu-deficient diet (0.6ppm Cu). After this period, all rats were fed the control diet for 12 days. Plasma, Cu, SOD-RBC, ceruloplasmin and DAO activities were assessed at 4, 8, 12 and 18 days of the control, supplemented or deficient diets and 4 and 12 days after the return to the control diet. SOD-RBC was significantly decreased at day-4 of the deficient diet while plasma Cu and plasma ceruloplasmin were decreased at day-8. DAO activity was significantly decreased at day-12 of the deficient diet. In Cu-deficient rats, SOD-RBC, plasma Cu and DAO activities were normalized after 4 days of the return to control diet while ceruloplasmin was normalized at day-12. Cu supplemented diet had no effect on the biomarkers of Cu status. In conclusion, the studied biomarkers of Cu status that were examined were 957

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affected rapidly by Cu deficiency and restored by return to control diet. Of the biomarkers studied, plasma DAO activity reacted less rapidly to Cu deficiency. Thus, further studies are necessary to understand the apparent contradictory response of DAO to modifications in Cu intake in man and experimental animals.

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EFFECT OF DIETARY COPPER INTAKE ON BIOCHEMICAL INDICES ASSOCIATED WITH COPPER METABOLISM L. Harvey1, G. Majsak-Newman1, S. Fairweather-Tait1, A. Baker2, K. Cashman2, and A. Flynn2 1

Institute of Food Research Colney, Norwich NR4 7UA UK 2 Department of Nutrition University College, Cork Ireland

Symptoms of severe copper deficiency in humans are well characterised, including anaemia and neutropenia. However, the full extent and effects of marginal copper deficiency within the population remain unknown. It has been suggested that chronic diseases such as osteoporosis and cardiovascular disease may be linked to marginal copper deficiency [1]. This study was designed to assess the effect of dietary copper intake on various biochemical parameters associated with copper metabolism and to provide data for the recommendation of optimum dietary intakes. Twelve healthy male volunteers (aged 18–60y) participated in a residential, longitudinal dietary intervention study investigating the effects of medium (1.6mg/d), low (0.7mg/d) and high (6.0mg/d) intakes of copper over eight week periods. The volunteers were fed low copper diets supplemented with copper as appropriate. All other nutrients met Dietary Reference Values and other dietary variables were within customary limits. During the course of each dietary period biochemical markers of bone metabolism, risk factors for cardiovascular disease and putative measures of copper homeostasis were monitored. Preliminary data for 11 out of the twelve volunteers suggested that bone metabolism was affected by dietary copper intake. Significant increases in the excretion of urinary pyridinoline/creatinine (Pyr/Cr) (30%) and deoxypyridinoline/creatinine (Dpyr/Cr) (25%) were found after equilibration on the low Cu diet when compared with the medium. Conversely, significant decreases were found for urinary Pyr/Cr (30%) and Dpyr/Cr (22%) following equilibration on the high when compared with the low copper diet. There were no significant differences in serum osteocalcin, a marker of bone 959

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formation, between the three dietary periods. Plasma caeruloplasmin and copper concentrations and Cu,Zn-superoxide dismutase activities were unaffected by dietary copper intake. In addition, risk factors for cardiovascular disease, including total, HDL and LDL cholesterol and triglyceride concentrations were also unaffected.

ACKNOWLEDGMENTS This collaborative project with CSL Norwich was funded by the Ministry of Agriculture Fisheries and Food, the European Commission and the BBSRC.

REFERENCE Danks, D.M., 1988, Ann. Rev. Nutr.: 8;235–237.

300

METALLOTHIONEINS Their Cellular Function and Relationship with Zinc

John H. Beattie and Ian Bremner Trace Element and Gene Expression Group Rowett Research Institute Bucksburn, Aberdeen AB21 9SB Scotland, UK

1. INTRODUCTION Low metal-binding proteins demonstrating the classic structural features of metallothionein (MT) have been found in many animals, plants and microorganisms. In mammals, 4 categories of MT gene have been identified, each coding for 61–68 amino acid proteins. MT-1 and MT-2 are expressed in many tissues but MT-3 is found predominantly in the brain and MT-4 in stratified squamous epithelia. Multiple isoforms with structure, charge and tissue expression characteristic of MT-1 or MT-2 have been identified in several species. Considerable progress in understanding the structure and expression of MT has been made over the last few years. In contrast however, the primary function of MTs remains elusive and the purpose of this review is to focus on recent discoveries that provide insight into the functional role of these enigmatic proteins.

2. STRUCTURAL CHARACTERISTICS OF MT The structural features of MT, which are conserved across all phylogenetic levels and are therefore likely to have functional significance, include the high content of cysteine. The cysteine residues are arranged in cys-X-cys, cys-X-X-cys or cys-cys-X-cys-cys motifs, where X is another residue, and the thiol ligands bind and/or Class I MTs, expressed in many animal species, bind 7 divalent metals in a 2-domain secondary structure but the significance of this conformation is unknown. A prominent view is that it provides an entropically stable binding arrangement for the metal ions, which are Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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considered to be the functional components of the protein. Release of for example zinc from MT may occur through a redox sensing mechanism, whereby reduced glutathione can associate with a cleft in the beta-domain, allowing access of oxidising agents, particularly glutathione disulphide, to the intradomain thiol ligands (Jiang et al., 1998). In this way, localised intracellular changes in redox balance may facilitate release from MT of Zn, which may then be utilised for activation of enzymes or transcription factors. However, recent evidence suggests that MT can itself bind to other proteins (Gasull and Hidalgo, 1996; Whitacre, 1996). The 2-domain structural feature may therefore have more significance than simply an appropriate conformation for efficient metal sequestration and delivery. MT-3 is reported to inhibit cortical neuron survival in culture and the biologically active sequence was found to be a cys-pro-cys-pro motif close to the Nterminus (Sewell et al., 1995). However, the activity found with the holo protein was also obtained with the apo-form, indicating that integrity of the secondary structure, which is dependent on metal-binding, was not necessary for biological function (Uchida and Ihara, 1995).

3. REGULATION OF MT GENE EXPRESSION Mammalian MT genes are clustered at a single locus on one chromosome, which in the case of mouse and human MT is chromosome 8 and 16, respectively. These genes contain 3 exons and 2 introns and their expression is regulated by a variety of different upstream cis-acting elements (Samson and Gedamu, 1998). In addition to basal promoters responsive to SP-1 and AP-1, perhaps the best characterised promoters are the metal response elements (MRE), several of which are required for effective activation of gene expression by MRE-specific transcription factors (Koizumi et al., 1999). Zinc upregulation of MT transcription is mediated by a zinc sensitive transcription factor MTF-1, which contains 6 zinc fingers (Bittel and Andrews, 1998a). The first finger acts as an activator and the binding of zinc is thought to change the conformation of the molecule, allowing fingers 2–4 to bind to DNA. Fingers 5 and 6 have no known function. In contrast to other Zn-finger transcription factors which have Zn dissociation constants in the picomolar to nanomolar range, MTF-1 is regulated at micromolar concentrations of Zn (Bittel et al., 1998b). Thus, MTF-1 seems to act as a zinc sensor, and is rapidly activated when zinc levels increase. Schaffner and colleagues have demonstrated that MTF-1 knock-out mice die at day 14 of embryonic development, showing liver decay and generalised edema (Gunes et al., 1998). Since MT knock-out mice do not die in utero, it is clear that MTF-1 must also regulate the expression of other genes. In fact, recent studies show that expression of synthetase and the zinc efflux transporter ZnT-1 are both regulated by MTF-1 (Gunes et al., 1998). Intriguingly, MTF-1 is also activated by reactive oxygen species, although this is thought to occur through liberation of cellular zinc that is bound to redox-sensitive ligands (Murphy et al., 1999). Cadmium is a classical and strong inducer of MT gene transcription and yet it seems that it does not activate MTF-1. Instead, cadmium appears to activate an independent transcription factor that also binds to MREs (Chu et al., 1999). Although the MT-3 and MT-4 gene promoters contain MREs, these are not activated in response to elevated zinc or cadmium. MT is induced in response to stress and mitogens and the mechanisms involved are being studied. Glucocorticoids stimulate MT expression through receptor binding to glucocorticoid response elements that have recently also been identified in the mouse MT-1 and MT-2 promoters (Kelly et al., 1997). Cytokines, such

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as IL-6, activate membrane receptors which signal through the JAK/STAT transduction pathway. Both STAT1 and STAT3 protein dimers that are activated in response to lipopolysaccharide treatment of mice, are now known to bind to an MRE in the MT promoter and amplify MT gene transcription (Lee et al., 1999). Activation of the MAPK signal transduction pathway through growth factor (e.g. EGF) binding to tyrosine kinase receptors initiates MT transcription, but the precise mechanism involved has yet to be demonstrated. Although MT expression is predominantly regulated at the level of transcription, there is now evidence suggesting translational regulation of at least some isoforms (Vasconcelos et al., 1995). Most MTs are co-translationally acetylated, but significant levels of unacetylated MT (>20% of total MT-2) has now been detected in rats treated with zinc (Beattie et al, 1999).

4. CELLULAR AND INTRACELLULAR LOCALISATION OF MT Mammalian MT-1 and MT-2 isoforms are expressed in many different cell types including liver parenchymal cells, pancreatic acinar cells, kidney proximal tubule cells, vascular endothelial cells, glial cells, intestinal epithelial cells and adipocytes of brown adipose tissue. In animals that are not subject to any form of stress, constitutive expression of MT is considerably higher in the pancreas and brain than in most other tissues (Liu et al., 1996a). The high levels classically associated with liver and kidney are usually found only in response to induction by e.g. metals such as zinc and cadmium. There is some controversy surrounding the cell type expressing MT-3 with evidence for both neuronal and astrocytic expression. Nevertheless, at a gross immunohistochemical level, there seems to be consensus that MT-3 is found predominantly in the cortex and hippocampus. New evidence shows that MT-3 is also expressed in other tissues, such as testis, prostate, epididymis, tongue, ovary, uterus, stomach, heart and seminal vesicles, albeit at lower levels than in the brain (Moffatt and Seguin, 1998). Although MT-1 and MT-2 isoforms were for many years considered to be cytoplasmic proteins, it is now clear that one or both of these isoforms concentrates in the nucleus during early S-phase of cells induced to proliferate (Tohyama et al., 1993; Tsujikawa et al., 1991). Indeed, rapidly dividing cells in culture contain higher levels of MT than do confluent cells which are not dividing (Studer et al., 1997). It is not yet clear how MT translocates into the nucleus but its site of translation on the cytoskeleton in a perinuclear localisation may be mechanistically critical for nuclear targeting (Mahon et al., 1997). It would appear that MT is bound or retained within the nucleus during S-phase because isolation of nuclei does not result in loss of this protein through the nuclear pores. As yet, there is no evidence that MT-3 is targeted to the nucleus.

5. FUNCTIONAL ROLES OF MT ISOFORMS 5.1. Function of MT-1 and MT-2 Since MT-1 and MT-2 are induced by a wide variety of different stress factors, including those of chemical, physical, biological and psychological origin, it is regarded as a stress response protein. In support of this contention, MT-null mice would appear to be more sensitive to the effects of stress (Klaassen and Liu, 1998). However, precisely because of the diversity of factors that stimulate its induction, defining a primary role

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for the protein is problematic. Its role in the detoxification of Cd has been confirmed in studies with MT-null and MT overexpressing mice. Cd appears to induce MT through a transcription factor which is independent of MTF-1 but which binds to MREs. Unless Cd binds to a transcription factor intended for another purpose, this evidence supports a specific role for MT in Cd detoxification. Nevertheless, Cd is excreted more rapidly in MT-null mice (Liu et al., 1996b), suggesting that the long biological half-life for Cd is due to its binding to MT. This would not appear to be advantageous for reducing Cd toxicity. MT is thought to have an important function in Zn homeostasis but it is clear that this role is not essential for life or reproduction, since MT-null mice do not seem to be adversely affected by lack of MT under controlled laboratory conditions. Nevertheless, the discovery that both MT and ZnT-1 gene expression is upregulated by Zn-activated MTF-1 would suggest that homeostasis is being maintained by both sequestration and removal of Zn from the cell. It is possible that MT could modulate activation of MTF-1 in the nucleus by sequestration of labile Zn, in which case MT may be a selfregulating protein. There is good evidence that MT can scavenge free radicals in vitro (Thornalley and Vasak, 1985) and studies with cells in culture which under- or over-express MT support the proposed role of this protein as an antioxidant (Lazo et al., 1995; Pitt et al., 1997). Nevertheless, there is a conspicuous absence of information concerning the sensitivity of MT-null and MT-overexpressing mice to oxidant stress although increased toxicity of paraquat to MT-null mice has been observed (Sato et al., 1996). Remarkably, there are also few reports concerning physiological oxidative stress in cell cultures or in animal tissues. Clearly, if MT has a role as an antioxidant, it should be effective in situations where reactive oxygen species are generated during a physiological process, such as during thermogenesis in brown adipose tissue (BAT) of cold-exposed animals. MT is in fact strongly induced in BAT of cold-exposed rats (Beattie et al., 1996) but its antioxidant role there has yet to be confirmed. We do however know that MT is present in adipocytes of BAT and, like uncoupling protein 1, which drives non-shivering thermogenesis, it is induced by catecholamines (J. Beattie et al., unpublished observations). Since MT is induced in proliferating cells and shows transitory nuclear localisation during the G1 to S phase transition, a role for MT in cell division has been proposed (Studer et al., 1997). The cells of MT-null mice appear to divide normally and so this role is not critical. Cells overexpressing MT following transfection with MT expression vectors tend to grow more rapidly than wild-type cells (Abdel-Mageed and Agrawal, 1997). This is consistent with the observation that some aggressive cancer cells overexpress MT and the prognosis for recovery from some, but not all forms of cancer has been linked to the degree of MT overexpression (Jasani and Schmid, 1997). Conversely, inhibition of MT in a breast cancer cell line (MCF7) using an antisense gene appears to inhibit cell growth (Abdel-Mageed and Agrawal, 1997). Apoptosis is more readily induced in MT-null embryonic fibroblasts subjected to anticancer drugs and oxidant stress than in wild-type cells (Kondo et al., 1997). Increased sensitivity to apoptosis may be linked to p53, because the level of this Zn-dependent transcription factor is elevated in MT-null cells. The mechanisms regulating cell proliferation and apoptosis which MT appears to influence are however unknown. MT-null mice were generated independently on 2 different genetic backgrounds and were originally described as being phenotypically normal (Michalska and Choo, 1993; Masters et al., 1994). However, we recently observed that some MT-null mice on a mixed C57BL/6J and 129Ola genetic background showed late-onset moderate obesity combined

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with hyperleptinaemia and hyperphagia (Beattie et al., 1998). The degree of obesity and high plasma leptin suggested that this phenotype was probably not related to the mixed genetic background and so the relationship between MT and leptin was further investigated. We have now shown that leptin regulates MT-1 gene expression in ob/ob mice, which do not produce functional leptin and are therefore obese, within hours of injection (J. Tavernier et al., unpublished observations). Thus, there is now evidence for an interaction between MT and leptin that may influence control of appetite and/or energy expenditure.

1.2. Function of MT-3 MT-3 was first discovered as a growth inhibitory factor which inhibited the formation of neurofibrillary tangles of neurons in culture and appeared to be deficient in the brains of Alzheimers disease patients. However, its role in protecting against this disease remains controversial due to conflicting results from different laboratories. Mice with targeted disruption of the MT-3 gene are described as being phenotypically normal and do not show age-related impaired brain function (Erickson et al., 1997). These mice are however more sensitive to kainic acid-induced seizures, which may indicate a possible role in maintaining sufficient amounts of vesicular Zn during sustained neuronal firing. Ectopic expression of MT-3 in mice where the MT-3 promoter is replaced by that of MT-1, resulted in mortality at 2–3 months of age (Quaife et al., 1998). The primary pathological lesion was necrosis of pancreatic acinar cells.

CONCLUSION The ubiquity of MT expression in response to a wide variety of inducing factors suggests that MT has a fundamental biological role in the cellular response to changing environmental conditions. Clearly, the presence of MT is not essential for life but its role in protecting against stress in particular is now established. Under normal physiological conditions, nuclear localisation of MT during the cell cycle would seem to indicate an important function during S-phase, and yet the growth and development of MT-null mice appears to be normal. One possible explanation for the apparent redundancy of MT is that an efficient compensatory mechanism takes on a regulatory or protective role in the absence of MT. It would seem that Zn activation of enzymes and transcription factors can proceed in the absence of MT, but its presence may modulate this process, particularly during periods of stress. Recent evidence that MT can bind to the p50 subunit of response element (Abdel-Mageed thus enhancing its association with the and Agrawal, 1998), may indicate another possible mechanism for modulating gene expression, through direct binding to transcription factors. The absence of MT may not prevent transcription proceeding, but regulation may be compromised when environmental conditions are less favourable.

REFERENCES Abdel-Mageed, A. and Agrawal, K.C., 1997, Antisense down-regulation of metallothionein induces growth arrest and apoptosis in human breast carcinoma cells. Cancer Gene Ther., 4:199–207. Abdel-Mageed, A.B. and Agrawal, K.C., 1998, Activation of nuclear factor kappaB: potential role in metallothionein-mediated mitogenic response. Cancer Res., 58:2335–2338.

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Beattie, J.H., Black, D.J., Duncan, J.S., Wood, A.M., and Trayhurn, P., 1996, Cold-induced expression of the metallothionein-1 gene in brown adipose tissue of rats. Am. J. Physiol, 270:R971–R977. Beattie, J.H., Wood, A.M., and Duncan, J.S., 1999, Rat metallothionein-2 contains and unacetylated isoforms. Electrophoresis, (In Press). Beattie, J.H., Wood, A.M., Newman, A.M., Bremner, I., Choo, K.H.A., Michalska, A.E., Duncan, J.S., and Trayhurn, P., 1998, Obesity and hyperleptinemia in metallothionein (-I and -II) null mice. Proc. Natl. Acad. Sci. U.S.A., 95:358–363. Bittel, D. and Andrews, G.K., 1998a, The Zn-fingers of MTF-1 contain a mechanism for sensing intracellular free Zn (Abstract). International Conference on Metal-binding Proteins in Biology, Banff, Alberta, Canada, p. 46. Bittel, D., Dalton, T., Samson, S.A., Gedamu, L., and Andrews, G.K., 1998b, The DNA binding activity of metal response element-binding transcription factor-1 is activated in vivo and in vitro by zinc, but not by other transition metals. J. Biol. Chem., 273:7127–7133. Chu, W.A., Moehlenkamp, J.D., Bittel, D., Andrews, G.K., and Johnson, J.A., 1999, Cadmium-mediated activation of the metal response element in human neuroblastoma cells lacking functional metal response element-binding transcription factor-1. J. Biol. Chem., 274:5279–5284. Erickson, J.C., Hollopeter, G., Thomas, S.A., Froelick, G.J., and Palmiter, R.D., 1997, Disruption of the metailothionem-III gene in mice: analysis of brain zinc, behavior, and neuron vulnerability to metals, aging, and seizures. J. Neurosci., 17:1271–1281. Gasull, T. and Hidalgo, J., 1996, Evidence for a high molecular weight cytosolic factor that binds brain and liver metallothionein. Neurochem. Res., 21:969–974. Gunes, C., Heuchel, R., Georgiev, O., Muller, K.H., Lichtlen, P., Bluthmann, H., Marino, S., Aguzzi, A., and Schaffner, W, 1998, Embryonic lethality and liver degeneration in mice lacking the metal-responsive transcriptional activator MTF-1. EMBO J., 17:2846–2854. Jasani, B. and Schmid, K.W., 1997, Significance of metallothionein overexpression in human tumours [In Process Citation]. Histopathology, 31:211–214. Jiang, L.J., Maret, W., and Vallee, B.L., 1998, The glutathione redox couple modulates zinc transfer from metallothionein to zinc-depleted sorbitol dehydrogenase [In Process Citation], Proc. Natl. Acad. Sci. U.S.A., 95:3483–3488. Kelly, E.J., Sandgren, E.P., Brinster, R.L., and Palmiter, R.D., 1997, A pair of adjacent glucocorticoid response elements regulate expression of two mouse metallothionein genes. Proc. Natl. Acad. Sci. U.S.A., 94:10045–10050. Klaassen, C.D. and Liu, J., 1998, Metallothionein transgenic and knock-out mouse models in the study of cadmium toxicity [In Process Citation]. J. Toxicol. Sci., 23 Suppl 2:97–102:97–102. Koizumi, S., Suzuki, K., Ogra, Y, Yamada, H., and Otsuka, F., 1999, Transcriptional activity and regulatory protein binding of metal-responsive elements of the human metallothionein-IIA gene. Eur. J. Biochem., 259:635–642. Kondo, Y., Rusnak, J.M., Hoyt, D.G., Settineri, C.E., Pitt, B.R., and Lazo, J.S., 1997, Enhanced apoptosis in metallothionein null cells. Mol. Pharmacol, 52:195–201. Lazo, J.S., Kondo, Y., Dellapiazza, D., Michalska, A.E., Choo, K.H.A., and Pitt, B.R., 1995, Enhanced sensitivity to oxidative stress in cultured embryonic cells from transgenic mice deficient in metallothionein I and II genes. J. Biol. Chem., 270:5506–5510. Lee, D.K., Carrasco, J., Hidalgo, J., and Andrews, G.K., 1999, Identification of a signal transducer and activator of transcription, (STAT) binding site in the mouse metallothionein-I promoter involved in interleukin-6-induced gene expression. Biochem. J, 337:59–65. Liu, J., Liu, Y., Michalska, A.E., Choo, K.H.A., and Klaassen, C.D., 1996a, Distribution and retention of cadmium in metallothionein I and II null mice. Toxicol. Appl. Pharmacol, 136:260–268. Liu, J., Liu, Y.P., Michalska, A.E., Choo, K.H.A., and Klaassen, CD., 1996b, Distribution and retention of cadmium in metallothionein I and II null mice. Toxicol. Appl. Pharmacol, 136:260–268. Mahon, P., Partridge, K., Beattie, J.H., Glover, L.A., and Hesketh, J.E., 1997, The 3´ untranslated region plays a role in the targeting of metallothionein-I mRNA to the perinuclear cytoplasm and cytoskeletal-bound polysomes. Biochim. Biophys. Acta, 1358:153–162. Masters, B.A., Kelly, E.J., Quaife, C.J., Brinster, R.L., and Palmiter, R.D., 1994, Targeted disruption of metallothionein I and II genes increases sensitivity to cadmium. Proc. Natl. Acad. Sci. U.S.A., 91:584–588. Michalska, A.E. and Choo, K.H.A., 1993, Targeting and germ-line transmission of a null mutation at the metallothionein I and II loci in mouse. Proc. Natl. Acad. Sci. U.S.A., 90:8088–8092. Moffatt, P. and Seguin, C., 1998, Expression of the gene encoding metallothionein-3 in organs of the reproductive system [In Process Citation]. DNA Cell Biol., 17:501–510.

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Murphy, B.J., Andrews, G.K., Bittel, D., Discher, D.J., McCue, J., Green, C.J., Yanovsky, M., Giaccia, A., Sutherland, R.M., Laderoute, K.R., and Webster, K.A., 1999, Activation of metallothionein gene expression by hypoxia involves metal response elements and metal transcription factor-1. Cancer Res., 59:1315–1322. Pitt, B.R., Schwarz, M., Woo, E.S., Yee, E., Wasserloos, K., Tran, S., Weng, W., Mannix, R.J., Watkins, S.A., Tyurina, Y.Y., Tyurin, V.A., Kagan, V.E., and Lazo, J.S., 1997, Overexpression of metallothionein decreases sensitivity of pulmonary endothelial cells to oxidant injury. Am. J. Physiol, 273:L856–L865. Quaife, C.J., Kelly, E.J., Masters, B.A., Brinster, R.L., and Palmiter, R.D., 1998, Ectopic expression of metallothionein-III causes pancreatic acinar cell necrosis in transgenic mice [Full text delivery]. Toxicol. Appl. Pharmacol., 148:148–157. Samson, S.L. and Gedamu, L., 1998, Molecular analyses of metallothionein gene regulation. Prog. Nucleic Acid Res. Mol. Biol., 59:257–288. Sato, M., Apostolova, M.D., Hamaya, M., Yamaki, J., Choo, K.H.A., Michalska, A.E., Kodama, N., and Tohyama, C., 1996, Susceptibility of metallothionein-null mice to paraquat. Environ. Toxicol. Pharmacol., 1:221–225. Sewell, A.K., Jensen, L.T., Erickson, J.C., Palmiter, R.D., and Winge, D.R., 1995, Bioactivity of metallothionein-3 correlates with its novel beta domain sequence rather than metal binding properties. Biochemistry, 34:4740–4747. Studer, R., Vogt, C.P., Cavigelli, M., Hunziker, P.E., and Kagi, J.H., 1997, Metallothionein accretion in human hepatic cells is linked to cellular proliferation. Biochem. J., 328:63–67. Thornalley, P.J. and Vasak, M., 1985, Possible role for metallothionein in protection against radiation-induced oxidative stress. Kinetics and mechanism of its reaction with superoxide and hydroxyl radicals. Biochim. Biophys. Acta, 827:36–44. Tohyama, C., Suzuki, J.S., Hemelraad, J., Nishimura, N., and Nishimura, H., 1993, Induction of metallothionein and its localization in the nucleus of rat hepatocytes after partial hepatectomy. Hepatology, 18:1193–1201. Tsujikawa, K., Imai, T., Kakutani, M., Kayamori, Y., Mimura, T., Otaki, N., Kimura, M., Fukuyama, R., and Shimizu, N., 1991, Localization of metallothionein in nuclei of growing primary cultured adult rat hepatocytes. FEBS Lett., 283:239–242. Uchida, Y. and Ihara, Y., 1995, The N-terminal portion of growth inhibitory factor is sufficient for biological activity. J. Biol.Chem., 270:3365–3369. Vasconcelos, H., Tam, S.C., Beattie, J.H., and Hesketh, J., 1995, Evidence for differences in the posttranscriptional regulation of rat metallothionein isoforms. Biochem. J., 314:665–671. Whitacre, C.M., 1996, Application of Western blotting to the identification of metallothionein binding proteins. Anal. Biochem., 234:99–102.

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301

ZINC AS A CELLULAR REGULATOR OF APOPTOSIS

Alain Favier LBSO, Research Laboratory on Biology of Oxidative Stress University of Grenoble Faculty of Pharmacy 38700 La Tronche, France

A large part of the essential effect of zinc has been attributed to its activity as a cofactor of numerous enzymes or proteins involved in DNA replication and gene expression. All these biochemical effects explain the cellular proliferative action of zinc that is necessary to cell growth and division. The disturbance of these mechanisms is for a large part responsive of the defect in the development of animals and organs. But the growth of a tissue is not only the result of the speed in cell proliferation. The size of its cell population results from the balance between cell proliferation and cell death. The concept of programmed cell death recently became preeminent and zinc appears to be as important for this process as it is for cell proliferation. Apoptosis, also named programmed cell death, is not a unique cascade of events but, according to the triggering factor, use different ways that converge to common effects such as activation of caspase, flip-flop of membrane phospholipid, chromatin condensation, DNA cleavage, nucleus and cell shrinkage. The final result is a clean death of the cell, without membrane disruption releasing the cytosol content in the extracellular matrix. The apoptotic cell being finally destroyed by macrophage phagocytosis? Since the pioneer research of Elmes, who observed an increased number of apoptotic bodies in the crypt region of the intestinal mucosa of zinc deficient rat (Elmes, 1977), a lot of cellular and molecular data let us speculate that zinc is involved as a regulator in the control of apoptosis.

1. EFFECT OF ZINC DEFICIENCY The influence of zinc depletion is rather difficult to investigate because zinc is an ubiquitous element very abundant in culture medium and fetal calf serum. For that reason many researchers create zinc depletion by adding a zinc chelator into the culture Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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medium. Such a treatment of thymocytes with the membrane-permeable zinc-chelator TPEN (tetrakis-(2-pyridylmethyl)ethylenediamine) induces nuclear condensation and oligonucleosomal ladders (Mc Cabe et al., 1993). TPEN was demonstrated to decrease the content in zinc when inducing apoptosis in lymphocytes (Treves et al., 1994) and we observed the same decrease of cell zinc in TPEN-treated keratinocytes, with a concomitant release in the culture medium (Fig. 1). At the same time we observed early event of apoptosis, such double DNA strand breaks, after 1 hour, before the appearance of apoptosis (after 6 hours) (Parat et al., 1997a). The chelator induces at the same time a decrease in intracellular glutathione secreted outside of the cell in the culture medium. As presented in Fig. 2, all these effects can be prevented by adding zinc simultaneously with the chelator. We also observed that TPEN-induced zinc deprivation leads to apoptosis in HeLa cells and exacerbates the pro-apoptotic effect of the tat protein of HIV1 (Seve et al., 1999). When treating Hela cells with the metal chelator TPEN we observed after a few hours the occurrence of nuclear fragmentation and a great number of apoptotic corps (Fig. 3). The effect of TPEN in inducing DNA fragmentation and decreasing cell viability occurs within 3 hours in our experiments.

2. EFFECT OF SUPPLEMENTATION WITH ZINC Reversibly, supplemental zinc addition is found able to inhibit various models of apoptosis induced by different agents: mastocytoma cells exposed to cytotoxic T lymphocytes (Duke et al., 1983), thymocytes exposed to dexamethasone, fibrosarcoma cells treated by actinomycin (Fieger et al., 1989), thymocytes treated by NO donors (Fehsel et al., 1995). We observed that addition of zinc in the culture medium prevents the rapid strand breaks of DNA induced by exposure to UVB and partially prevents apoptosis

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as measured by the increase in histone-associated DNA fragments (Parat et al., 1997b). A supplementation with zinc protects also fibroblasts totally from the UV-A induced cell death, which is more related to oxidative stress; when a supplemental selenium gives only a partial protection (Leccia et al., 1993). The total suppression of apoptosis by zinc (Fig. 4), can be observed by supplementing cells before 8 hours after irradiation.

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3. MECHANISMS EXPLAINING THE ANTIAPOPTOTIC EFFECT OF ZINC Zinc has an impressive number of potential metabolic effects, being cofactor of 200 enzymes as well as cofactor of thousands of transcription factors. But some of these effects can be more directly related to the apoptotic pathways. Many actions of zinc can be explained by its antagonistic effect of cellular calcium that is a strong inductor of apoptosis. But zinc seems even more important as Travers demonstrated that the apoptosis of lymphocytes is dependent on the cellular content on zinc and independent to calcium.

3.1. Zinc Enzymes Among the hundred of different zinc enzymes, many are supposed to play a part in the apoptotic process such as proteases or superoxide dismutase. A zinc depletion in cultured epithelial cells deeply decreases activites of catalase, alkaline phosphatase, alpha mannosidase and the level of metallothionein, but has no effect on acid phosphatase, superoxide dismutase or glutathione peroxidase (Tate et al., 1995). We confirmed recently that data by using TPEN. The chelator decreases the zinc and copper content of HaCaT cells by releasing the metals in the culture medium, but don’t modify the Cu-Zn or Mn superoxide dismutase activities.

3.2. Inhibition of Caspases Some recent studies indicate that zinc inhibits caspase-3, a cell death protease implicated in apoptosis (Perry et al., 1997). Furthermore cell line resistant to TPEN-induced apoptosis, present a complete loss of caspase 3 expression demonstrating the strong

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relationship between zinc and caspase3 (Kolenko et al., 1999). We observed that zinc is efficient to prevent cleavage of PARP, procaspase or Sp1 by caspase3. 3.3. Zinc as Antioxidant Oxidative stress is a part of the apoptotic cascade, and many antioxidants prevent this phenomenon (Buttke and Sandstrom, 1994). Zinc as been found to exert an antioxidant effect as well in chemical system as in organelle-based system as in cell or animal models. The mechanisms explaining the effect of zinc are numerous and complex as zinc differently of copper is unable to dismutate or destroy radicals (Favier, 1995). We observed that the TPEN-induced apoptosis is exacerbated by paraquat, a redox cycling generator of superoxide anions. 3.4. Zinc and Transcription Factors Zinc is an essential component in thousand of nuclear proteins involved in transcription of genes. The zinc finger family is a very large group of transcription factors for who zinc is necessary to stabilize all their structural motif. But zinc may also protect these sensitive proteins from oxidation. Zinc can particularly protect nuclear factors involved in the cell cycle and apoptosis as p53, NF-KB or Sp1, which are thiol proteins sensitive to oxidation. The deprivation of the p53 protein from zinc increases its sensitivity to oxidation and suppresses the binding to its consensus sequence on DNA (Hainaut and Milner, 1993). Its efficiency is restored by reducing agents. By treating HeLa cells with the zincchelator TPEN we observed a fast decrease of the activity of the transcription factor Sp1 in the nucleus. Activation of the CD40 receptor at the surface of B lymphocytes induces the zinc-finger protein A20, that inhibits apoptosis (Sarma et al., 1995). The induction of A20 is mediated by the anti-apoptotic factor NF-KB who bind to the A20 promoter. 3.5. Prevention of Megachannels Opening in the Mitochondrion Zinc inhibits calcium movements across mitochondrion membrane and decreases mitochondrial potential. It is supposed to enter by the same uniporter transporter as calcium, and can protect thiol groups of the megachannels that open during apoptosis releasing oxygen radicals, calcium and cytochrome c inside the cytosol (Saris and Niva, 1994). 3.6. Endonuclease of Apoptosis The effect of zinc seems particularly important at the nucleus level. The formation of apoptotic bodies and cleavage of DNA into nucleosomal ladder from isolated nuclei in a cell-free medium is inhibited by zinc ions (Lazebnik et al., 1993). This effects is explained by the total inhibition by zinc of the Ca-Mg dependent endonuclease activity whereas acid endonuclease was only partially inhibited (Beletsky et al., 1989). The DNA fragmentation by endonuclease was inhibited by zinc but also by addition of and (Lohmann and Beyersmann, 1993).

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3.7. Antagonism to Calcium Inside the Nucleus Calcium induces an intracellular acidification that can be blocked by zinc (Morana et al., 1994). Incubation of isolated nuclei with Ca2+ induces DNA fragmentation in a concentration dependent manner (Lohmann and Beyersmann, 1994). Addition of calcium and ATP to liver nuclei caused an important increase in nuclear DNA fragmentation, which is inhibited by zinc (Yamaguchi and Oishi, 1995).

4. REGULATION OF CELLULAR ZINC The relationship between the intracellular content in labile or free zinc and sensitivty to apoptosis has been demonstrated by Zalewski using fluorescent probes for measuring zinc (zinquin) and DNA fragmentation in CLL cells treated with colchicine or thymocytes from aged rats spontaneously apoptotic (Zalewski et al., 1994). It exists an homeostatic control of the free concentration of zinc in the nucleus. The level of free zinc in the nucleus is low comparatively to the extracellular level, suggesting a regulatory process. In contrast with calcium the accumulation of intra nuclear zinc is not ATP dependent and is not inhibited by tharpsigargin a calcium-pump inhibitor (Hechtenberg and Beyersmann, 1993). Using the patch clamp technique we just identified zinc permeant channels inside the inner membrane of nucleus. These channels display following characteristic: conductance 11pS, open time 3.7ms, close time 4.8ms) (Longin et al., 1997).

5. PARADOXAL PRO-APOPTOTIC EFFECT OF ZINC We are to be careful considering the anti-apoptotic effect of zinc first as apoptosis is often useful to eliminate dangerous damaged cell, secondly because zinc has often a biphasic effect in various physiological functions. It is not surprising that in some circumstances it can favor apoptosis. Neurons are particularly sensitive to zinc-induced apoptosis, may be because they contain zinc rich vesicles that are released after ischemia –reperfusion (Koh et al., 1996). Addition of 30 micromolar zinc leads to a mixed apoptotic and necrotic death (Kim et al., 1999).

REFERENCES Beletsky, P., Matyasova, J., Nikonova, L.V., Shalka, M., and Umansky, S.R. 1989 On the role of Ca, Mg-dependent nuclease in the post-irradiation degradation of chromatin in lymphoid tissues. Gen. Physiol. Biophys. 8, 381–398. Buttke, T.M. and Sandstrom, P.A. 1994 Oxidative stress as a mediator of apoptosis. Immunol Today 15, 7–10. Duke, R.C., Chervenak, R., and Cohen, J.J. 1983 Endogenous endonuclease-induced DNA fragmentation: an early event in cell-mediated cytolysis. Proc. Natl. Acad. Sci. U S A 80, 6361–6365. Elmes, M. 1977 Apoptosis in the small intestine of zinc-deficient and fasted rats. J. Pathol. 123, 219–223. Favier, A. 1995 Zinc-ligand interactions and oxygen free radicals formation in,. Handbook on metal-ligand interactions in biological fluids. New-York: Marcel Dekker Inc. Fehsel, K., Kroncke, K., Meyer, K., Huber, H,, Wahn, V., and Kolb-Bachofen, V. 1995 Nitric oxide induces apoptosis in mouse thymocytes. J. Immunol 155, 2858–2865, Fieger, D., Riethmuller, G., and Ziegler-Heitbrock, H. 1989 Zn ++ inhibits both tumor necrosis factor-mediated DNA fragmentation and cytolysis. Int. J. Cancer 44, 315–319.

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Hainaut, P. and Milner, J. 1993 Redox modulation of p53 conformation and sequence-specific DNA binding in vitro. Cancer Res. 53, 4469–473. Hechtenberg, S. and Beyersmann, D. 1993 Differential control of free calcium and free zinc levels in isolated bovine liver nuclei. Biochem. J. 289, 757–760. Kim, Y., Kim, E., Gwag, B., Sohn, S., and Koh, J. 1999 Zinc-induced cortical neuronal death with featuures of apoptosis and necrosis: mediation b y free radicals. Neuroscience 89, 175–182. Koh, J., Suh, S., Gwag, B., He, Y, Hsu, C., and Choi, D. 1996 The role of zinc in selective neuronal death after transient global cerebral ischemia. Science 272, 1àl3–1016. Kolenko, V., Uzzo, R.G., Bukowski, R., Bander, N.H., Novick, A.C., Hsi, E.D., and Finke, J.H. 1999 Dead or dying: necrosis versus apoptosis in capase-deficient human renal cell carcinoma [In Process Citation], Cancer Res. 59, 2838–2842. Lazebnik, Y.A., Cole, S., Cooke, C.A., Nelson, W.G., and Earnshaw, W.C. 1993 Nuclear events in vitro in cell-free mitotic extracts: a model system for analysis of the active phase of apoptosis. J. Cell Biol. 123, 7–22. Leccia, M.T., Richard, M.J., Beani, J.C., Faure, H., Monjo, A.M., Cadet, J., Amblard, P., and Favier, A. 1993 Protective effect of selenium and zinc on UV-A damage in human skin fibroblasts. Photochem. Photobiol. 58, 548–553. Lohmann, R. and Beyersmann, D. 1994 Effects of zinc and cadmium on apoptotic DNA fragmentation in isolated bovine liver nuclei. Env. Health Perspect. 102, 269–271. Lohmann, R.D. and Beyersmann, D. 1993 Cadmium and zinc mediated changes of the Ca(2+)-dependent endonuclease in apoptosis. Biochem. Biophys. Res. Commun 190, 1097–1103. Longin, A.S., Mezin, P., Favier, A., and Verdetti, J. 1997 Presence of zinc and calcium permeant channels in the inner membrane of the nuclear envelope. Biochem. Biophys. Res. Commun 235, 236–241. Mc Cabe, M., Jiang, S., and Orrenius, S. 1993 Chelation of intracellular zinc triggers apoptosis in mature thymocytes. J. Lab. Invest. 69, 101–110. Morana, S., Li, J., Springer, E., and Eastman, A. 1994 The inhibition of etoposide-induced apotosis by zinc is associated with modulation of intracellular pH. Int. J. Oncol. 5, 153–158. Parat, M.O., Richard, M.J., Beani, J.C., and Favier, A. 1997a Involvement of zinc in intracellular oxidant/antioxidant balance. Biol. Trace. Elem. Res. 60, 187–204. Parat, M.O., Richard, M.J., Pollet, S., Hadjur, C., Favier, A., and Beani, J.C. 1997b Zinc and DNA fragmentation in keratinocyte apoptosis: its inhibitory effect in UVB irradiated cells. J. Photochem. Photobiol. 37, 101–106. Perry, D.K., Smyth, M.J., Stennicke, H.R., Salvesen, G.S., Duriez, P., Poirier, G.G., and Hannun, Y.A. 1997 Zinc is a potent inhibitor of the apoptotic protease, caspase-3. A novel target for zinc in the inhibition of apoptosis. J. Biol. Chem. 272, 18530–18533. Saris, N. and Niva, K. 1994 Is Zn2+ transported by the mitochondrial calcium uniporter. FEBS Let. 356, 195–198. Sarma, V., Lin, Z., Clark, L., Rust, M., Tewari, M., Noelle, R.J., and Dixit, V.M. 1995 Activation of the B-cell surface reptor CD40 induces A20, a novel zinc finger protein that inhibits apoptosis. J. Biol. Chem. 2705, 12343–12346. Seve, M., Favier, A., Osman, M., Hernandez, D., Vaitaitis, G., Flores, N.C., McCord, J.M., and Flores, S.C. 1999 The Human Immunodeficiency Virus-1 Tat Protein Increases Cell Proliferation, Alters Sensitivity to Zinc Chelator-Induced Apoptosis, and Changes Spl DNA Binding in HeLa Cells. Arch. Biochem. Biophys. 361, 165–172. Tate, D., Miceli, M., Newsome, D., Alcock, N., and Oliver, P. 1995 Influence of zinc on selected cellular funcions of cultured human retinal pigment epithelium. Curr. Eye. Res., 897–903. Treves, S., Trentini, P.L., Ascanelli, M., and Bucci, G. 1994 Apoptosis is dependent on intracellular zinc and independent of intracellular calcium in lymphocytes. Exp. Cell Res. 211, 339–343. Yamaguchi, M. and Oishi, K. 1995 Effect of nuclear Ca2+ uptake inhibitors on Ca(2+)-activated DNA fragmentation in rat liver nuclei. Mol. Cell Biochem. 148, 33–37. Zalewski, P.D., Forbes, I.J., Seamark, R.F., Borlinghaus, R., Belts, W.H., Lincoln, S.F., and Ward, A.D. 1994 Flux of intracellular labile zinc during apoptosis (gene-directed cell death) revealed by a specific chemical probe, Zinquin. Chem. Biol. 3, 153–161.

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ZINC AND GROWTH

Michael Hambidge and Nancy Krebs Section of Nutrition Department of Pediatrics University of Colorado

While each of the micronutrients that has a role in mammalian physiology is necessary for normal growth and development, none is more intimately involved in these processes than zinc. Results of studies of animal models, ranging from rodents to primates, have emphasised the deleterious effects of zinc deprivation on both pre- and postnatal growth. Recent advances in our understanding of the biology of zinc at a sub-cellular and molecular level have served to illustrate, if not yet completely elucidate, why zinc is so important for growth (Cousins, 1998). Not only are zinc metalloenzymes involved in multiple aspects of nucleic acid metabolism and protein synthesis, it appears that this metal may have an intracellular regulatory role in cellular growth and differentiation. The zinc finger motif of many transcription proteins is likely to be involved in this role and there are, currently, rapid advances in identification of the involvement of zinc in gene expression. Among many other potential examples of how cellular growth and differentiation are dependent on zinc, are the zinc finger motifs of receptors for steroid hormones and maintenance of the integrity of ion channels. It appears that the deleterious effects of zinc on linear, ie. bone, growth may be mediated in part through interference with the effects of IGF-1 at a post-receptor level by loss of the integrity of a calcium ion channel (MacDonald, 1999). This is just one example of how zinc deficiency may impair linear growth. It is also known that bone cell growth and differentiation are diminished by zinc deficiency (Schmidmayer, 1999). This brief review will focus on zinc and growth in the human, outlining our growing appreciation of the prevalence and significance of growth-limiting zinc deficiency in children. Growth failure is a prominent feature of Adolescent Nutritional Dwarfism, which was the focus of the first hypothesis and investigations of human zinc deficiency (Prasad et al., 1961) in the early 1960s. This pioneering research merits further recognition as the Address all correspondence to: Michael Hambidge MD, Box C225, University of Colorado Health Sciences Center, 4200 East Avenue, Denver, Colorado 80262, USA Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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global public health significance of zinc deficiency becomes increasingly apparent. Perhaps surprisingly, however, very little subsequent zinc research has been directed to the adolescent. Later in the 1960s, severe life-threatening zinc deficiency syndromes were identified, the result of one or more inborn errors of zinc metabolism and of the use of intravenous feeds that lacked zinc. Again, growth failure is an early and prominent feature of these syndromes including acrodermatitis enteropathica (Hambidge and Walravens, 1982); adequate zinc therapy results in rapid catch-up growth. In the 1970s and 1980s a small series of placebo-controlled, randomized studies of dietary zinc supplementation were undertaken in Colorado in which growth variables were selected as the principal end-points (Hambidge, 1989). Their purpose was to test the hypotheses that dietary zinc deficiency was present among and was a factor contributing to failure to thrive in otherwise healthy older infants and young children in North America. An important premise, based on earlier observations in animal models (Williams and Mills, 1970) was, and is, that zinc has no pharmacological effect on growth. Therefore, a demonstrable growth response to dietary zinc supplements under placebocontrolled, blinded, randomized study conditions provides prima facie evidence, as in these studies, of a pre-existing growth-limiting zinc deficiency state. A review of data from the most recent of those studies (Walravens et al., 1989) suggested a prevalence of growth-limiting zinc deficiency in otherwise healthy young children in the United States at that time of approximately 3.5%. There is evidence for a similar syndrome elsewhere in North America (Gibson et al., 1989) and it appears that this can still occur (Egger et al., 1999). Our current clinical experience suggests that one group at risk is the older infant or toddler who continues to be breast-fed and does not receive adequate zinc-containing complementary foods. As is the case for iron, breast milk provides only a limited source of zinc after 6 months lactation (Krebs et al., 1994). A typical presenting feature of these subjects, apart from failure to thrive and the dietary history, is a remarkably poor appetite not explicable on the basis of organic disease or family dynamic issues. Zinc supplements have been found previously to increase food intake under placebo-controlled conditions (Krebs et al., 1984). The 1990s have been remarkable for a flurry of large-scale, well designed intervention studies with quite modest zinc supplements in young children in Central and South America, the Indian Sub-continent, Asia and Africa. A major initial incentive for these studies was recognition that major causes of morbidity and mortality in young children in the developing world, notably persistent diarrhea and pneumonia, were associated with growth failure that was not readily explicable by macronutrient deficiencies. Reliance on zinc for the integrity of the immune system, together with either low intakes of zinc or/and high intakes of phytic acid, lent plausibility to the hypothesis that zinc deficiency was a significant etiologic factor. Results of these studies have given strong support to this hypothesis (Bhutta et al., 1999). However, meta-analysis of all the growth data from these and other intervention trials has provided confirmatory evidence for the widespread occurrence of growth-limiting zinc deficiency in young children especially in the developing world (Brown et al., 1998). The latter is important not only in its own right, but because it provides strong evidence that the other concurrent beneficial effects of zinc supplementation, especially those on host defense mechanisms, are also attributable to correction of underlying zinc deficiency. There remains a pathetic lack of adequate biomarkers to detect zinc deficiency in the individual and to estimate its prevalence. Recent estimates, however, (K. Brown,

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personal communication, 1999) have indicated that more than 50% of young children globally are at risk. Not all intervention trials in populations at apparent risk from zinc deficiency have been associated with significant increases in growth velocity, but changes in body composition have been seen in some of these “negative” studies (Kikafunda et al., 1998). One plausible explanation is the likely concurrent occurrence of other micronutrient deficiencies (Solomons et al., 1999). It is apparent that the full potential benefits of correcting zinc deficiency cannot be anticipated in the presence of more generalized malnutrition (beyond any extent to which the latter may be secondary to reduced food intake as a consequence of zinc deficiency). It is remarkable how much has been learned recently about the public health importance of zinc deficiency in young children from single nutrient intervention studies. The growth response to zinc supplements has provided a vital cornerstone to these studies. Finally, it appears that deleterious effects of a limited supply of zinc to the human conceptus, as has previously been observed in animal models, can effect post-natal growth, morbidity and mortality (Castillo Duran et al., 1995; Sazawal et al., 1999). Beneficial effects of very early zinc supplementation of intra-uterine growth retarded infants has been observed. In conclusion, growth retardation (linear growth or/and weight gain) is a very common though not necessarily a universal early feature of mild zinc deficiency states in children. More severe zinc deficiency states occurring before closure of the epiphyses are inevitably associated with growth failure. As the biology of zinc at a molecular level is elucidated, the relative importance of disturbances of different aspects of this complex biology in the etiology of the growth failure of human zinc deficiency are likely to be clarified within the next few years. At the same time, this progress will provide new insights into the variable growth responses to correction of zinc deficiency in children. Apart from improving poor growth, correction of growth-limiting zinc deficiency states has been very useful in confirming the existence of specific deficiency syndromes of this micronutrient and, thence, helping to elucidate their broader role in childhood morbidity and mortality.

REFERENCES Bhutta Z.A., Black R.E., Brown K.H., Gardner J.M., Gore S., Hidayat A., Khatun E, Martorell R., Ninh N.X., Penny M.E., Rosado J.L., Roy S.K., Ruel M., Sazawal S., and Shankar A., 1999, Prevention of Diarrhea and Pneumonia by Zinc Supplementation in Children in Developing Countries: Pooled Analysis of Randomized Controlled Trials. Journal of Pediatrics in press. Brown K.H., Peerson J.M., and Allen L.H., 1998, Effect of Zinc Supplementation on Children’s Growth: A Meta-Analysis of Intervention Trials. Bibl Nutr Dieta 54:76–83. Castillo-Durán C., Rodríquez A., Venegas G., Alvarez P., and Icaza G., 1995, Zinc supplementation and growth of infants born small for gestational age. J Pediatr 127:206–211. Cousins R.J., 1998, A role of zinc in the regulation of gene expression. Proc Nutr Soc (2):307–311. Eberle J., Schmidmayer S., Erben R.G., Stangassinger M., and Roth H.P., 1999, Skeletal effects of zinc deficiency in growing rats. J Trace Elem Med Biol 1–2:21–26. Egger N., Sandstead H., Penland J., Alcock N., Plotkin R., Rocco C., Dayal H., and Zavaleta A., 1999, Zinc supplementation improves growth in Mexican-American children. The FASEB Journal 13:A246. Gibson R.S., Vanderkooy P.D., MacDonald A.C., Goldman A., Ryan B.A., and Berry M., 1989, A growthlimiting, mild zinc deficiency syndrome in some Southern Ontario boys with low height percentiles. Am J Clin Nutr 49:1266–1273. Hambidge K.M., 1989, Mild Zinc Deficiency in Human Subjects. Zinc in Human Biology 281–296.

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Hambidge K.M. and Walravens P.A., 1982, Disorders of mineral metabolism. Clinics of Gastroenterology 11:87–118. Kikafunda J.K., Wlaker A.P., Allan E.F., and Tumwine J.K., 1998, Effect of zinc supplementation on growth and body composition of Ugandan preschool children: a randomized, controlled, intervention trial. Am J Clin Nutr 68:1261–1266. Krebs N.F., Hambidge K.M., and Walravens P.A., 1984, Increased food intake of young children receiving a zinc supplement. Am J Dis Childh 138:270–273. Krebs N.F., Reidinger C.J., Hartley S., Robertson A.D., and Hambidge K.M., 1995, Zinc supplementation during lactation: Effects on maternal status and milk zinc concentrations. Am J Clin Nutr 61:1030–1036. MacDonald R.S., Browning J.D., Wollard-Biddle L., and O’Dell B.L., 1999, Calcium Uptake and Proliferation of IGF-I-Stimulated 3T3 Cells are impaired by DTPA-Induced zinc deprivation. The FASEB Journal 13:A570. Prasad A.S., Halsted J.A., and Nadimi M., 1961, Syndrome of iron deficiency anemia, hepatosplenomegaly, hypogonadism, dwarfism and geophagia. Am J Med 31:532. Sazawal S., Black R.E., Menon V.P., Dhingra U., Dhingra P., Mazumder S., Caulfleld L., and Khosla S., 1999, Effects of Zinc and Mineral Supplementation in Small for Gestational Age Infants on Growth and Mortality. The FASEB Journal 13:A376. Solomons N.W., 1999, Single-nutrient intervention with zinc. Am J Clin Nutr 65:111–112. Walravens P.A., Hambidge K.M., and Koepfer D.M., 1989, Zinc supplementation in infants with a nutritional pattern of failure to thrive: a double-blind, controlled study. Pediatrics 83:532–538. Williams R.B. and Mills C.F., 1970, The Experimental Production of Zinc Deficiency in the Rat. British Journal of Nutrition 24:989.

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ZINC NUTRITURE AS RELATED TO BRAIN Harold H. Sandstead1, Christopher J. Frederickson2, and James G. Penland3 1

Division of Human Nutrition Department of Preventive Medicine and Community Health 2 Center for Bioengineering Department of Neuroscience the University of Texas Medical Branch Galveston, Texas 77555, and 3 the US Department of Agriculture Agricultural Research Service Grand Forks Human Nutrition Research Center Grand Forks, North Dakota, 58201

Zn’s effect on neurotransmission is in part related to its role in a special class of glutaminergic neurons that have Zn containing vesicles in their presynaptic terminals. With a few exceptions, such as certain cerebrocortical systems, these neurons are located in the telencephalon (Frederickson and Moncrieff, 1994). Glutaminergic systems that do not have Zn containing vesicles include the brain stem, thalamus, and cerebellum. Within the telencephalon, fiber systems with Zn containing vesicles form an associational network that reciprocally interconnects isocortical, allocortical and ‘limbic’ structures. Hippocampal, amygdalar, and perirhinal regions are prominent components of this network. In hippocampus about 8% of Zn in the hippocampus is in vesicles (Frederickson et al., 1982). Vesicle uptake and release of Zn is impulse-dependent (Howell et al., 1984). Uptake by vesicles is facilitated by a Zn-transporter (ZnT-3) membrane protein (Palmiter et al., 1996). Zn released from vesicles modulates the excitability of postsynaptic N-methyl-D-aspartate (NMDA)-specific (Peters et al., 1987) receptors for glutamate in a dose-dependent and reversible manner. Dietary Zn deficiency decreases the number of glutamate activated NMDA mediated calcium channels in post-synaptic terminals (Browning and O’Dell, 1995). In addition reversible chelation of Zn in the hippocampus impairs spatial-working-memory (Frederickson et al., 1990). Processes that control release of vesicle Zn are disrupted by seizures (Frederickson et al., 1989) or ischemia (Tender et al., 1990). Subsequent post-synaptic Zn uptake causes post-synaptic neuronal degeneration. Excess Zn released from vesicles also enters the extra-cellular fluid space (Perez-Clausell and Danscher, 1986). In vitro studies show that Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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high concentrations of Zn destabilize amyloid protein precursor and A-beta-l–40 in the extra-cellular fluid, to form amyloid (Bush et al., 1994). Metal binding proteins, metalothioneins 1, 2, and 3, sequester Zn (Gasull et al., 1994; Hao et al., 1994; Masters et al., 1994). In vitro studies have shown that oxidation of MT by glutathione disulfide (GSSG) releases Zn to Zn binding ligands and that reduced glutathione (GSH) and ATP facilitate the process (Jiang et al., 1998a; Jiang et al., 1998b). Certain selenium compounds also oxidize MT and release Zn (Jacob et al., 1999). Relevant to neurotransmission, Zn released from MT is bound by ATP. Zn-ATP reacts with pyridoxal kinase and pyridoxine to form pyridoxal-5-phosphate (PLP) (Churchich et al., 1989) the co-enzyme for synthesis of biogenic amines (Dakshinamurti et al., 1990). Zn-ATP also reacts with flavo-kinase and riboflavin to form FMN the precursor of FAD (Yamada et al., 1990). FAD is the co-enzyme for MAO degradation of biogenic amines (Hsu et al., 1988). Zn nutriture affects growth and development of brain. The mechanisms include synthesis of nucleic acids and proteins (Duerre et al., 1977; Fosmire et al., 1974; Lieberman and Ove, 1962; Sandstead et al., 1975). Zn deprivation in early gestation causes neural tube defects (Hurley and Swenerton, 1966; Swenerton et al., 1969; Warkany and Petering, 1972). Zn deprivation during late gestation (McKenzie et al., 1975) and postnatal development impair brain growth (Buell et al., 1977) and cause histologic teratology (Dvergsten, 1984). Abnormalities include a 60% decrease in the number of granule cells relative to Purkinje cells and an associated decrease in the dendritic growth of Purkinje, basket and stellate cells. Height of the dendrite arbor is reduced and there were fewer branches. The number of asymmetric synapses between parallel fibers (axons of granule cells) and dendrites of the Purkinje, basket and stellate cells are decreased about 40%. Severely Zn deficient rats display poor performance of a simple water maze (Caldwell et al., 1970; Hesse et al., 1979) and impairs mossy fiber evoked potentials (Hesse, 1979). Moderate acute Zn deprivation impairs complex behaviors of rats (Massaro et al., 1982). In rhesus monkeys “moderate” Zn deprivation impairs prepubertal and adolescent behaviors such as activity, attention, and memory tasks before growth is impaired and other overt signs of deficiency are evident (Golub et al., 1995). Zn deprivation during brain development adversely affects subsequent adult behaviors (Golub et al., 1995; Halas and Eberhardt, 1987). Abnormalities in rat offspring include poor maze learning, poor shock avoidance, increased aggression after shock, and poor working memory of a radial maze task. In rhesus monkeys mild Zn deprivation throughout gestation and the first year of life caused lethargy, apathy, and hypo-activity (Golub et al., 1995). Severe Zn deprivation in humans impairs taste and smell, and causes ataxia and abnormal mentation including depression, hallucinations and paranoia (Henkin et al., 1975). Patients with acrodermatitis enteropathica may behave abnormally (Moynahan, 1976) and patients given insufficient Zn in long term intravenous or oral feeds may show abnormal behaviors (Kay et al.,1976; Pekarek et al., 1979). Severe maternal Zn deficiency during gestation from acrodermatitis enteropathica has been associated with brain malformations (Hambidge et al., 1975). Findings from Turkey suggest low maternal Zn status during gestation may be associated with fetal anencephaly (Çavdar et al., 1983; Çavdar et al., 1988). Limited data suggest postnatal performance of infants can be related to maternal diets during gestation. In Egypt infants of women whose diets contained more animal protein and Zn had higher scores on the Brazelton Neonatal Development Assessment

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Scales soon after birth (Kirksey et al., 1991). Six months later their motor development, by the Bayley Scales of Infant Development, was inversely related to maternal intake of diets that were low in animal protein and Zn (Kirksey et al., 1994). Limited observations in infants suggest Zn nutriture affects performance. For example, subjects from a group of 52 very-low-birth-weight (<1,500g) infants who were given 11 mg Zn/L in formula displayed superior growth and motor development (Griffiths Developmental Assessment Test) compared to subjects given 6.7mg (Friel et al., 1993). In another study 205 low birth weight low-income Brazilian infants given placebo, 1 mg, or 5 mg Zn 6 days per week for the first 8 weeks of life had similar scores on Bayley Scales of Infant Development at 6 and 12 months of age (Ashworth et al., 1998). However at 12-months of age the five behavior ratings were significantly higher in the infants who had been supplemented with 5mg Zn. In a third study, a double-blind randomized controlled trial (RCT) of 85 Guatemalan infants, aged 6–9 months, found similar effects on motor development after placebo or 10 mg Zn daily for 7 months (Bentley et al., 1997). However after 7 months the Zn-group sat up, played more, and was less likely to cry or whine. Few studies of children have been reported. In about 150 children from the Eastern Shore of Maryland, the concentration of Zn in hair was directly related to reading performance and frontal lobe EEG coherence (Thatcher et al., 1984). Three double blind RCTs found that Zn repletion, given with other potentially limiting nutrients, improved neuropsychological function (see details below). In contrast two double blind RCTs found no improvement in performance of tasks that focused on attention (Cavan et al., 1993; Gibson et al., 1989). One suspects that limitations in the methods for assessment contributed to these findings. The first RCT, above, studied 740 low-income urban Chinese children, aged 6–9 (Sandstead et al., 1998). The ZnM group had fewer missed targets and more hits than either Zn or M groups on the Continuous Vigilance task (a measure of sustained attention). The ZnM group needed fewer trials and less time to learn concepts on the Oddity task (a measure of reasoning). The ZnM group had more time on targets following the most difficult trajectories on the Tracking task (a measure of eye-hand coordination). Both the Zn and ZnM groups had more taps on multiple-key sequences than did the M groups on the Tapping task (a measure of gross motor speed, fatigue and anticipation). The ZnM group had more taps than M, but not Zn, on single key trials that measured fine motor speed and fatigue. The second RCT studied 540 low – income semi-rural Chinese children aged 6–9 years (Penland, 1999). The findings were generally similar to those in the urban children. The third RCT measured neuropsychological performance in 240 low-income Mexican-American children, aged 6–9 years (Penland et al., 1999). The tasks were similar to those administered in China. In contrast to the Chinese subjects only one task was significantly affected. Greatest improvement (decrease in number of errors) in the reasoning task (solving oddity problems) occurred after treatment with ZnM. The ANOVA for interaction, change x treatment, was significant (p = 0.041). Changes by treatment were P, –10%; M, –11%; ZnM, –27%; FeM, –10%. Change after ZnM was greater than all others (p < 0.03). Likely causes for the differences from China include less severe Zn deficiency, and the smaller number of subjects. Few studies examined effects of Zn nutriture on neuropsychological functions of adults. An example is a double-blind randomized depletion-repletion study of 11 men who lived under highly controlled conditions (Penland, 1991). In random order they were fed 1, 2, 3, or 4mg Zn daily for intervals of 35 days. Then they were repleted with 10mg

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Zn daily for 35 days. During depletion performance of 9 of 15 neuropsychological tasks deteriorated. Effects of the four low Zn diets were similar. Relevant to the elderly Burnet (Burnet, 1981) suggested low Zn nutriture can contribute to the occurrence of dementia. He based his thesis on Zn’s essentiality for synthesis and repair of DNA. Supportive of this thesis Tully et al. (Tully et al., 1995) found in 12 elderly women that their plasma Zn concentrations one year before death were significantly inversely related to the number of senile and other plaques present in their brains at autopsy.

ACKNOWLEDGMENT Research support was provided by the International Lead Zinc Research Organization, the Thrasher Research Foundation, the Gerber Company Foundation, the General Nutrition Products Company and the US Department of Agriculture. LABCATAL supported the first author’s participation in the meeting.

REFERENCES Ashworth, A., Morris, S.S., Lira, P.I., and Grantham-McGregor, S.M. (1998). Zinc supplementation, mental development and behavior in low-birth-weight-term-infants in northeast Brazil. Eur. J. Clin. Nutr. 52, 223–227. Bentley, M.E., Caulfield, L.E., Ram, M., Santizo, M.C., Hurtado, E., Rivera, J.A., Ruel, M.T., and Brown, K.H. (1997). Zinc supplementation affects the activity patterns of rural Guatemalan infants. J. Nutr. 127, 1333–1338. Browning, J. and O’Dell, B. (1995). Zinc deficiency decreases the concentration of N-methyl-D-aspartate receptors in guinea pig cortical synaptic membranes. J. Nutr. 125, 2083–2089. Buell, S.J., Fosmire, G.J., Ollerich, D.A., and Sandstead, H.H. (1977). Effects of postnatal zinc deficiency on cerebellar and hippocampal development in the rat. Exp. Neurol 55, 199–210. Burnet, F.M. (1981). A possible role of zinc in the pathology of dementia. Lancet 1, 186–188. Bush, A.I., Pettingell, W.H., Multhaup, G., d Paradis, M., Vonsattel, J.P., Gusella, J.F., Beyreuther, K., Masters, C.L., and Tanzi, R.E. (1994). Rapid induction of Alzheimer A beta amyloid formation by zinc [see comments]. Science 265, 1464–1467, Caldwell, D.F., Oberleas, D., Clancy, J.J., and Prasad, A.S. (1970). Behavioral impairment in adult rats following acute zinc deficiency. Proc, Soc, Exp. Biol Med, 133, 1417–1421. Cavan, K.R., Gibson, R.S., Grazioso, C.F., Isalgue, A.M., Ruz, M., and Solomons, N.W. (1993). Growth and body composition of periurban Guatemalan children in relation to zinc status: a cross-sectional study. Am. J. Clin. Nutr. 57, 334–343. Çavdar, A.O., Babacan, E., Asik, S., Arcasoy, A., Ertem, U., Himmetoglu, O., Baycu, T., and Akar, N. (1983). Zinc levels of serum, plasma, erythrocytes and hair in Turkish women with anencephalic babies. Prog. Clin. Biol. Res. 129, 99–106. Çavdar, A.O., Bahceci, M., Akar, N., Erten, J., Bahceci, G., Babacan, E., Arcasoy, A., and Yavuz, H. (1988). Zinc status in pregnancy and the occurrence of anencephaly in Turkey. J. Trace. Elem. Electrolytes Health Dis. 2, 9–14. Churchich, J., Scholz, G., and Kwok, F. (1989). Activation of pyridoxal kinase by metallothionein. Biochim. Biophys. Acta. 996, 181–186. Dakshinamurti, K., Paulose, C., Viswanathan, M., Siow, Y., and Sharma, S. (1990). Neurobiology of pyridoxine. Ann. NY Acad. Sci. 585, 128–144. Duerre, J.A., Ford, K.M., and Sandstead, H.H. (1977). Effect of zinc deficiency on protein synthesis in brain and liver of suckling rats. J. Nutr. 107, 1082–1093. Dvergsten, C. (1984). Retarded synaptogenesis and differentiation of cerebellar neurons in zinc-deficient rats. In The Neurobiology of Zinc Part B: deficiency, Toxicity, and Pathology, vol. 11 (ed. C. Fredericksen, G. Howell and E. Kasarkis), pp. 17–31. New York: Alan R Liss, Inc.

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Fosmire, C.J., al-Ubaidi, Y.Y., and Sandstead, H.H. (1974). The effect of zinc deprivation on the brain. Adv. Exp. Med Biol. 48, 329–345. Frederickson, C.J., Hernandez, M.D., and McGinty, IF. (1989). Translocation of zinc may contribute to seizure-induced death of neurons. Brain. Res. 480, 317–321. Frederickson, C.J., Manton, W.I., Frederickson, M.H., Howell, G.A., and Mallory, M.A. (1982). Stable-isotope dilution measurement of zinc and lead in rat hippocampus and spinal cord. Brain. Res. 246, 338–341. Frederickson, C.J. and Moncrieff, D.W. (1994). Zinc-containing neurons. Biol. Signals 3, 127–139. Frederickson, R.E., Frederickson, C.J., and Danscher, G. (1990). In situ binding of bouton zinc reversibly disrupts performance on a spatial memory task. Behav. Brain. Res. 38, 25–33. Friel, J.K., Andrews, W.L., Matthew, J.D., Long, D.R., Cornel, A.M., Cox, M., McKim, E., and Zerbe, G.O. (1993). Zinc supplementation in very-low-birth-weight infants. J. Pediatr. Gastroenterol. Nutr. 17, 97–104. Gasull, T., Giralt, M., Hernandez, J., Martinez, P., Bremner, I., and Hidalgo, J. (1994). Regulation of metallothionein concentrations in rat brain: effect of glucocorticoids, zinc, copper, and endotoxin. Am. J. Physiol. 266, E760–E767. Gibson, R.S., Vanderkooy, P.D., MacDonald, A.C., Goldman, A., Ryan, B.A., and Berry, M. (1989). A growthlimiting, mild zinc-deficiency syndrome in some southern Ontario boys with low height percentiles. Am. J. Clin. Nutr. 49, 1266–1273. Golub, M.S., Keen, C.L., Gershwin, M.E., and Hendrickx, A.G. (1995). Developmental zinc deficiency and behavior. J. Nutr. 125, 2263S–2271S. Halas, E.S. and Eberhardt, M.J. (1987). A behavioral review of trace element deficiencies in animals and humans. Nutr. Behav. 3, 257–271. Hambidge, K.M., Neldner, K.H., and Walravens, P.A. (1975). Letter: Zinc, acrodermatitis enteropathica, and congenital malformations. Lancet 1, 577–578. Hao, R., Cerutis, D.R., Blaxall, H.S., Rodriguez-Sierra, J.F., Pfeiffer, R.F., and Ebadi, M. (1994). Distribution of zinc metallothionein I mRNA in rat brain using in situ hybridization. Neurochem. Res. 19, 761–767. Henkin, R.I., Patten, B.M., Re, P.K., and Bronzert, D.A. (1975). A syndrome of acute zinc loss. Cerebellar dysfunction, mental changes, anorexia, and taste and smell dysfunction. Arch. Neurol. 32, 745–751. Hesse, G.W. (1979). Chronic zinc deficiency alters neuronal function of hippocampal mossy fibers. Science 205, 1005–1007. Hesse, G.W., Hesse, K.A., and Catalanotto, F.A. (1979). Behavioral characteristics of rats experiencing chronic zinc deficiency. Physiol. Behav. 22, 211–215. Howell, G.A., Welch, M.G., and Frederickson, C.J. (1984). Stimulation-induced uptake and release of zinc in hippocampal slices. Nature 308, 736–738. Hsu, Y.P., Weyler, W., Chen, S., Sims, K.B., Rinehart, W.B., Utterback, M.C., Powell, J.F., and Breakefield, X.O. (1988). Structural features of human monoamine oxidase A elucidated from cDNA and peptide sequences. J. Neurochem. 51, 1321–1324. Hurley, L.S. and Swenerton, H. (1966). Congenital malformations resulting from zinc deficiency in rats. Proc. Soc. Exp. Biol. Med. 123, 692–696. Jacob, C., Maret, W., and Vallee, B.L. (1999). Selenium redox biochemistry of zinc-sulfur coordination sites in proteins and enzymes. Proc. Natl. Acad. Sci. USA 96, 1910–1914. Jiang, L.J., Maret, W., and Vallee, B.L. (1998a). The ATP-metallothionein complex. Proc. Natl. Acad. Sci. USA 95, 9146–9149. Jiang, L.J., Maret, W., and Vallee, B.L. (1998b). The glutathione redox couple modulates zinc transfer from metallothionein to zinc-depleted sorbitol dehydrogenase. Proc. Natl. Acad. Sci. USA 95, 3483–3488. Kay, R.G., Tasman-Jones, C., Pybus, J., Whiting, R., and Black, H. (1976). A syndrome of acute zinc deficiency during total parenteral alimentation in man. Ann. Surg. 183, 331–340. Kirksey, A., Rahmanifar, A., Wachs, T.D., McCabe, G.P., Bassily, N.S., Bishry, Z., Galal, O.M., Harrison, G.G., and Jerome, N.W. (1991). Determinants of pregnancy outcome and newborn behavior of a semirural Egyptian population. Am. J. Clin. Nutr. 54, 657–667. Kirksey, A., Wachs, T.D., Yunis, F., Srinath, U., Rahmanifar, A., McCabe, G.P., Galal, O.M., Harrison, G.G., and Jerome, N.W. (1994). Relation of maternal zinc nutriture to pregnancy outcome and infant development in an Egyptian village. Am. J. Clin. Nutr. 60, 782–792. Lieberman, I. and Ove, P. (1962). Deoxyribonucleic acid synthesis and its inhibition in mammalian cellscultured from the animal. J. Biol. Chem. 237, 1634–1642. Massaro, T.F., Mohs, M., and Fosmire, G. (1982). Effects of moderate zinc deficiency on cognitive performance in young adult rats. Physiol. Behav. 29, 117–121. Masters, B.A., Quaife, C.J., Erickson, J.C., Kelly, E.J., Froelick, G.J., Zambrowicz, B.P., Brinster, R.L., and Palmiter, R.D. (1994). Metallothionein III is expressed in neurons that sequester zinc in synaptic vesicles. J. Neurosci. 14, 5844–5857.

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McKenzie, J.M., Fosmire, G.J., and Sandstead, H.H. (1975). Zinc deficiency during the latter third of pregnancy: effects on fetal rat brain, liver, and placenta. J. Nutr. 105, 1466–1475. Moynahan, E.J. (1976). Letter: Zinc deficiency and disturbances of mood and visual behavior. Lancet 1, 91. Palmiter, R.D., Cole, T.B., Quaife, C.J., and Findley, S.D. (1996). ZnT-3, a putative transporter of zinc into synaptic vesicles. Proc. Natl. Acad. Sci. USA 93, 14934–14939. Pekarek, R.S., Sandstead, H.H., Jacob, R.A., and Barcome, D.F. (1979). Abnormal cellular immune responses during acquired zinc deficiency. Am. J. Clin. Nutr. 32, 1466–1471. Penland, J.G. (1991). Cognitive performance effects of low zinc (Zn) intakes in healthy adult men. FASEB J. 5, A938, abstract. Penland, J.G. (1999). Behavioral Data and Methodology Issues in Studies of Zinc Nutrition in Humans. FASEB J, in press. Penland, J.G., Sandstead, H.H., Egger, N.G., Dayal, H.H., Alcock, N.W., Plotkin, R., Rocco, C., and Zavaleta, A. (1999). Zinc, iron and micronutrient supplementation effects on cognitive and psychomotor function of Mexican-American school children. FASEB J. 13, A921, abstract 683.4. Perez-Clausell, J. and Danscher, G. (1986). Release of zinc sulphide accumulations into synaptic clefts after in vivo injection of sodium sulphide. Brain. Res. 362, 358–361. Peters, S., Koh, J., and Choi, D.W. (1987). Zinc selectively blocks the action of N-methyl-D-aspartate on cortical neurons. Science 236, 589–593. Sandstead, H.H., Fosmire, G.J., McKenzie, J.M., and Halas, E.S. (1975). Zinc deficiency and brain development in the rat. Fed Proc. 34, 86–88. Sandstead, H.H., Penland, J.G., Alcock, N.W., Dayal, H.H., Chen, X.C., Li, J.S., Zhao, F., and Yang, J.J. (1998). Effects of repletion with zinc and other micronutrients on neuropsychologic performance and growth of Chinese children. Am. J. Clin. Nutr. 68, 470S–475S. Swenerton, H., Shrader, R., and Hurley, L.S. (1969). Zinc-deficient embryos: reduced thymidine incorporation. Science 166, 1014–1045. Thatcher, R.W., McAlaster, R., Lester, M.L., and Cantor, D.S. (1984). Comparisons among EEG, hair minerals and diet predictions of reading performance in children. Ann. NY Acad. Sci. 433, 87–96. Tonder, N., Johansen, F.F., Frederickson, C.J., Zimmer, J, and Diemer, N.H. (1990). Possible role of zinc in the selective degeneration of dentate hilar neurons after cerebral ischemia in the adult rat. Neurosci. Lett. 109, 247–252. Tully, C.L., Snowdon, D.A., and Markesbery, W.R. (1995). Serum zinc, senile plaques, and neurofibrillary tangles: findings from the Nun Study. Neuroreport. 6, 2105–2108. Warkany, J. and Petering, H.G. (1972). Congenital malformations of the central nervous system in rats produced by maternal zinc deficiency. Teratology 5, 319–334. Yamada, Y., Merrill, A.H., Jr., and McCormick, D.B. (1990). Probable reaction mechanisms of flavokinase and FAD synthetase from rat liver. Arch. Biochem. Biophys. 278, 125–130.

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ZINC IN PRETERM AND TERM INFANTS Parallel Studies on Metabolic Balances and Plasma Concentrations

E. Sievers, U. Schleyerbach, *T. Arpe, *D. Garbe-Schönberg, and J. Schaub Department of Pediatrics *Institute of Geology University of Kiel, Kiel Germany

Most infant formulas are supplemented with zinc (Zn). The capacity of very low birthweight preterm infants to retain Zn and the optimal concentration in their formulas are still controversial. Due to the very rapid postnatal growth the supply with essential nutrients is of special importance for their optimal development. Therefore Zn retention and Zn plasma concentration were investigated longitudinally in preterm infants and compared to results of healthy term infants.

1. METHODS AND RESULTS In three consecutive Zn balance studies over 72 hours preterm infants were fed different formulas appropriate for their respective age: I. Formula with hydrolyzed protein (Alfaré®, Nestlé Co.); II. Preterm infant formula (Prematil®, Milupa Co.); III. Adapted infant formula (PreAptamil® Milupa Co.). Healthy term infants were fed either human milk or formula III. All specimens (formulas, human milk, drugs, faeces, urine etc.) were analysed by atomic absorption spectrometry. At the balance studies I and III, plasma specimens of the majority of the infants were analysed by high resolution inductively coupled plasma mass spectrometry (ICP-MS; Institute of Geology). No clinical signs of Zn deficiency were observed in the infants studied. The range of the results in preterm infants exceeded that of term infants by far (Table 1). Negative Adress all correspondence to: Dr. Erika Sievers, Dept. of Pediatrics, Univ. of Kiel, 24105 Kiel, Germany, telephone 49-431-5971700, FAX 49-431-5971831 Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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results were obtained for some of the balance studies. Breast-fed infants had the lowest Zn intake and retention. Extremely negative balance results (Table 1) were mainly attributable to higher fecal Zn excretion. The median plasma concentration of breast-fed was the highest in the groups studied and differed significantly from preterm infants at the comparable gestational age (p < 0.05, Table 1).

2. DISCUSSION The Zn intake of infants fed supplemented formulas may exceed the absolute intake by breast-milk (Salmenperä et al., 1994). It is dependent on the choice of the product, only small variations are to be expected with time unless the formula is changed. Due to the decline of the Zn concentration during lactation, the Zn intake observed at one investigation period cannot be representative for the Zn intake by human milk during infancy (Sievers et al., 1992). Zn concentrations in the plasma of breast-fed and formula-fed infants did not differ significantly despite negative results in the balance studies of the breast-fed infants. Three extreme values in this group are attributable to excessive fecal Zn excretion. Though this is suggestive of contamination during the collecting procedures, we were not able to find evidence for this. It is known that Zn resorption from human milk is better than from infant formulas (Sandström et al., 1983). Adequate Zn retention from infant formulas can be achieved if they are supplemented sufficiently (Table 1, Sievers et al., 1992). Plasma concentrations observed in the term groups were in accordance with the literature (Rossipal et al., 1998). The range of the plasma Zn concentrations of preterm infants in this study urges investigations focussing on the Zn supply and the clinical course of the infants with the lowest results. They may be due to insufficient nutritional support or the severeness of the diseases during the preceeding period of life. Clinical symptoms of nutritional zinc deficiency in preterm infants are well known (Connors et al., 1983). Though prevention by adequate Zn supply seams to be easy, typical severe clinical deficiency symptoms were

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recently again observed in a very low birth weight infant (Obladen et al., 1998). Zn supplementation of formulas can result in retention and thus may improve the Zn status of preterm infants. It has been shown, that formulas containing 4.8–6.1mg/l Zn resulted in a median retention of 0.33 mg/kg (Table 1). Supplementation up to 12.5 mg/l lead to a daily retention of 0.66 mg/kgZn (Tyrala 1986). In view of the results observed in breast-fed infants this seems to be excessive. However, in preterm infants supplementation has to take into consideration the possibility of interactions with other minerals and trace minerals in the nutrition. As no increase of plasma Zn was observed within the study period, it is likely, that preterm infant formulas should contain at least 5–6 mg/l. In summary, supplementation of preterm infant formulas is supported: The lowest Zn concentration in plasma was observed in preterm infants initially and Zn is retained from the formulas fed. Despite the lower Zn intake, however, breast-feeding resulted in plasma concentrations comparable to feeding the supplemented formulas. This may be indicative of differences in the Zn metabolism of the groups studied.

ACKNOWLEDGMENTS The authors are especially thankful to the families participating in the project. The study has been funded by the Deutsche Forschungsgemeinschaft (Si 514-1), formulas were supplied by the companies Milupa and Nestlé, Germany. This support is greatfully acknowledged by the authors.

REFERENCES Connors, T.J., Czarnecki, D.B., and Haskett, M.I., 1983, Aquired zinc deficiency in a breast-fed premature infant. Arch. Dermatol. 119:319–321. Krebs, N.F., Reidinger, C.J., Miller, L.V., and Hambidge, K.M., 1996, Zinc homeostasis in breast-fed infants. Pediatr. Res. 39:661–665. Obladen, M., Loui, A., Kampmann, W., and Renz, H., 1998, Zinc deficiency in rapidly growing preterm infants. Acta. Paediatr. 87:685–691. Rossipal. E., Krachler M., and Micetic-Turk, D., 1998, Concentrations of trace elements in sera of young infants fed breast milk or infant formula, in: Metal Ions in Biology and Medicine (P. Collera, V. Negretti de Brätter, I. Khassanova, J.C. Etienne, and J. Libbey, eds.): Band 5, Eurotext, Paris pp. 511–515, Eurotext, Paris, France. Salmenperä, L., Perheentupa, J., Pakarinen, P., and Siimes, M.A., 1994, Zinc supplementation of infant formula. Am. J. Clin. Nutr. 59:985–989. Sandström, B., Cederblad, A., and Lönnerdal, B., 1983, Zinc absorption from human milk, cow’s milk, and infant formulas. Am. J. Dis. Child. 137:726–729. Sievers, E., Oldigs, H.D., Dörner, K., and Schaub, J., 1992, Longitudinal zinc balances in breast-fed and formula-fed infants. Acta. Paediatr. 81:1–6. Tyrala, E.E., 1986, Zinc and copper balances in preterm infants. Pediatrics 77:513–517.

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ACTIVITY OF CALMODULIN-REGULATED ENZYMES IN TISSUES OF ZINC-DEFICIENT RATS

H.-P. Roth, Schmidmayer, S., and Kirchgessner, M. Institute of Nutrition Physiology Technical University Munich D-85350 Freising-Weihenstephan Germany

1. INTRODUCTION Intracellular functions of calcium ions are mediated primarily by calmodulin. This small intracellular calcium-binding protein possesses four binding domains for calcium ions; two with a high and two with a low affinity. An increase in cytosolic free calcium causes a concentration dependent occupation of the binding domains followed by a conformational change in the protein structure as the result that the enzyme is transformed from an inactive to an active form. This activated calcium-calmodulin complex has a high affinity for a variety of cellular target proteins including key regulatory enzymes which increase their activity state after calmodulin binding. Such calcium-calmodulin dependent or regulated enzymes are for example phosphodiesterase, adenylate cyclase, membrane-bound Ca-Mg-ATPase and phosphorylase kinase. Although calmodulin is considered to be calcium-specific Baudier et al. (1984) were able to show, that calmodulin can bind zinc ions with varying affinity, which suggested that zinc also modulates calmodulin activity. We previously demonstrated (Roth and Kirchgessner, 1991) that calmodulin activity was indeed affected in zinc-deficient rats. However, the alterations showed tissue specificity. Whereas alimentary zinc deficiency significantly reduced calmodulin activity in muscle by 42% and in testes by 80% no effects were obtained in rat brain. The present study was designed to investigate whether calmodulin-dependent enzymes also show this tissue specific alteration in activity state in rats submitted to an alimentary zinc deficiency. We therefore determined the activity of the calmodulin-regulated enzymes Ca-MgATPase in erythrocyte membranes, adenylate cyclase in liver, phosphodiesterase in liver, kidney, heart, brain and testes and phosphorylase kinase in muscle, liver, heart and brain. Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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2. MATERIAL AND METHODS 36 male Sprague-Dawley rats with an average weight of 110–120g were divided into 3 groups of 12 animals each. Two groups of rats were fed ad libitum an AIN-93G based diet with casein as source of protein made either deficient in zinc (1mgZn/kg diet) or providing full zinc supply (60 mgZn/kg diet). A third group, named pair-fed group, received the zinc supplemented diet but restricted to the average feed intake of the zincdeficient rats. The rats were housed in an fully air-conditioned room at 23 °C, 60% relative humidity and on a 12-hour light-dark cycle. After 42 experimental days all animals, having been fasted for 10 h, were anesthetized with ether, decapitated and dissected. Sample of plasma, femur, liver, kidney, heart, brain, testes and skeleteal muscle (quadriceps) were stored at –20 °C until analyzed.

3. RESULTS AND DISCUSSION 3.1. Growth All three groups showed typical curves of weight gain. While the control animals gained on average weight of 6.3 g/day, the zinc-depleted animals showed a sharp growth depression from day 7 onwards, with an average weight gain of only 0.4 g/d. The pairfed control animals responded four days later with an reduced weight gain of 4.2 g/d. As the diet was provided in restrictive amounts a normal physiological growth was not possible but still was substantially better than that of zinc-deficient rats. Starting with an average live mass of 109 g at the beginning of the experiment, control rats reached a mean live mass of 372 g during the 42-d study period, the pair-fed control rats 254 g, whereas the zinc-deficient animals only grew to 156 g. The first zinc deficiency symptoms like sparse and rough hair could be noticed already after two weeks on the zinc-deficient diet.

3.2. Zn Supply Status The zinc supply status of the rats was estimated with reference to the activity of alkaline phosphatase in the plasma and the zinc content in plasma and femur. The zinc concentration in plasma of the zinc-depleted rats was on average 75% lower than that of both control groups (1.24 ± 0.08 and Likewise was the femur zinc content reduced significantly by 80% in comparison with the control animals (241 ± 18 and The alkaline phosphatase activity in the plasma of the zinc-deficient rats (229 ± 59 mU/ml) was reduced significantly by 56% when compared to control rats (533 ± 88 and 516 ± 118 mU/ml). This distinct differences in the three parameters of zinc status clearly established the presence of a manifest zinc deficiency in depleted animals.

3.3. Activity of Calmodulin-Regulated Enzymes 3.3.1. Ca-Mg-ATPase. Zinc deficiency caused a significant decline in the activity of the Ca-Mg-ATPase in erythrocytes membranes by 80% when compared to that in control animals (Table 1). As we could demonstrate earlier, the erythrocyte membrane is a sensitive compartment that responds rapidly with a decrease in zinc content during

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zinc deficiency. This depletion obviously also affects membrane-bound Ca-Mg-ATPase activity. 3.3.2. Adenylate cyclase. The activity of adenylate cyclase in isolated plasma membranes of the liver showed no dependence on alimentary zinc intake of the rats. This is in accordance with previous results that showed that even severe zinc-deficiency in rats retains an almost normal zinc concentration in liver tissue. 3.3.3. Phosphorylase kinase. The phosphorylase kinase activity was on average increased by 34% in livers of zinc-deficient as well as pair-fed control rats in comparison to ad libitum fed control animals. Consequently the increased activity of the phosphorylase kinase is mainly the result of the reduced feed intake in both groups rather than being caused by the zinc deficiency per se. Similarely activity of phosphorylase kinase in quadriceps and heart muscle also responded to reduced feed intake but not to the zinc status and this is in agreement to previous studies demonstrating that muscle tissue zinc concentration—similar to liver—does not decline in alimentary zinc deficiency. As already mentioned in the introduction, the calmodulin concentration in rat brain was not altered by alimentary zinc deficiency, explaining that the activity of the calmodulin-regulated enzyme phosphorylase kinase in brain showed also no dependence on zinc intake in the present study. 3.3.4. Phosphodiesterase. The activity of the phosphodiesterase in liver remained unaltered during zinc deficiency and indicated no significant differences to that in pairfed control animals. The difference in comparison with that of the ad libitum fed control animals is again mainly an effect of the reduced feed intake. Other organs such as kidney, brain and heart also showed no changes in the activity of phosphodiesterase when compared with the activity in ad libitum fed control rats. With the exception of the kidney, no differences were obtained also in comparison with the pair-fed control rats. Testes, however, are sensitive to the zinc status and respond to zinc deficiency with both, a significant reduction of zinc and calmodulin concentrations as observed in preceeding studies. In the present experiments (Table 1) the calmodulin-regulated activity of phosphodiesterase showed as a functional correlate a significant reduction by 35–40% in the cytosolic as well as particle fraction prepared from testes of zinc-deficient rats compared to both control groups. In summary, we here demonstrate that in erythrocyte membranes and testes in which alimentary zinc deficiency reduces the zinc concentrations, the activity of

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calmodulin-regulated enzymes also declines. No reduction of the activity of calmodulindependent enzymes was observed in brain, muscle or liver in which neither zinc nor calmodulin levels are decreased by a deficient alimentary zinc supply. Sensitive tissues may therefore respond via calmodulin-dependent enzymes to zinc deficiency with impared metabolic control.

REFERENCES Baudier, J., Haglid, K., Haiech, J., and Gerard, D., 1983, Zinc ion binding to human brain calcium binding proteins. Calmodulin and protein, Biochem. Biophys. Res. Commun. 114:1138–1146. Roth, H.-P. and Kirchgessner, M., 1991, Calmodulin activity in tissues of Zn- and Ca-deficient rats, J. Trace Elem. Electrolytes Health, Dis. 5:213–217.

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ZINQUIN ESTER—A REAGENT FOR THE INVESTIGATION OF THE ROLE OF AVAILABLE ZN(II) IN LIVING SYSTEMS David Ward*, Stephen Lincoln*, Henry Betts‡, Peter Zalewski‡, Ian Forbes‡, Indumathy Mahadevan*, Marc Kimber*, and Kym Hendrickson* *Department of Chemistry University of Adelaide Adelaide, South Australia, 5005 ‡Queen Elizabeth Hospital Woodville, South Australia, 5011

1. INTRODUCTION Zn(II) has an important role in biochemical and nutritional processes and about 300 enzymes contain Zn(II) as an essential component. Most of this intracellular Zn(II) is strongly complexed and hence not generally available to the cell. However the cell has small amounts [about 10% of the total cellular Zn(II)] of readily exchangeable [i.e. less strongly bound] Zn(II) which are important in many situations associated with cell activation, cell growth and cell death. Zinquin ester, a reagent we have developed specifically for the measurement of this less strongly bound cellular Zn(II), selectively forms a highly fluorescent complex with zinc(II). It is capable of detecting intracellular Zn(II) down to low nanomolar levels. Estimates of the concentration of intracellular available Zn(II) range from about M in the cytoplasm of many cells to about M in some vesicles. This paper describes some of the ways that Zinquin ester has been used to examine the roles of available Zn(II) in cell processes.

2. DISCUSSION We have synthesised (Mahadevan et al., 1996) a number of reagents which are specific for Zn(II) amongst the cations commonly present within a living cell. These reagents are weakly fluorescent themselves but form highly fluorescent complexes with Zn(II). Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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They can therefore be used to detect and measure this available Zn(II) within cells. These complexes have stability constants for Zn(II) such that they do not remove the tightly bound Zn(II) from zinc containing enzymes. One of these ligands, which we have named Zinquin ester, can detect Zn(II) at low nanomolar levels and is commercially available. $F Obtainable from Dr. A. D. Ward, Department of Chemistry, University of Adelaide, Adelaide, South Australia, Australia 5005. (email “[email protected]”) A basic requirement of these fluorophores is that not only should they be selective for Zn(II) but that they should readily traverse the cell membrane and not leak from the cell once they have entered it. Zinquin ester fulfills these requirements. Thus lipophilic zinquin ester traverses the cell membrane and is rapidly hydrolysed by cell esterases to zinquin acid, which at the cell pH of about 7 exists in the less lipophilic and charged carboxylate form (Fig. 1). Because of the charge, Zinquin acid does not cross through the cell membrane and hence is retained within the cell. A potientiometric and fluorescence study of the complexation of divalent metal ions by Zinquin acid in aqueous ethanol shows that the stabilities of the [ZnL] and complexes are 9.65 ± 0.02 and 19.11 ± 0.06, where the first and second figures are and respectively. (Hendrickson et al., 1997) Figure 2 shows the relative fluorescence of Zinquin acid with a range of metal cations. The relative fluorescence of 1.25 mM Zinquin acid in the absence of any added metal ions is about 10. However, since Zinquin acid is so sensitive to the presence of Zn(II) great care has to be taken to ensure the absence of any adventitious Zn(II). The relative fluorescence increased to 358 in the presence of 1 mM Zn(II) and 96 in the presence of 1 mM Cd(II). With all the other metal cations tested the relative fluorescence was similar to that of Zinquin acid alone. Zinquin ester has been used to investigate the spatial and temporal distribution of labile pools of available intracellular Zn(II) in a variety of biological tissues. Control

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studies have used the intracellular Zn(II) chelator TPEN to quench Zinquin fluorescence and the Zn(II) ionophore pyridinethione to increase the fluorescence. Pancreatic islets fluoresced intensely with Zinquin indicating their relatively very high concentration of available Zn(II). The fluorescence was particularly bright in the region of the insulin containing secretory granules and absent in the nucleus. A marked decrease in fluorescence was observed after treatment of the islets with relatively high concentrations of glucose in vitro to (1 mM) in HBSS, measured at 37°C, excitation 364 nm, emission 485 nm. induce insulin release. Substantial fluxes of Zn(II) were shown to occur in rat hepatocytes, both in vivo and in vitro, in response to the acute stress hormone and hepatocyte growth factor and to be mediated by changes in the intracellular Zn(II)-binding protein, metallothionein. Although lymphocytes fluoresced weakly with Zinquin the fluorescence increased more than five fold in lymphocytes undergoing the early stages of apoptosis (programmed cell death). Using Zinquin we have shown that there is an inverse correlation between the levels of available Zn(II) and the susceptibility of the cells to undergo DNA fragmentation in apoptosis. Substantial fluxes of available Zn(II) have also been revealed by Zinquin during critical events in the function of the reproductive tissues. Loss of available Zn(II) pools and change in the pattern of fluorescence labelling of the sperm head accompanied maturation of spermatozoa in vivo. Much of the fluorescence of oocytes was associated with the lipid granules and may represent storage pools of Zn(II) for use by the developing embryo. This pool is mobilised following fertilization of the egg and during early cleavage events. In sheep oocytes there is a major flux of available Zn(II) between the 8-cell and 16-cell stages of development. Zinquin ester has been used to monitor Zn(II) concentrations in human urine. Normally, there is little Zn(II) in urine since the kidneys have little role in regulating Zn(II) homeostasis. However, excessive excretion of Zn(II) may occur in some diseases and result in Zn(II) deficiency. We have used Zinquin ester to show that urinary Zn was abnormally high in about 30% of patients with type II (non-insulin-dependent) diabetes

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mellitus. Since Zn(II) is anti-inflammatory, it may have beneficial therapeutic effects in chronic inflammatory diseases such as asthma. We are currently using Zinquin ester to visualize and quantify Zn(II) in cells lining the conducting airways of the human and animal (pig, sheep) respiratory tract. Both normals and patients with asthma will be studied. Our preliminary findings suggest higher Zn(II) concentrations in the upper part of the normal respiratory tract, which is exposed to more pro-inflammatory agents. Zn(II) may be high in the upper respiratory tract to protect the epithelial cells from damage by noxious agents. Zinquin ester is better than TS-Q for these studies because it has less tendency to crystallize. These, and other areas that show changes in the levels of available biological Zn(II), within cells continue to be investigated by us and others using Zinquin ester.

ACKNOWLEDGMENTS We thank the National Health and Medical Research Council, the Australian Research Council and the University of Adelaide for their support of this work. We also acknowledge the ongoing collaboration with others in the biological area.

REFERENCES Mahadevan, I.M., Kimber, M.C., Lincoln, S.F., Tiekink, E.R.T., Ward, A.D., Betts, W.H., Forbes, I.J., and Zalewski, P.D., 1996, The synthesis of Zinquin ester and Zinquin acid, zinc(II)-specific fluorescing agents for use in the study of biological zinc(II), Aust. J. Chem., 49:561–568. Hendrickson, K.M., Rodopoulos, T., Pittet, P.-A., Mahadevan, I., Lincoln, S.F., Ward, A.D., Kuruscev, T., Duckworth, P.A., Forbes, I.J., Zalewski. P.D., and Betts, W.H., 1997, Complexation of zinc(II) and other divalent metal ions by the fluorophore [2-methyl-8-(p-toluenesulfonamido)-6-quinolyloxy]acetic acid in 50% aqueous ethanol, J. Chem. Soc., Dalton Trans., 1997, 3879–3882.

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COGNITIVE EFFECTS OF ADAPTATION TO A LOW ZINC DIET IN HEALTHY MEN

M. J. Kretsch, A. K. H. Fong, J. G. Penland, B. Sutherland, and J. C. King USDA, ARS, Western Human Nutrition Research Center University of California Davis, California 95616, U.S.A. and USDA, ARS, Grand Forks Human Nutrition Research Center Grand Forks, North Dakota 58202, U.S.A.

1. INTRODUCTION Zinc is known to be essential for brain development and function in animals (Wallwork and Sandstead, 1993) but considerably less is known about the role of zinc in the behavior and cognition of humans. Only a few studies on experimentally induced zinc deprivation and cognitive performance have been conducted with adults. In one such study, memory and perceptual deficits were observed in patients treated with histidine, a zinc chelating agent (Henkin et al., 1975). In another study with healthy men, short-term zinc deprivation (l–4mg/day) under metabolic unit conditions resulted in diminished performance on a range of cognitive and psychomotor tests, particularly those emphasizing attention, perception and memory (Penland, 1991). However, a four month study with men in which a diet was fed containing between 3 to 4mg zinc/day did not report adverse neuropsychological effects (Tucker and Sandstead, 1984). Thus, further research is needed to determine the zinc intake threshold at which cognitive performance is impaired. The objective of this research was to measure the cognitive consequences of sustained low zinc intake in healthy men. The level of zinc fed was slightly less than the World Health Organization’s recommendation for adult men consuming a diet of highly available zinc sources (WHO, 1996).

Address all correspondence to: Dr. Mary Kretsch, USDA, Western Human Nutrition Research Center, University of California, One Shields Avenue, Davis, California 95616, USA; email: [email protected]. Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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2. METHODS Eight normal-weight healthy men, ranging between 27 to 47 years of age, participated in a metabolic unit study consisting of 34 days of baseline (13.7 mg zinc/day), 70 days of low zinc intake intervention (4.6 mg zinc/day), and 36 days of zinc repletion (13.7 mg zinc/day). A controlled conventional food diet (3 day rotational menu) calculated to contain 5.0 mg zinc/day (4.6 mg zinc/day, analyzed value) was fed throughout the study. In addition, a 9.1 mg zinc gluconate supplement was administered daily during the baseline and repletion periods and a placebo supplement during the intervention period. All other dietary nutrients were adequate. A standardized, computerized, battery of tests (CPAS-revised, Grand Forks, ND) was used to assess a variety of cognitive and psychomotor skills after 25 days of baseline, after 19 and 61 days of low zinc intake (Zn1 and Zn2, respectively), and after 26 days of zinc repletion. Tests included measures of fine and gross motor skills, eye-hand coordination, sustained attention, visual perception, concept formation and abstract reasoning, and spatial, object and verbal memory. In the verbal memory test, 20 short words one-second intervals, and the subject was asked to remember as many words as possible. Next, a mix of previously presented words and new words were shown individually on the computer screen. The subject’s task was to determine which words were new words and which were previously presented words. The percentage of words correctly identified and the mean reaction time required for correct identifications was calculated for each subject. Plasma zinc concentrations were measured at times coinciding with the cognitive tests. Statistics were performed with the personal computer version of the Statistical Analysis System (SAS) for Windows 95, version 6.12 (SAS Institute, Inc., Cary, NC).

3. RESULTS AND DISCUSSION The most pronounced cognitive function finding was for short-term verbal memory. Using the SAS MIXED Procedure with repeated measures, a highly significant effect (p < 0.0001) of period was found for the reaction time needed for correct word recall (a measure of cognitive processing efficiency). In contrast to the baseline period, reaction time slowed significantly at Zn1, was significantly faster at Zn2, and was equivalent to baseline levels at repletion (Table 1). This pattern of change was evident for all subjects. Although word recall response time declined with the low zinc diet, no significant effect of period was found for the percentage of words accurately recalled at the four time points. Mean plasma zinc did not change significantly during the study.

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These findings suggest that within the first weeks of adaptation to a low zinc diet, the latency of short-term verbal memory is slowed. However, given sufficient time, the human body appears to adjust to the lower level of zinc intake and the cognitive impairment is no longer detectable. These findings would also appear to support the results of Tucker and Sandstead (1984) who fed a low zinc diet (3–4mg/day) to healthy men for four months and found neither significant changes in plasma zinc status nor adverse cognitive consequences. It should be noted, however, that both of these studies were relatively short-term. Long-term studies are needed to firmly establish that a zinc intake of about 5 mg/day does not impair the cognition of men.

REFERENCES Henkin, R.I., Patten, B.M., Re, P.K., and Bronzert, D.A., 1975, A syndrome of acute zinc loss. Cerebellar dysfunction, mental changes, anorexia, and taste and smell dysfunction, Arch. Neurol. 32:745–751. Penland, J.G., 1991, Cognitive performance effects of low zinc intakes in healthy adult men, FASEB J. 5:A938. Tucker D.M. and Sandstead, H.H., 1984, Neuropsychological function in experimental zinc deficiency in humans, in: The Neurobiology of Zinc, Part B. (C.J. Frederickson, G.A. Howell, and E.F. Kasarskis, eds), pp. 139–152, Alan R. Liss, New York. Wallwork, J.C. and Sandstead, H.H., 1993, Zinc and brain function, in: Essential and Toxic Trace-Elements in Human Health and Disease: An Update, Progress in Clinical and Biological Research, Volume 380 (A.S. Prasad, ed), pp. 65–80, Wiley-Liss, New York. Trace elements in human nutrition and health, 1996, World Health Organization, Geneva.

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INTRACELLULAR ZINC CHELATION INDUCES APOPTOSIS, CASPASES ACTIVATION, AND TRANSCRIPTION FACTORS DEGRADATION IN JURKAT AND HELA CELLS

Sève Michel, Chimienti Fabrice, Richard Sandrine, Mathieu Jacques, and Favier Alain Laboratoire de Biologie du Stress Oxydant (LBSO) UJF, Grenoble, France Unité de Radiobiologie et inflammation CRSSA, Grenoble, France

1. INTRODUCTION Zinc is an important component of cell survival. It participates in several proteins structure and its intracellular concentration seems to be precisely regulated. Zinc is involved in apoptosis regulation and has been described as an inhibitor of the DNA fragmentation in many cell lines, in part by inhibiting the calcium-dependent endonuclease (Cohen and Duke, 1984). Zinc chelation has been previously reported to induce apoptosis in different cell lines (McCabe Jr et al., 1993). Recent studies indicate that zinc inhibits caspase-3 enzyme, a cell death protease implicated in apoptosis (Perry et al., 1997). Caspases (standing for cystein activated aspartate-directed proteases) are proteolytic proteins activated by different pathways, at the early stage of apoptosis, either through auto-activation or proteolytic cascade. The activation of caspase-3 and -7 by caspase-8 and -9 seems to be an essential step in the execution of apoptosis (Porter and Janicke, 1999). Caspase-3 is responsible for the activation of DNA fragmentation factor, but also degrades several proteins such as poly (ADP-ribose) polymerase (PARP) (Lazebnik et al., 1994) or lamin (Lazebnik et al., 1995).

Address all correspondance to: Dr M. Sève; LBSO, UJF; UFR de Pharmacie; Domaine de la Merci; 38700 La Tronche; France; EMail: [email protected] Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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2. MATERIAL AND METHODS Jurkat cells and HeLa cells were treated with various inducers of apoptosis: the intracellular zinc chelator TPEN (N,N,N',N' tetrakis-(2pyridylmethyl) ethylene diamine), etoposide or TNF plus cycloheximide, with or without Zinc. The apoptotic effect was confirmed by direct observation or by measurement of free nucleosomes. At 0, 3, 6, 9, 12 hours after treatment, cells were harvested and a total protein extract was prepared and analysed by western blot, using antibodies against the proteins Sp1, Sp2, Sp3, Sp4, PARP, caspase-3 or actin and ECL detection. The caspase assay was performed by adding for one hour at 37°C to the cell lysate a specific fluorogenic substrate in order to determine separately each caspase activity. The specific fluorogenic substrates used in the assay were: Ac-DEVD-AMC (Becton-Dickinson, USA) for caspase-3, Ac-IETD-AMC (Calbiochem, USA) for caspase-8 and Ac-LEHD-AMC (Euromedex, France) for caspase-9. The activated caspase cleaves the specific substrate releasing the fluorogenic AMC (excitation 380nm, emission wavelength 460nm). Specific caspase inhibitors were used to control the specificity of substrate degradation. Sp1 cleavage assay was performed in vitro by mixing 50 ng purified baculovirus expressed Sp1 with 100 units of pure recombinant caspase-3 (Biomol, USA) in 50 mM Hepes pH 7.4, 100mM NaCl, 10% glycerol buffer with or without 5 mM DTT or 1 mM EDTA. After incubation at 37°C (2 hours), the protein was analyzed by western-blot.

3. RESULTS We show that deprivation of zinc by TPEN induces apoptosis in both cell lines with the typical morphology: loss of adhesion for Hela cells, condensation of the nucleus, membrane blebbing and formation of apoptotic bodies. These changes are visible after 3 hours of and reversible by zinc. We observe that the zinc finger proteins Sp1, Sp3 and Sp4 are affected by intracellular zinc chelation with a decrease of their DNA binding after 3 h and their specific degradation (Fig. 1). After 9 hours in HeLa cells and 3 hours in Jurkat cells, a 68 KDa proteolytic fragment is specifically recognized by anti-sp1 anti-

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body. We also observe in the two cell lines, the degradation of Sp3 and Sp4 which are others proteins of the Sp family with great homology with Sp1. The PARP protein, a well documented “cell death substrate” is also degraded during this apoptosis, with a faster rate when compared to Sp1. The kinetics of Sp1 and PARP specific proteolysis is faster in Jurkat than in HeLa cells. We measured the caspases activity in HeLa and Jurkat cells treated by TPEN. Caspase-3 like activity is increased by zinc depletion with a maximum of activity occurring after 12 hours in HeLa cells (Fig. 2) and after 9 hours in Jurkat cells. Adding zinc in the medium reverses this increase in caspase-3 like activity. This reversibility is observed until 6 hours after addition of TPEN. The kinetic of caspase activation in the two cell lines is slightly different with a shift in time. The maximal activity appears earlier for Jurkat cells than for HeLa cells with a time-correlation with the protein degradation. Caspase-1 and caspase-8 activities remain very low after induction of zinc depletion and we don’t observe any caspase-9 activity. In both cell lines, the appearance of caspase fragments is inhibited by zinc. To prove a direct degradation of Sp1 by caspase-3, we incubated recombinant Sp1 produced by expression in insect cells infected by a recombinant baculovirus with caspase-3 produced in E. coli. Sp1 protein is partially degraded by the protease. We observe a 68 KDa fragment of Sp1 corresponding to the proteloytic fragment appearing in cells during TPEN-induced apoptosis. The degradation process requires the presence in the buffer of 1 mM EDTA and very reductive condition (5 mM DTT).

4. DISCUSSION We have previously shown that treatment with 20 to of TPEN induces a dose-dependent depletion in intracellular zinc (Seve et al., 1999; Parat et al., 1997). This depletion induces apoptosis in the both cell lines with a different sensitivity, Jurkat cells being the more sensitive. The temporal relationship between zinc depletion and induc-

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tion of apoptosis suggests that the modification of cellular zinc is a good candidate as a causal factor to switch the control of cell cycle towards apoptosis. Concomitantly to morphological signs of apoptosis, the major biochemical event observed is the caspase-3 activation. Addition of an excess of zinc was recently described to inhibit the activation of this proteolytic enzyme (Perry et al., 1997). However, we observed for the first time the role of intracellular zinc to maintain the enzyme in the inactive form. Cell line resistant to TPEN-induced apoptosis presents a complete loss of caspase-3 expression demonstrating the strong relationship between zinc and caspase-3 (Kolenko et al., 1999). We confirm that transcription factor Sp1 is cleaved after zinc depletion-induced apoptosis. Such a caspase-dependent cleavage of Sp1 has been previously described in other models of apoptosis (Piedrafita and Pfahl, 1997; Rickers et al., 1999). The same process cleaves other members of the Sp family (Sp2, Sp3 and Sp4) in Jurkat cells as well as in HeLa cells. We explain the difference of PARP and Sp proteins proteolysis by a differential kinetic of caspase cascade activation, confirmed by direct measurement of caspase activity. The maximum of activity of caspase-3 is reached at 9 hours in Jurkat cells and 12 hours in HeLa cells. The cleavage of Sp proteins and PARP is totally reversed by the addition of zinc even after TPEN treatment, demonstrating the responsibility of zinc depletion in the mechanism. Zinc addition continues to be efficient in reversing TPEN-induced Sp1 cleavage as long as 6 hours after TPEN-induced zinc depletion in HeLa cells, at a time similar to the increase in caspase-3 activity. We conclude that apoptosis induced by zinc deprivation involved specific pathways activation and not only endonuclease activation. We show that DNA is fragmented, caspases are activated, but also transcription factors are proteolysed in this type of apoptosis, defining an other irreversible step in cell death. From these results, we have drawn a model for the regulatory role of zinc in which the latter is protecting cells from apoptosis by maintaining caspase-3 and Ca-Mg endonuclease in an inactive form at the resting state of cell. A change in the zinc localization in the cell constitutes a major step that will initiate the cascade of events leading to the cell death.

REFERENCES J.J. Cohen and R.C. Duke, 1984, Glucocorticoid activation of a calcium-dependent endonuclease in thymocyte nuclei leads to cell death, J. Immunol. 132:38–42. M.J. McCabe Jr, S. Jiang, and S. Orrenius, 1993, Chelation of intracellular zinc triggers apoptosis in mature thymocytes., Lab. Invest. 69:101–110. D.K. Perry, M.J. Smyth, H.R. Stennicke, G.S. Salvesen, P. Duriez, G.G. Poirier, and Y.A. Hannun, 1997, Zinc is a potent inhibitor of the apoptotic protease, caspase-3. A novel target for zinc in the inhibition of apoptosis, J. Biol. Chem. 272:18530–18533. A.G. Porter and R.U. Janicke, 1999, Emerging roles of caspase-3 in apoptosis, Cell. Death Differ 6:99–104. Y.A. Lazebnik, S.H. Kaufmann, S. Desnoyers, G.G. Poirier, and W.C. Earnshaw, 1994, Cleavage of poly (ADPribose) polymerase by a proteinase with properties like ICE, Nature 371:346–347. Y.A. Lazebnik, A. Takahashi, G.G. Poirier, S.H. Kaufmann, and W.C. Earnshaw, 1995, Characterization of the execution phase of apoptosis in vitro using extracts from condemned-phase cells, J. Cell. Sci. Suppl. 19:41–49. M. Seve, A. Favier, M. Osman, D. Hernandez, G. Vaitaitis, N.C. Flores, J.M. McCord, and S.C. Flores, 1999, The human immunodeficiency virus-1 tat protein increases cell proliferation, alters sensitivity to zinc chelator-induced apoptosis, and changes Sp1 DNA binding in HeLa cells, Arch. Biochem. Biophys. 361:165–172.

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M.O. Parat, M.J. Richard, J.C. Beani, and A. Favier, 1997, Involvement of zinc in intracellular oxidant/antioxidant balance, Biol. Trace. Elem. Res. 60:187–204. V. Kolenko, R.G. Uzzo, R. Bukowski, N.H. Bander, A.C. Novick, E.D. Hsi, and J.H. Finke, 1999, Dead or dying: necrosis versus apoptosis in capase-deficient human renal cell carcinoma [In Process Citation], Cancer Res. 59:2838–2842. F.J. Piedrafita and M. Pfahl, 1997, Retinoid-induced apoptosis and Sp1 cleavage occur independently of transcription and require caspase activation, Mol. Cell. Biol. 17:6348–6358. A. Rickers, N. Peters, V. Badock, R. Beyaert, P. Vandenabeele, B. Dörken, and K. Bommert, 1999, Cleavage of transcription factor Sp1 by caspases during anti-IgM-induced B-cell apoptosis, Eur. J. Biochem. 261:269–274.

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EFFECT OF ZINC SUPPLEMENTATION ON BIOLOGICAL PARAMETERS OF BONE TURNOVER IN HEALTHY MEN

Anne Peretz, Pierre Bergmann, Théodore Papadopoulos, Vania Siderova, and Jean Nève CHU Brugmann, Place A. Van Gehuchten 4, B-1020 Brussels, Belgium Pharmacy Institute Université Libre de Bruxelles Campus Plaine 205-5, B-1050 Brussels Belgium

1. INTRODUCTION Bone is a reservoir of minerals and also of trace elements such as zinc which accounts for 28% of total body zinc. In addition, the element is involved in bone turnover as a cofactor for metalloenzymes lysyloxydase, collagenase. Zinc increases the activity of bone alkaline phosphatase activity and activates osteoblast tyrosine kinase, RNA synthetase suggesting a role in bone formation (Yamaguchi et al., 1994). This is further demonstrated by in vitro and in vivo studies showing that zinc stimulates bone growth and mineralization, osteoblast proliferation, differentiation and increases IgF-1 activity (Yamaguchi and Hashizume, 1994; Yamaguchi et al., 1996). On the other hand, zinc has an inhibitory effect on bone resorption in osteoblast cell cultures (Yamaguchi et al., 1995; Holloway et al., 1997) and prevents bone loss after ovariectomy or corticosteroid administration (Kishi et al., 1994; Segawa et al., 1992). In humans, zinc deficiency is associated with delayed bone growth (Nishi, 1992) but few studies have been performed to elucidated the potential role of zinc in the regulation of bone turnover. The aim of the present study was to evaluate the effect of zinc supplementation at supraphysiological doses on parameters of bone formation and bone resorption in humans. Healthy men were chosen to eliminate the possible interference of hormonal cycle. Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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2. MATERIALS AND METHODS Study Design and Subjects Nineteen healthy male volunteers with normal BMI aged 24 ± 5 years participated in this double-blind, placebo-controlled randomised trial, after written informed consent. Subjects had normal hepatic and renal functions. No vitamin, mineral supplements or drugs were allowed during the study period. Subjects were selected at random order to receive either a daily oral dose of 50 mg zinc under the form of gluconate or a placebo for 12 weeks. Fasting blood and urine samples were taken at 0 and at 12 weeks.

Parameters Serum and urine zinc levels were measured by atomic absorptiometry. Urine zinc was expressed as the ratio zinc/creatinine Parameters of bone formation were determined in serum: total alkaline phosphatase activity (AP), bone specific alkaline phosphatase (BAP) activity (BAP-E, Alkphase®, Metra), BAP mass concentration (BAP-M, Ostase®, Hybritech), intact osteocalcin (BGP, Osteo SP®, Incstar). The C-terminal collagen telopeptide (CTX, Cross Laps®, Hybritech) and N-terminal collagen telopeptide (NTX, Osteomark®, Kodak) were measured in urine as parameters of bone resorption.

Statistics Results were expressed as means ± standard deviation. The difference for each parameter within groups (zinc-treated and placebo-treated) was assessed by ANOVA followed by paired-t test. The difference between groups was assessed using unpaired t test at the beginning of the study (time 0) and +12 weeks.

3. RESULTS A significant increase in urine zinc/creatinine ratio was observed within zinc-treated group at time 0 vs at 12 week, p = 0.03). The difference remains significant when zinc-treated subjects were compared to the placebotreated subjects (p = 0.003). A non significant increase in serum zinc concentration was observed in zinc-supplemented subjects at 12 weeks. The level of serum and urine zinc remained unchanged in the placebo group. A significant increase in total AP was observed in the zinc-treated group at week 12 (p = 0.005), while no significant change was observed in the control group. This increase was not significant when compared to the placebotreated group. BAP-E increased in zinc-treated subjects from at time 0 to at week 12 (p = 0.04). The difference was also significant when zinc-treated subjects were compared to placebo-treated subjects (p = 0.05). BAP-M remained unchanged in both groups. Serum BGP concentration increased in zinc supplemented group from 5.2 ± 2.6 to 6.5 ± ng/mL at +12 weeks vs time 0 (p = 0.04) and from 4.5 ±1.8 to 5.7 ± 1.3 ng/mL in the placebo group (p = 0.001). However, there was no difference

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between groups. Urine CTX and NTX remained unchanged during the study in both groups.

4. DISCUSSION A supplementation with supraphysiological dose of zinc in healthy men led to significant modifications in parameters of bone turnover, especially parameters of bone formation: total AP, BAP-E and BGP. The dose of 50 mg of elemental zinc under the form of an organic salt, gluconate, appeared as non toxic and well tolerated. The choice of this dose was based on previous data showing that 45 mg of zinc element under the form of gluconate in patients with rheumatoid arthritis increased AP in leukocytes (Peretz et al., 1993). The BAP increase is in agreement with previous studies performed in animals. An increase in BAP activity has been reported in the femoral diaphyses of rats fed with zinc-rich diet (Yamaguchi et al., 1986) while a decrease of BAP activity was observed in bones of rats during zinc deficiency (Prasad et al., 1967). A dose-dependent relationship between zinc intake and BAP was found in the serum or the tibia in adult female mice (Dimai et al., 1996). In humans, zinc stimulated BAP activity in monolayer cell culture (Hall et al., 1999). According to our knowledge, no data are available in humans about the effect of in vivo zinc supplementation on BAP. The absence of modification in BAPM suggests that zinc could act through the activation of the enzyme rather through a direct effect on its synthesis. A significant increase of BGP was observed in both groups. Whether this modification represents physiological variations with time is not elucidated. A 30% variation in BGP levels might be observed in the absence of any intervention (Peretz et al., 1997). Dimai reported that serum osteocalcin level increases when serum zinc level reaches twice the normal values in adult female mice (Dimai et al., 1998). Few subjects in the zinctreated group reached this level. Moreover a dose-response effect on BGP levels by supplementation with zinc has not been reported in humans. The absence of modifications in parameters of bone resorption together with an increase of bone formation parameters supports the hypothesis that zinc might be a stimulant of bone formation. A relationship between zinc deficiency and bone loss is suggested by the observation that osteoporotic subjects have lower serum zinc level than age-matched controls and

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increased urinary zinc excretion (Herzberg et al., 1990; Relea et al., 1995). Few data are available in humans concerning the role of zinc supplementation in these conditions (Strause et al., 1994). In conclusion, zinc supplementation could have a possible application in the treatment of osteoporosis. Further studies should be performed in old people and in postmenopausal women.

REFERENCES Dimai, P., Hall, S., and Farley, J.,1996, Effect of dietary zinc on bone formation and resorption indices in adult female mice. J. Bone Miner. Res. 11(suppl):s228. Dimai, H.P., Hall, S.L., Stilt, S.B., and Farley, J.R., 1998, Skeletal response to dietary zinc in adult female mice. Calcif. Tissue Int. 62:309–315. Hall, S.L., Dimai H.P., and Farley, J.R., 1999, Effects of zinc on human skeletal alkaline phosphatase activity in vitro. Calcif. Tissue Int. 64:163–172. Herzberg, M.J., Foldes, Steinberg, R., and Menczel, J., 1990, Zinc excretion in osteoporotic women. J. Bone Miner. Res. 5:251–257. Holloway, W.R., Collier, F.M., Herbst, R.E., Hodge, J.M., and Nicholson, G.C., 1996, Osteoblast-mediated effects of zinc on isolated rat osteoclasts: inhibition of bone resorption and enhancement of osteoclast number. Bone 19:137–142. Kishi, S., Segawa, Y., and Yamaguchi, M., 1994, Histomorphological confirmation of the preventive effect of beta-alanyl-L-histidinato zinc on bone loss in ovariectomized rats. Biol. Pharm. Bull. 17:862–865. Nishi, Y., 1996, Zinc and growth. J. Am. Coll. Nutr. 15:340–344. Peretz, A., Nève, J., Jeghers, O., and Pelen, F., 1993, Zinc distribution in blood components (plasma, leucocyte fractions and erythrocytes), inflammatory status and clinical parameters of disease activity during zinc supplementation in inflammatory rheumatic diseases. Am. J. Clin. Nutr. 57:690–694. Peretz, A., Willems, D., Siderova, V., Karmali, R., Bergmann, P., and Hotimsky, A., 1997, Effect of long term corticosteroid (CS) treatment on the biochemical markers of bone formation. 19th annual meeting ASMBR, Cincinnati, Ohio. Prasad, A., Oberleas, D., Wolf, P., and Horwitz, J.P., 1967, Studies on zinc deficiency: changes in trace elements and enzyme activities in tissues of zinc-deficient rats. J. Clin. Invest. 46:217–224. Relea, P., Revilla, M., Ripoll, E., Arribas, I., Villa, L.F., and Rico, H., 1995, Zinc, biochemical markers of nutrition, and type I osteoporosis. Age Ageing. 24:303–307. Segawa, Y., Tsuzuike, N., Itokazu, Y., Tagashira, E., and Yamaguchi, M., 1992, Bta-alanyl-L-histidinato zinc prevent hydrocortisone induced disorder of bone metabolism in rats. Res. Exp. Med. 192:317–322. Strause, L., Saltman, P., Smith, K.T., Bracker, M., and Andon, M.B., 1994, Spinal bone loss in postmenopausal women supplemented with calcium and trace minerals. J. Nutr. 124:1060–1064. Yamaguchi, M., Inamoto, K., and Suketa, Y., 1986, Effect of essential trace metals on bone metabolism in weanling rats: comparison with zinc and other metals actions. Res. Exp. Med. (Berl) 186:337–342. Yamaguchi, M., 1995, Beta-alanyl-L-histidinato zinc and bone resorption. Gen. Pharmacol. 26:1179–1183. Yamaguchi, M. and Hashizume, M., 1994, Effect of beta-alanyl-L-histidinato zinc on protein components in osteoblastic MC3T3-E1 cells: increase in osteocalcin, insulin-like growth factor-I and transforming growth factor-beta. Mol. Cell. Biochem. 136:163–169. Yamaguchi, M. and Matsui, T., 1996, Stimulatory effect of zinc-chelating dipeptide on deoxyribonucleic acid synthesis in osteoblastic MC3T3-E1 cells. Peptides 17:1207–1211. Yamaguchi, M., Kishi, S., and Hashizume, M., 1994, Effect of zinc-chelating dipeptides on osteoblastic MC3T3-E1 cells: activation of aminoacyl-RNA synthetase. Peptides 15:1367–1371.

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BETAINE-HOMOCYSTEINE SMETHYLTRANSFERASE IS AN ABUNDANT ZINC METALLOENZYME IN LIVER

Andrew P. Breksa III and Timothy A. Garrow Department of Food Science and Human Nutrition University of Illinois Urbana, Illinois 61801 USA

1. INTRODUCTION BHMT (EC 2.1.1.5) is a cytosolic enzyme found in the pathway of choline oxidation. This enzyme is a hexamer of identical 45 kDa subunits and catalyzes a methyl transfer from betaine to homocysteine to form dimethylglycine and methionine, respectively. It is expressed at very high levels in the liver and kidney cortex. A cDNA encoding human liver BHMT was cloned several years ago (Garrow, 1996). Shortly thereafter, the recombinant enzyme was shown to contain Zn (Millian and Garrow, 1998). Zn could be removed from the protein using the thiol-specific reagent methylmethane thiosulfonate suggesting that Zn was ligated to BHMT by one or more Cys residues. After cloning the human BHMT cDNA, Garrow (1996) reported that the deduced amino acid sequence of the enzyme shared limited homology to E. coli’s vitamin dependent methionine synthase another enzyme that methylates homocysteine. One of the strongest regions of homology included a GGCCG sequence. The Cys residues in this sequence correspond to 299 and 300 in human BHMT, and 310 and 311 in Subsequently, Goulding et al. (1997) identified Cys310 and Cys 311 as ligands for Zn in and later it was determined that Cys247 was a third ligand (Peariso et al., 1998). Cys247 of did not align with any Cys residue in BHMT when analyzed by a number of computer-based alignment programs; however, when BHMT was aligned by hand with the sequence ALGP, the following BHMT sequence HFDP appeared to be similar. The goal of our current study

Address all correspondence to: Dr. Timothy A. Garrow, 905 South Goodwin Avenue, 463 Bevier Hall, Urbana IL 61801 USA; telephone: 217-333-8455; fax: 217-333-9368; email: [email protected] Trace Elements in Man and Animals 10, edited by Roussel el al., Kluwer Academic / Plenum Publishers, New York, 2000.

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was to use site-directed mutagenesis to determine which residues were required for Zn binding to BHMT and we were guided by our choice of which residues to mutate by the limited homology between E. coli and BHMT.

Experimental Procedures Recombinant human liver BHMT was overexpressed and purified using the (Intein Mediated Purification with a Affinity Chitin-binding Tag) T7 system (New England Biolabs). In brief, the cDNA sequence of human liver BHMT was cloned into pTYB4, the vector for the T7 system, by standard procedures and the construct named pTYB4-hBHMT. This plasmid was then subjected to site-directed mutagenesis to change Cys217, Cys299, and Cys300 to Ala residues. The mutagenesis was performed using a kit (Stratagene) using the manufacturer’s instructions. The wild-type and mutant BHMT proteins were then overexpressed and purified using the procedures recommended by the T7 manufacturers. Cells were grown in culture media supplemented with 250mM Zn chloride. Following purification, the BHMT proteins were homogenous as judged by sodium dodecylsulfate polyacrylamide electrophoresis in combination with coomassie blue staining. Fractions containing BHMT were analyzed for protein content by a coomassie dye-binding assay using bovine serum albumin as standard. BHMT activity was measured as described previously (Garrow, 1996). The Zn content of proteins were determined by inductively coupled plasma emission spectrometry (ICP). In brief, PD-10 columns (Pharmacia BioTech) were equilibrated with buffer that had been scrubbed of metals by passage over Chelex 100 resin (Bio-Rad Laboratories). Protein samples (1.5 ml) that had been previously affinitypurified were loaded into these PD-10 columns and eluted with the addition of metalfree buffer.

2. RESULTS AND DISCUSSION Although we had previously developed a purification protocol for recombinant human liver BHMT (Millian and Garrow, 1998), the method employed required multiple chromatographic steps and several days to complete. In order to more rapidly evaluate the effects of site-directed mutagenesis on Zn binding, we prepared a BHMTintein/chitin-binding fusion construct (pTYB4-hBHMT) to facilitate the purification of the enzyme. In this construct, the C-terminus of BHMT is fused to the N-terminus of S. cerevisiae’s intein gene, which in turn is fused to a B. circulans’ chitin-binding domain, the latter of which is required for affinity purification. Upon autocleavage of the BHMT-intein fusion, BHMT is left with an additional C-terminal glycine residue (residue 407) which is not found on the native 406 residue protein. Using this system, 10–15 mg of pure human BHMT could be recovered from one liter of cultured cells. The pTYB4-hBHMT construct was used as the template for site-directed mutagenesis to create the following mutant proteins: Cys217Ala, Cys299Ala, and Cys300Ala. Following the purification of the wild-type and mutant enzymes, analysis for BHMT activity revealed that the mutant proteins were inactive, and ICP analysis indicated a significant loss of protein-bound Zn (Table 1). The wild-type enzyme has a specific activity similar to those enzymes previously purified from the livers of various species (data not shown).

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The studies reported here indicate that Cys217, Cys299, and Cys300 were critical for BHMT activity and Zn binding. These amino acids align to Cys247, Cys310, and Cys311 of E. coli and, as in the case of BHMT, when any of these conserved Cys residues were mutated to Ala in E. coli the protein lacked methyltransferase activity and retained only residual levels of Zn (Goulding et al., 1997; Peariso et al., 1998). In addition, extended X-ray fine structure analysis of E. coli showed that Zn was bound by three thiolate ligands and a fourth ligand that was either a nitrogen or oxygen (Peariso et al., 1998). These data are consistent with their Cys-to-Ala mutagenesis studies. Although tenuous in the absence of crystal structure, it can be concluded that these conserved Cys residues are required for Zn binding in both BHMT and

REFERENCES Garrow, T.A., 1996, Purification, kinetic properties, and cDNA cloning of mammalian betaine-homocysteine methyltransferase, J. Biol. Chem. 271:22831–22838. Goulding, C.W. and Matthews, R.G., 1997, Cobalamin-dependent methionine synthase from Escherichia coli: involvement of zinc in homocysteine activation, Biochemistry 36:15749–15757. Millian, N.S. and Garrow, T.A., 1998 Human betaine-homocysteine methyltransferase is a zinc metalloenzyme, Arch. Biochem. Biophys. 356:93–98. Peariso, K., Goulding, C.W, Huang, S., Matthews, R.G., and Pennerhahn J.E., 1998, Characterization of the zinc binding site in methionine synthase enzymes of Escherichia coli: the role of zinc in the methylation of homocysteine, J. Amer. Chem. Soc. 120:8410–8416.

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NOURISHMENT To Moderate Constantly or to Indulge Occasionally—the Message of Zinc Dose-Rate Idiorrhythm to the Needy

Berislav Momcilovic Institute for Medical Research and Occupational Health Ksaverska cesta 2, Zagreb, Croatia

1. INTRODUCTION Our knowledge of nutrient requirements and metabolism is based on studies in which a constant diet of known composition is fed daily; not quite a real life situation (Momcilovic, 1995). The natural rhythm of feeding is also taken for granted, although random rhythmic oscillations are the essential control factor in regulating the constant homeostasis of the body (Hyndman, 1974; Momcilovic, 1988; Moss and Wiesenfeld, 1995). The idiorrhythmic dose-rate variability in feeding dietary Zn brings us the missing dimension of time and periodicity to the conventional dose-response model (Momcilovic, 1988; Momcilovic, 1997). The response pattern of Zn metabolism to the idiorrhythmic feeding was different from that of conventional dose-response feeding as shown by considerably modified body growth and Zn deposition in the calcified tissue (Momcilovic and Reeves, 1997; Momcilovic, Reeves and Blake, 1997). The aim of this work was to study the paradigmatic case on how to divide a limited amount of Zn, an essential non-caloric nutrient, over the period of 48 days when only one fourth of daily requirements is available on the average.

2. MATERIALS AND METHODS Materials The effect of idiorrhythmic dose-rate variability in low dietary Zn intake on body growth and Zn deposition in the femur, a Zn sensitive indicator tissue (Momcilovic, Belonje, Giroux and Shah, 1975) was studied in young growing male Sprague-Dawley rats. Body weight of the animals was recorded daily. Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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Idiorrhythmic Experimental Model An idiorrhythmic approach requires that the average dietary Zn concentration (modulo, M) is kept constant across different groups throughout the whole experiment (epoch, E). This is done by adjusting the Zn concentration of the supplemented diet supplied to compensate for the reduction in the number of days on which Zn-supplemented diet is fed, the latter being spread evenly over the experiment. Idiorrhythms involve offering the diet with n times the overall Zn concentration (M) only every nth day with Zndeficient diet offered on other days. The relationship between the dose-rate idiorrhythm (I), the selected dose-time equivalent modulo level and the sequential number of the day on which the peak dose is administered (dosing day; is formally expressed as: Low Zn idiorrhythmic diets provide a daily average of over a 48-day epoch. Thus, I = 15/5 means feeding the diet containing (nominator) every day (denominator) with no Zn in the diet on the other four days between the dosing days.

Analytical Methods Zn in the idiorrhythmic diets and femur was determined by the ICAP-AES (Nielsen et al., 1988).

Statistical Analysis The difference between the treatments was considered to be significant at p < 0.05 (Einot and Gabriel, 1975).

3. RESULTS Idiorrhythmic Zn dose-rate feeding generates a regular sequence of distinct idiorrhythm-dependent catch-up body growth cycles (data not shown). The cyclic changes in the body weight gain (BWG) and body weight loss (BWL) were analyzed by summing the daily BWGs separately for days when BW decreased (cumulative body weight loss; CBWL) from days when BW increased (cumulative body weight gain; CBWG), i.e., BWG = CBWG—CBWL, for each animal and idiorrhythm over the entire epoch (Table 1). All animals experienced 40–60% CBWL, whereas CBWG was changing with the progression of the idiorrhythmic intervals in a complex bi-modal pattern with a peak at I = 15/5. The data on combined CBWG and CBWL showed major increase in the metabolic turnover induced by Zn dose-rate feeding by a low dose-time equivalent idiorrhythmic diets. Similar bi-phasic pattern and secondary peak at I = 15/5 was observed for Zn deposition in the femur (Table 2).

4. DISCUSSION Evidently, the metabolic efficiency of dietary Zn varies with the Zn dose-rate idiorrhythm. Thus, if the non-caloric nutrient, like Zn, is in a short supply in the diet over a given period of time, it would be better to indulge in Zn ample diet from time to time than to moderate on zinc deficient diet providing the same total amount of Zn all

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the time. Such temporary indulging should not be random, but rhythmic, if better growth results are to be achieved. In other words, metabolic efficiency of nutrients like Zn is an interplay between the relative nutritional density of the respective minerals in the diet and adequate time spacing in the delivery of the diet. Feeding of a every day gave the best response; stressing the primary importance of a well balanced diet.

Both balanced and unbalanced diets may vary in their total available amount, but only the unbalanced diets can be of either high and/or low density with respect to their relative nutrient proportions. In fact, the unbalanced diet can be at the same time of high density for one element and of low density for the other. Meaning that conventional division into an element deficient and an element excessive diet is an oversimplified description of an endless array of unbalanced diets. What may be the response of other elements and to more complex idiorrhythms is not known. The phenomenon of catch-up growth (Dullo and Girardier, 1993) is considered to be a very important biological adaptation to the inadequate supply of the nutrients (James, McNeill, and Ralph, 1990). The gut is a central organ in nutrient

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requirements and metabolism (McBurney, 1994), but how does it adapt to the different metabolic fuels in the diet (MacDonald and Webber, 1995) and what may be the health effects of adaptive metabolic cycling remains to be elucidated (Brownell and Rodin, 1994; Rozen, Brigant, and Apfelbaum, 1994). REFERENCES Brownell, K.D. and Rodin, J., 1994, Medical, metabolic, and psychological effects of weight cycling, Arch. Intern. Med. 154:1325–1330. Dullo, A.G. and Girardier, L., 1993, Adaptive role of energy expenditure in modulating body fat and protein deposition during catch-up growth after early undernutrition, Am. J. Clin. Nutr. 58:614–621. Einot, I. and Gabriel, K.K., 1975, A study of the powers of several methods of multiple comparisons, J. Am. Stat. Assoc. 70:574–583. Hyndman, B.W., 1974, The role of rhythms in homeostasis, Kybernetik, 15:227–236. James, W.P.T., McNeill, G., and Ralph, A., 1990, Metabolism and nutritional adaptation to altered intakes of energy substrates, Am. J. Clin. Nutr. 51:264–269. McBurney, I.M., 1994, The gut: central organ in nutrient requirements and metabolism, Can. J. Physiol. Pharmacol. 72:260–265. MacDonald, I.A. and Webber, J., 1995, Feeding, fasting, and starvation: factors affecting fuel utilization, Proc. Nutr. Soc. 54:267–274. Momcilovic, B., 1988, The epistemology of trace element balance and interaction, in: Trace Elements in Man and Animals, Volume 6 (L.S. Hurley, C.L. Keen, B. Lonnerdal, and R.B. Rucker, eds.), pp. 173–177, Plenum Press, New York. Momcilovic, B., 1995, Coupling of zinc dose to frequency in a regularly recurrent pattern shows a limited capacity of excessive dietary zinc to compensate for a previously deficient intake, J. Nutr. 125:2687–2699. Momcilovic, B., 1997, Dose-rate idiorrhythm is a powerful tool for the detection of subtle mineral interactions. A case for the expression of Recommended Dietary Allowances (RDAs) and Safety Limits (RFDs) in the range format, in: Trace Elements in Man and Animals, Volume 9 (P.W.F. Fischer, M.R. L’Abbe, K.A. Cockell, and R.S. Gibson, eds.), pp. 403–405, NRC Research Press, Ottawa, Canada. Momcilovic, B., Belonje, B., Giroux, A., and Shah, B.G., 1975, Total femur zinc as a parameter of choice for a zinc bioassay, Nutr. Rep. Intl. 12:197–203. Momcilovic, B. and Reeves, P.G., 1997, Quantitative assessment of the effects of variability in dietary zinc doserate idiorrhythms upon zinc deposition in bone of weanling rats by using a slope-ratio assay, J. Nutr. Biochem. 8:256–264. Momcilovic, B., Reeves, P.G., and Blake, M.J., 1997, Idiorrhythmic dose-rate variability in dietary zinc intake generates a different response pattern of zinc metabolism than conventional dose-response feeding, Brit. J. Nutr. 78:173–191. Moss, M. and Wiesenfeld, K., 1995, The benefits of background noise, Sci. Am. August:66–69.4. Nielsen, F.H., Shuler, T.R., Zimmermann, T.J., and Uthus, E.O., 1988, Magnesium and methionine deprivation after the response of rats to boron deprivation, Biol. Trace Elem. Res. 17:91–107. Rozen, R., Brigant, L., and Apfelbaum, M., 1994, Effects of cycles of food restriction followed by ad libitum refeeding on body composition and energy expenditure in obese rats, Am. J. Clin. Nutr. 59:560–565.

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CONCENTRATION OF GROWTH HORMONE, IGF-1, INSULIN AND C-PEPTIDE IN RAT SERUM DURING DEVELOPMENT OF ALIMENTARY ZINC DEFICIENCY

H.-P. Roth and M. Kirchgessner Institute of Nutrition Physiology Technical University Munich D-85350 Freising-Weihenstephan Germany

1. INTRODUCTION In an earlier study, the growth hormone (GH) concentration in the serum of zincdeficient rats was greatly reduced compared to the ad libitum fed control animals. However the same reduction in GH levels, was also observed in pair-fed control animals. This means that the reduced GH concentration in the serum of the Zn-deficient and the pair-fed control rats is mainly the consequence of the reduced feed intake and the associated protein and energy depletion and not by the Zn deficiency per se. In a following study rats showed again that zinc deficiency as well as strongly restricted feed intake reduced the growth hormone concentration in the serum (Kirchgessner and Roth, 1985). In a marginal zinc deficiency status however (6–12mg Zn/kg diet), when serum zinc concentration was significantly reduced, but feed intake was not impaired or only slightly reduced, serum growth hormone levels were also low when compaired to pair-fed animals. Therefore, the influence of Zn deficiency on serum GH concentration only becomes apparent if it is not masked by the effect of the reduced feed intake. In order to be able to distinguish clearly between the effect of reduced feed intake with the associated energy and protein depletion and the consequences of Zn deficiency as such, a new experiment was conducted in which rats were fed by gastric tube (Roth and Kirchgessner, 1994). This made it possible to supply the rats adequately with nutrients even in Zn deficiency while at the same time synchronising feed intake. In this study however, at the end of the experiment after 12 days, the concentration of the growth Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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hormone in the serum of zinc-deficient rats was significantly increased by 78% in comparison with control rats. Because high levels of zinc ions inhibit the release of growth hormone, one might hypothesize that the circulating zinc concentration, which is already considerable reduced in incipient alimentary zinc deficiency, first leads to an enhanced release of growth hormone from the secretory granules while at the same time reducing the storage capacity for newly synthesized growth hormone. As the experiment progresses and the severity of zinc deficiency increases, this can ultimately also lead to a reduction in the concentration of growth hormone in the serum as we have shown previously in several conventionally designed trials. In an new study (Roth and Kirchgessner, 1997), we determined the concentration changes of zinc, growth hormone, insulin-like growth factor-1, insulin and C-peptide in the serum of rats throughout the experiment until a severe alimentary zinc deficiency was established.

2. MATERIAL AND METHODS One hundred and forty-four Sprague-Dawley rats with an average live weight of 130 g were divided into 16 groups of 9 animals each. At the beginning of the experiment one basic group was killed. Five groups received ad libitum a basal low-zinc AIN-93G based diet with casein as source of protein and a zinc concentration of about 1 mgZn/kg diet. Further five groups were provided ad libitum the zinc supplemented control diet with 60 mgZn/kg diet and finally the last five groups named pair-fed received the control diet restricted to the feed intake of the zinc-deficient rats. On days 2, 7, 12, 22 and 32 of the experiment one group from each of the three feeding regimes, having been fasted for 12 hours, were anesthetized and killed. Beside zinc, the concentrations of growth hormone, insulin-like growth factor-1, insulin and C-peptide were determined in the serum of the rats by radioimmunological methods.

3. RESULTS AND DISCUSSION

Growth All three groups showed typical curves of weight gain. While the control rats showed an average weight gain of 6.2 g/d, the zinc-depleted animals experienced growth depression from day 6 onwards, with an average weight gain of only 1 g/d. The pair-fed control animals indicated 5 days later a reduced weight development of 2.4 g/d. After two weeks on the zinc-deficient diet, first zinc deficiency symptoms like sparse and rough hair could be noticed.

Serum-Zn Just after two days on the experimental zinc-deficient diet the zinc concentration in the serum of the zinc-deficient rats was significantly reduced by 12% compared to both control groups After 7 days on the zinc-deficient diet, the zinc concentration of the serum was decreased dramatically to 28% of the control level and lowered further to 23% at the end of the study. This demonstrates that this rats being in a severe zinc deficiency status.

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Growth Hormone The growth hormone concentration of the serum from the zinc deficiency rats was also significantly reduced after two days respectively 7 days compared with the two control groups (90–110 ngGH/ml), by 50% on average. From day 12 until the end of the experiment, the growth hormone concentration in the serum of the zinc deficiency rats amounted only one third of the ad libitum fed control values. The reduced feed intake of the pair-fed control rats, decreased the growth hormone concentration in the serum somewhat, too.

IGF-1 The IGF-1 concentration of the serum from the zinc deficiency rats was after two experimental days significantly increased by 42% respectively 51% in comparison with both control groups (75–80 ng IGF-1/ml); however a time at which the zinc and growth hormone concentration of the serum were significantly decreased. After seven experimental days there was no difference in the IGF-1 concentration in the serum between the 3 groups of the study. During the subsequent course of the trial, the IGF-1 concentration in the serum of the zinc deficiency rats permanent declined while it increased in the serum of the ad libitum fed control rats. At the end of the study after 32 days, the IGF1 concentration in the serum of the zinc-deficient rats amounted only one fifth of the ad libitum fed control values. Like before for zinc and growth hormone showed the pair-fed control rats due to the reduced feed intake a reduced IGF-1 concentration of the serum compared with the ad libitum fed control rats, but it was significantly higher again compared with the zinc-deficient rats.

Insulin Like IGF-1, the insulin activity was significantly increased by 40% in the serum after two days on the zinc-deficient diet in comparison with both control groups After seven experimental days again, the insulin activity of the serum showed no significant differences between the three groups of the study. From day 12 until the end of the experiment, the insulin activity of the serum was reduced by 50% respectively 64% compared with the ad libitum fed control rats. However, the same reduction in insulin activity of the serum could be find out in this time by pair-fed control rats. This suggest that the reduced feed intake, rather than zinc deficiency per se might well have been the reason for the reduced insulin activity in the serum.

C-peptide Also the C-peptide concentration (0.31 ± 0.14nmol/l) in the serum of the zinc deficiency rats was like the IGF-1 concentration and the insulin activity before after two experimental days significantly increased by 41% respectively 48% compared with both control groups (0.21 ± 0.05nmol/l). On day seven a significant difference in C-peptide concentration could be demonstrated only between zinc-deficient and pair-fed control rats. On day 12 and 22 the C-peptide concentration differed not in comparison with both control groups. At the end of the experiment the C-peptide concentration of the zincdeficient serum was only compared with the pair-fed control rats some what higher.

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Conclusion The serum zinc represents a small but highly dynamic pool which is significantly reduced within two days on a zinc-deficient diet; thus long before the first zinc deficiency symptoms like loss of appetite and growth depression could be observed. At his time the serum growth hormone concentration of the zinc-deficient rats was less by half, compared with both control groups. The growth promoting effect of growth hormone is largely mediated via insulin like growth factors like IGF-1 and by insulin. In this study led alimentary zinc deficiency in the first experimental days, before feed intake was reduced, to an increase of IGF-1, insulin and C-peptide. With the beginning of the reduction in feed intake and the development of alimentary zinc deficiency they began to decrease however. After prolonged zinc deficiency nutrition a decreased concentration of growth hormone and IGF-1 in the serum could be demonstrated again. The often very contradictory data in the literature on the influence of an alimentary zinc deficiency on the concentration of growth hormone, IGF-1 and insulin are not only attributed to the duration and severity of zinc deficiency but also to feed intake, because the supply of energy and protein influences the concentration of growth hormone, IGF-1 and insulin in serum, too.

REFERENCES Kirchgessner, M. and Roth, H.-P., 1985, Influence of zinc depletion and zinc status on serum growth hormone levels in rats, Biol. Trace El Res. 7:263–268. Roth, H.-P. and Kirchgessner, M., 1994, Influence of alimentary zinc deficiency on the concentration of growth hormone (GH), insulin-like growth factor I (IGF-1) and insulin in the serum of force-fed rats, Horm. Metab. Res. 26:404–408. Roth, H.-P. and Kirchgessner, M., 1997, Konzentrationsverlauf an Wachstumshormon, IGF-1, Insulin und CPeptid in Serum, Hypophyse und Leber von Zn-Mangelratten, J. Anim. Physiol. Anim. Nutr. 77:91–101.

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MODERATELY HIGH ZINC INTAKE IMPAIRS VERBAL MEMORY OF HEALTHY POSTMENOPAUSAL WOMEN ON A LOW COPPER DIET

James G. Penland, David B. Milne, and Cindy W. Davis USDA, ARS, Grand Forks Human Nutrition Research Center Grand Forks, ND 58202

1. INTRODUCTION The relationship between copper nutrition in adulthood and brain function and behavior is unclear. In mature animals, copper deprivation resulted in increased latencies in brainstem auditory evoked potentials (Olkowski et al., 1990), increased high-frequency activity and right-left hemisphere asymmetries in electrocorticograms (Penland, Sawler and Klevay, 1989), and reduced amplitudes of auditory startle responses (Prohaska and Hoffman, 1996). Penland, Speaker and Moulton (1996) found that low copper intakes increased locomotor activity and impaired maze (memory) performance; however, Thorne et al. (1983) saw no effects of low intakes between weaning and 10 weeks on subsequent locomotor activity, maze performance or responsiveness to shock. Research with humans has been limited primarily to studies of copper status indicators related to pathological behavior in clinical populations. For example, copper deficiency has been related to depression in adults (Hansen et al., 1993), while copper excess has been related to hyperactivity and autism in children (Raiten, Massaro and Zuckerman, 1984). Akil and Brewer (1995) recently reviewed the type and incidence of psychiatric disturbances in patients with Wilson’s disease.

Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products that may also be suitable. The U.S. Department of Agriculture, Agricultural Research Service, Northern Plains Area, is an equal opportunity/affirmative action employer and all agency services are available without discrimination. Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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Zinc is essential for brain development and function in animals (Halas and Eberhardt, 1987) and appears to play a role in motor development and activity in infants (Ashworth et al., 1998), and in cognitive function of children (Penland et al., 1997) and adults (Darnell and Sandstead, 1991). Zinc deficiency continues to be a problem in both developing and developed countries (Gibson, 1994; Sandstead, 1995). Findings that zinc supplementation may benefit cognitive function and other aspects of behavior lead to the possibility that some individuals will supplement their diets with excessive amounts of zinc. However, moderately high zinc intakes may interfere with absorption and availability of other elements, including copper (Walsh et al., 1994).

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This study experimentally investigated the effects of copper and zinc intakes on cognitive function (memory) of older adult women. In addition to low intakes of both copper and zinc, the effects of a moderately high zinc intake were examined.

2. MATERIALS AND METHODS Subjects and Diets Twenty-three healthy postmenopausal women aged 64 ± 6y (mean ± SD) participated in a 200-d metabolic unit study. Diets consisted of 10 d of equilibration followed by 90 d of low zinc intake (3 mg/2,000kcal/d) followed by 10 d of equilibration, and con-

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eluding with 90 d of high zinc intake (53 mg/d). Thirteen women were fed low copper (1 mg/2000kcal/d) while 10 women were fed high copper (3 mg/d) throughout both 90-d dietary periods. The equilibration diet contained 9 mg/d zinc and 1.6 mg/d copper. Diets consisted of conventional foods fed as a 3-d rotating menu, and were adequate in all other nutrients. Energy was distributed as 57% carbohydrates, 34% fats and 9% proteins.

Assessments The Memory Assessment Scale (MAS; Williams, 1991) was used to assess verbal and visual short- and long-term memory at the end of each 90-d dietary period. The MAS yielded several subscale scores describing immediate recall of numbers presented verbally, immediate and delayed recall of words and prose presented verbally, and immediate and delayed recognition of face-name pairs and designs presented visually, as well general scores summarizing short-term memory, verbal memory and visual memory, and a global memory score. Raw scores were standardized for age and level of education. The MAS was designed for use with both normal and clinical populations ranging in age from 18 to 80 y. Copper status was assessed by measuring plasma copper, erythrocyte Cu/Zn superoxide dismutase (SOD), and ceruloplasmin (enzymatic to immunoloreactive ratio). Zinc status was assessed by measuring plasma zinc, extracellular SOD, and alkalin phosphatase in bone.

Statistical Analysis MAS scores and biochemical status indicators were analyzed for dietary effects with a copper x zinc x cohort ANOVA for repeated measures (SAS GLM; version 6.12). In addition, MAS scores were correlated with copper and zinc indicators, and multiple regressions were run by using copper and zinc indicators separately as predictors of MAS scores (SAS REG).

3. RESULTS ANOVAs Significant (p < 0.05) ANOVA findings are depicted graphically in Fig. 1. An interaction between copper and zinc affected the general measure of short-term memory, immediate recall of numeric sequences presented verbally (verbal span), and immediate recall of words presented verbally (list recall). Subsequent planned pair comparisons with Student’s t-tests showed that short-term memory (p = 0.007) in general and immediate recall of numeric sequences presented verbally (p = 0.005) in particular, were worse when women on the low copper diet were fed high zinc compared to low zinc diets. Immediate recall of words presented verbally was improved with high zinc compared to low zinc, when diets were low in copper (p = 0.027). Contrasted with high copper intake, low copper intake was associated with increased intrusions during recall (p = 0.003), which suggests difficulty discriminating between relevant and irrelevant responses.

Correlations and Regressions Copper status indicators (plasma Cu and ceruloplasmin) were positively correlated with and predicted improved verbal memory, improved long-term memory, and increased

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clustering of verbal material (strategy), but fewer intrusions during recall (reduced distraction). Cu/Zn SOD was not related to or an effective predictor of memory performance, nor was any measure of zinc status.

3. DISCUSSION Low copper intakes in the presence of moderately high zinc intakes may negatively affect short-term verbal memory in otherwise normal healthy postmenopausal women. The effect on short-term verbal memory is more characteristic of a specific brain insult to one hemisphere than of non-specific dementia. The effect on verbal span is consistent with an insult to the dominant hemisphere. These effects of low copper intake exacerbated by moderately high zinc intake on verbal rather than visual memory are consistent with our previous observation of decreased left hemisphere (specialized for verbal processing) activity in copper deprived adult rats (Penland et al., 1989). Irrespective of zinc intake, low copper intakes may lead to increased difficulty discriminating between relevant and irrelevant responses, consistent with forced responses in the presence of a real memory impairment. However, performance on at least one task indicates that moderately high zinc intakes may also improve long-term memory for verbal material. Plasma copper and ceruloplasmin were the copper status indicators most predictive of memory performance, at least in this population. Future studies should specifically examine the relationship between memory and copper and zinc status and intakes in clinical populations. Because time and season confounded zinc treatment, no conclusion can be drawn about zinc effects in the absence of an interaction with copper intakes. This study extends previous findings that the negative effects of low copper intakes can be exacerbated by moderately high zinc intakes, showing consequences on cognitive function, specifically short-term memory for verbal material.

ACKNOWLEDGMENTS Rebecca Stadstad administered and scored the Memory Assessment Scales. Thanks also to Jeff Holm and the psychology staff, Bonnie Hoverson and the dietary staff, and Sandra Gallagher, clinical, nursing and metabolic unit staff at the Grand Forks Human Nutrition Research Center.

REFERENCES Akil, M. and Brewer, G.J., 1995, Psychiatric and behavioral abnormalities in Wilson’s disease, Adv. Neurol. 65:171–178. Ashworth, A., Morris, S.S., Lira, P.I., and Grantham-McGregor, S.M., 1998, Zinc supplementation, mental development and behaviour in low birth weight term infants in northeast Brazil, Eur. J. Clin. Nutr. 52:223–227. Darnell, L.S. and Sandstead, H.H., 1991, Iron, zinc and cognition, Am. J. Clin. Nutr. 53:P16. Gibson, R., 1994, Zinc nutrition in developing countries, Nutr. Res. Rev. 7:151–173. Halas, E.S. and Eberhardt, M.J., 1987, A behavioral review of trace element deficiencies in animals and humans, Nutr. Behav. 3:257–271. Hansen, C.R., Malecha, M., Mackenzie, T.B., and Kroll, J., 1993, Copper and zinc deficiencies in association with depression and neurological findings, Biol. Psychiatr. 18:395–401.

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Olkowski, A.A., Gooneratne, S.R., Crichlow, E.G., Rousseaux, C.G., and Christensen, D.A., 1990, Effects of high dietary sulfur on brain functions using evoked potentials technique, Can. J. Vet. Res. 54:113–118. Penland, J.G., Sawler, B.G., and Klevay, L.M., 1989, Brain electrophysiology in adult rats fed copper deficient diets, J. Trace. Elem. Exp. Med. 2:239–256. Penland, J.G., Speaker, K.S., and Moulton, PL., 1996, Dietary copper and magnesium effects on activity, learning, memory and anxiety in rats, N. Dak. Acad. Sci. Proc. 50:57. Penland, J.G., Sandstead, H.H., Alcock, N.W., Dayal, H.H., Chen, X.C., Li, J.S., Zhao, R, and Yang, J.J., 1997, A preliminary report: effects of zinc and micronutrient repletion on growth and neuropsychological function of urban Chinese children, J. Am. College Nutr. 16:268–272. Prohaska, J.R. and Hoffman, R.G., 1996, Auditory startle response is diminished in rats after recovery from perinatal copper deficiency, J. Nutr. 126:618–627. Raiten, D.J., Massaro, T.F., and Zuckerman, C., 1984, Vitamin and trace element assessment of autistic and learning disabled children, Nutr. Behav. 2:9–17. Sandstead, H.H., 1995, Is zinc deficiency a public health problem? Nutrition,11:87–92. Thome, B.M., Ker-Neng, L., Weaver, M.L., Be, N.W., and Medeiros, D.M., 1983, Postweaning copper restriction and behavior in the Long-Evans rat, Pharmacol. Biochem. Behav. 19:1041–1044. Walsh, C.T., Sandstead, H.H., Prasad, A.S., Newberne, P.M., and Franker, P.J., 1994, Zinc: health effects and research priorities for the 1990s, Environ, Hlth. Perspect. 102:5–46. Williams, J.M., 1991, Memory Assessment Scales Manual, Psychological Assessment Resources, Inc., Odessa, FL.

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IDENTIFICATION OF THE ZINC BINDING PROTEIN IN A CHILD WITH HYPERZINCAEMIA AS CALPROTECTIN (MRP8/MRP14) B. Sampson1, P. Richmond2, B. E. Golden3, M. K. Fagerhol4, J. H. Beattie5, and I. Z. Kovar6 1

Department of Clinical Chemistry Charing Cross Hospital, London UK 2 Immunobiology Unit, Institute of Child Health London, UK 3 Department of Child Health University of Aberdeen UK 4 Department of Immunology and Transfusion Medicine Ullevaal Hospital Oslo, Norway 5 Rowett Research Institute Aberdeen, UK 6 Department of Paediatrics Chelsea & Westminster Hospital London, UK

1. INTRODUCTION We previously reported the case of a child with raised plasma zinc (Zn) (Sampson et al., 1997). Clinical features include growth failure, hepatosplenomegaly, anaemia, impaired immune function, chronic vasculitis and osteoporosis. Symptomatic therapy includes prednisolone and immunoglobulin infusions. Abnormal Zn metabolism was demonstrated by stable isotope studies. Plasma Zn was shown to be bound to a high molecular weight protein fraction (180–250 kDa). The Zn-binding protein was partially purified, but not identified. We now report the identification of this protein as calprotectin (MRP8/MRP14 complex). The protein has been purified and characterised by mass spectrometry. Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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2. IDENTIFICATION OF THE ZINC-BINDING PROTEIN AS CALPROTECTIN By serendipity, the Zn-binding protein was identified after one of the authors (PR) heard a lecture by another author (BEG) on the subject of calprotectin. This is not a protein which would otherwise have been considered a likely candidate for the unidentified protein. Calprotectin was identified in the patient’s plasma by immunodiffusion and Western blotting. Plasma from the patient contains large amounts of calprotectin, showing a reaction of immunological identity with granulocyte calprotectin. Plasma calprotectin concentration measured by ELISA was 6,500 mg/L (normal <1mg/L). Urine and faecal calprotectin concentrations were normal: (reference range faecal homogenate, (upper limit normal On Western blots specific staining for calprotectin was seen in a broad area from the to the region with four stronger zones. The staining intensity of the 28 mg/L standard was comparable to patient’s plasma diluted 1/100.

3. MOLECULAR WEIGHT DISTRIBUTION OF CALPROTECTIN Size exclusion chromatography was performed with normal plasma and patient’s plasma. In normal plasma with calcium (Ca) in the buffer approximately 86% of the calprotectin was in a low molecular weight fraction and 14% in a high molecular weight fraction. With the addition of EDTA calprotectin in normal plasma shifted almost entirely to the low molecular weight fraction. In the presence of EDTA about 85% of the calprotectin in normal plasma eluted after the albumin peak, corresponding to a mass of about 35 kDa and about 15% eluted in fractions corresponding to mass of about 100 to 300 kDa. When patient’s plasma was run with Ca in the buffer, most of the calprotectin eluted in the higher mass fractions, suggesting an unusual tendency to complex formation. The elution profile of calprotectin is the same as that of Zn (Sampson et al., 1997). With EDTA in the buffer, the calprotectin elution profile showed a shift to lower molecular weight, similar to that of normal plasma, except that the post albumin fraction was broader and not a distinct peak. Plasma from the patient’s mother also showed a higher molecular weight distribution of calprotectin, but with normal concentrations.

4. PURIFICATION OF CALPROTECTIN Calprotectin was purified by a combination of size exclusion and ion exchange chromatography (Johne et al., 1997; Sampson et al., 1997). The calprotectin containing fractions were purified on a column of Zn loaded chelating Sepharose and eluted with buffer containing 20 mM EDTA. Impurities were removed by repeating the ion exchange separation in the presence of EDTA, eluting the bound calprotectin with buffer containing 5 mM Ca. SDS-acrylamide gel electrophoresis showed that the calprotectin isolated from the patient’s plasma had the same molecular weight as normal calprotectin.

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5. MASS SPECTROMETRY Mass spectra of the calprotectin isolated from the patient and of pure calprotectin were obtained as before. The reference preparation showed main peaks at m/ez 10844 and 12697 with minor components at m/ez 10920–10933, 13280 and 15582 and some diffuse peaks at m/ez 24051–24059 and 33143–33277. Calprotectin isolated from the patient showed peaks at m/ez 10456, 10848, 12705 and 13169 and a diffuse peak at m/ez 38636. Lower mass peaks may be consistent with the presence of the P6 protein (N Hogg, personal communication).

6. DISCUSSION This patient has significantly increased concentrations of Zn and calprotectin in his plasma. The high molecular weight suggests an unusual tendency to form polymers or complexes. The apparent anomalies in the molecular weight distribution of calprotectin in the mother’s plasma suggests a genetic origin for the condition. Incomplete studies of the calprotectin gene have failed to show any mutations. All data seems to suggest that this patient has structurally normal calprotectin in abnormal amounts. It is not known whether this may be due to increased synthesis of calprotectin or to defective catabolism. Calprotectin is found in many cells, but is primarily associated with myelomonocytes and keratinocytes. It is a major component of the soluble proteins in neutrophil cytosol. A precise biological function has yet to be defined, but its plasma concentration is increased in many inflammatory conditions. In vitro, calprotectin shows marked antimicrobial and anti-proliferative actions (Johne et al., 1997; Yui et al., 1995). The antiproliferative actions of calprotectin are probably responsible for the clinical condition of the patient. Calprotectin is a trimer of two chains of the MRP14 protein and one of the MRP8 protein (Johne et al., 1997). It is a member of the S100 group of Ca binding proteins.. These proteins are characterised by 2 EF hands which contain the Ca binding domains. It has recently been suggested that a third low molecular weight protein, P6 may also be associated with the calprotectin complex (VandenBos et al., 1998). The mass spectrometry data may support the presence of this protein in the protein isolated from our patient. Calprotectin differs from other S100 proteins in its Zn binding capacity. MRP 14 has a higher Zn binding capacity than other S100 proteins such as CP10 or S100B, and the Zn binding capacity is not affected by the binding of calcium. The C-terminal region of the protein which contains the putative Zn binding domains has significant homology with part of the sequence of neutrophil-immobilising factor. Both the MRP8 and MRP 14 chains contain the HEXXH motif (Clohessy and Golden, 1996; Loomans et al., 1996) which has been characterised as the Zn-binding structure in Zn-dependent metalloproteases. The MRP14 chain also contains a triple histidine sequence at position 100 which has been suggested to be a Zn binding site responsible for the Zn-dependent antimicrobial activity of calprotectin. The Zn binding capacity of calprotectin has been linked to it’s anti microbial properties, the suggestion being high calprotectin concentrations reduce Zn availability to pathogens.

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REFERENCES Clohessy, P.A. and Golden, B.E., 1996, His-X-X-X-His motifs in S100 protein, calprotectin: relation to microbiostatic activity, J. Leukoc. Biol. 60:674. Clohessy, P. and Golden, B.E., 1995, Calprotectin mediated zinc chelation as a biostatic mechanism in host defence, Scand. J. Immunol. 42:551–556. Johne, B., Fagerhol, M.K., Lyberg, T., Prydz, H., Brandtzaeg, P., Naess-Andresen, C.F., and Dale, I., 1997, Functional and clinical aspects of the myelo-monocyte protein calprotectin, Molecular Pathology, 50:113–123. Loomans, H.J., Hahn, B.L., Li, Q.Q., Phadnis, S.H., and Sohnle, P.G., 1998, Histidine-based zinc-binding sequences and the antimicrobial activity of calprotectin. J. Infect. Dis. 177:812–814. Sampson, B., Kovar, I.Z., Rauscher, A., Fairweather-Tait, S., Beattie, J., McArdle, H.J., Ahmed, R., and Green, C., 1997, A case of hyperzincaemia with functional zinc depletion: a new disorder? Pediatr. Res. 42:219–225. VandenBos, C., Rammes, A., Vogl, T., Boynton, R., Zaia, J., Sorg, C., and Roth, J., 1998, Copurification of P6, MRP8, and MRP14 from human granulocytes and separation of individual proteins. Protein Expression and Purification 13:313–318. Yui, S., Mikami, M., and Yamazaki, M., 1995, Induction of apoptotic cell death in mouse lymphoma and human leukemia cell lines by a calcium-binding protein complex, calprotectin, derived from inflammatory peritoneal exudate cells, J. Leukoc. Biol. 58:650–658.

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CHARACTERISATION OF THE DIFFERENT STEPS DURING ZINC CHELATION INDUCED APOPTOSIS IN JURKAT AND HELA CELLS F. Chimienti1, M. Sève1, S. Richard2, J. Mathieu2, and A. Favier1 1

Laboratoire de Biologie du Stress Oxydant (LBSO) UJF Domaine de la Merci 38700 La Tronche 2 Unité de Radiobiologie et inflammation CRSSA, Avenue des Maquis du Grésivaudan 38700 La Tronche

Zinc is an important component of the cell survival. It participates in several proteins structure and its intracellular concentration seems to be precisely regulated. Zinc is involved in apoptosis regulation and has been responsible for DNA fragmentation during apoptosis. We try to define the mechanism of apoptosis induced by zinc deprivation and to determine the kinetic of the different steps in this type of apoptosis: morphological changes, nuceus condensation, DNA fragmentation, protein activation,.... Jurkat and HeLa cells were treated by the intracellular zinc chelator TPEN (N, N, N', N' tetrakis-(2 pyridylmethyl) ethylene diamine). The cells were then observed by phase-contrast and fluorescence microscopy, and harvested after 3, 6, 9, 12 or 24 hours of treatment. We show that deprivation of zinc induces apoptosis in Jurkat and HeLa cells with a typical morphology. Zinc deprivation by TPEN treatment was monitored in HeLa cells, using a new Zn-specific fluorescent probe: Zinquin-ethyl-ester. Observation by phase contrast microscopy provide evidence that membrane blebbing occurs after 3 hours of TPEN treatment Studies by fluorescence microscopy were assessed with DNA staining (Hoechst 33342). Nuclear condensation is visible after 6 hours of TPEN treatment andagarose gel electrophoresis confirms that DNA is fragmented after 6 hours. Additionally, the caspase-3 activity shows a huge increase with a maximum at 12 hours. By western-blot, we reveal the specific degradation of several transcription factors implicated in cell proliferation and differentiation. We conclude that zinc seems to be involved in the regulation of apoptosis not only in endonuclease protection, but in upstream specific activation pathways. 1035

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EFFECT OF ION ZINC ON THE METABOLISM OF PHOSPHATIDYLCHOLINE AND SPHINGOMIELIN IN LUNG OF RAT

Gomez N. N., Fernandez M. R., Ojeda M. S., and Gimenez M. S. Fac. de Qca. Bioquimica y Farmacia U.N.S.L. 5700, San Luis, Argentina

Zinc is an essential oligoelement for cellular metabolic equilibrium; its deficiency produces many disturbances. It is known that Zn is essential for the activity of numerous enzymes, either as a constitutent part of their molecules or as a required cofactor. Zn-requiring enzymes are related to energy utilization, protein synthesis and oxidative protection. Zn plays a fundamental role in stabilization of certain macromolecules and cellular membranes. Our aim was to assess whether Zn deficiency causes also alterations of the biosynthesis of phosphatidylchole (PC) and sphingomielin (Spm) with the incorporation of in slices in rat lung. Wistar male rats (200–230 g), were separated in two groups; with a treatment of two months and four months: Each group was separated into two lots: (Co) control fed with AIN-93 diet containing 30 Zn mg/kgabd (ZD) Zinc completely deficient, fed with the same diet without Zn. The animals were kept in cages with water and food ad libitum. Tissue collection and analysis: Rats were decapitated, the lungs were excised, rinsed in ice-cold saline, the lung slice (l00 mg) were rapidly cut and were immediately placed in individual flasks containing BHT medium, were preincubated with for 5 min at 37°C in Dubnoff metabolic shaking incubator. One chloride was added to initiate the reactions. After 60min of incubation, the reaction was stopped by dilution with medium. Immediately, after the incubation period, tissue slice were removed from the medium. Lipids extraction was performed by the methods of Hara et al. A portion of the lipid phase was used for the determination of total phospholipid content and total radioactivity. The remainder was evaporated under nitrogen gas. The duplicate samples were spotted on thin-layer chromatography, one spot was used to determine phosphorus by the method of. Rousser et al. In all cases the other spots were used for the determination of radioactivity in a Beckman LAS 100 C scentillation counter. Data were analysed by Student’s t test for unpaired samples. e-mail: [email protected]

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Results showed: Analyses of total radioactivity incorporated in form of cpm/g. tissue in: (two months) Co 3,746 ± 160 ZD 4,285 ± 174 p < 0.01 and in (four months); Co 1,183 ± 135 ZD 2,785 ± 120 p < 0.03. In two months; PC P Co 3,331 ± 175 ZD 4,332 ± 422 p < 0.01; Spm Co 720 ± 12 ZD 440 ± 44 p < 0.01; in four months PC: Co 1,390 ± 259 ZD 4,147 ± 169 p < 0.01 Spm Co 439 ± 148 ZD 801 ± 410 p = n.s. We can conclude that in lung of ZD rats, the sunthesis of PC increased after 2 and 4 months of treatment, while the Spm decreased after 2 months but it did not change at 4 months. Zinc deficiency alters the metabolism of phospholipids in lung, but the mechanisms must be studied.

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METALLOTHIONEIN, CALMODULIN, AND TRACE ELEMENTS IN HEALING SKIN WOUNDS B. Sampson1, A. Lansdown1, and A. Rowe2 Clinical Chemistry, Charing Cross Hospital London, UK1 and Dermatology, Imperial College London, UK2

Repair processes following injury involve a variety of proteolytic, biosynthesic and cell proliferative stages dependant on trace elements and metalloenzymes. The various enzyme activities and trace element requirements can be expected to vary according to the stage of wound healing. The present studies were designed to examine sequential changes in trace element concentrations and metal binding protein (metallothionein, MT and calmodulin, CAL) distribution in experimental incisional wounds in the rat. Standard incisional wounds were induced surgically in the mid dorsal skin of 6week old Sprague Dawley rats. Wounds were sutured with polyamide monofibre and left unoccluded. Trace element concentrations were measured in excised wound sites at up to 10 days post-wounding. Wound sites were also excised for routine histology and for immunocytochemistry. Zn concentrations increase through the inflammatory phase to 5 days post wounding. MT follows a similar pattern. In intact skin MT is located in the basal epidermis, in wounded skin highest concentrations are found in the papillary dermis proximal to the wound margin. Milder increases in MT in the deeper dermis may be consistent with increases seen in copper concentration. MT and Zn concentrations decline to normal by 10 days after wounding. CAL is more specifically associated with keratinocyte proliferation and development. Increases in dermal MT may suggest an accumulation of a zinc reservoir for metalloenzymes necessary for the repair process.

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ZINC MODULATION OF MACROPHAGES INTERLEUKIN 1 GENE EXPRESSION AND SECRETION

A. E. Aguilar, R. Pastelin, S. Pérez, and M. D. Lastra Laboratorio de Investigación en Inmunología Departamento de Biología Facultad de Química, UNAM Circuito Escolar, Ciudad Universitaria, México DF, CP 04510

Trace elements particularly Zn, have a great influence in the development and maintenance of the immune system. The role of Zn in the improvement of impaired immunity is well documented. The macrophage appears to be more susceptible to Zn intervention. IL-1 secretion is characteristic of macrophage activation, thus its modulation by Zn could result in the immune response elevation. We addressed this question by designing a model of BALB/c mice supplemented with Zn (500 mg/L) during gestation, lactation and postweaning. We investigated the possibility of zinc modulation of IL-1. We report that Zn can enhance the IL-1 gene expression on macrophages activated by LPS. Kinetic studies of IL-1 gene expression evaluated by RT-PCR, showed a maximum level of induction 4h after treatment with zinc, followed by a decrease to basal levels within l0h. IL-1 secretion evaluated by an ELISA assay, showed an increment in LPS stimulated peritoneal macrophages supernatant after the lactation period (6 weeks treatment) with a drop after the postweaning period (9 weeks treatment). IL-1 serum concentrations showed a significant increment after 6 weeks of zinc supplementation with no variation after 9 weeks. Results suggest an important role of zinc in macrophages stimulation, which carefully monitored could result in modulation of the monokine secretion and even modification of its seric concentrations.

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ASSESSMENT OF Zn ROLE IN MURINE EXPERIMENTAL CYSTICERCOSIS CAUSED BY TAENIA CRASSICEPS

M. D. Lastra., E. Sciutto, R. Pastelin, A. E. Aguilar, and G. Fragoso Laboratorio de Investigacion en Inmunología Departamento de Biología Facultad de Química, UNAM Circuito Escolar, Ciudad Universitaria México, DF, CP 04510

Zn is a trace element with a pivotal role in the immune system influencing T cells maturation and functions, elevating the antibody response and increasing the phagocytic capacity in mice perinatal stages Infection status often agrees with zinc concentrations. Zn supplementation could improve immune responses to parasite infections. To further improve knowledge about parasite infections and its zinc relation, this study assesses zinc (500 ppm) supplementation impact, in an experimental T.crassiceps model of cysticerci, evaluating the metal effects over lymphocytes subpopulations and parasite load. Female BALB/cAnN mice were distributed in four groups: Group I mice Zn supplemented (Zn+), non infected Group II mice Zn supplemented (Zn+), infected with 10 T.crassiceps cysticerci Group III control mice non-supplemented (Zn–) non-infected Group IV control mice non-supplemented (Zn–) infected Results show that significantly fewer parasites were recovered from Zn+(II) than from mice that did not receive Zn (IV), although this increased resistance was not related to antibody response. T.crassiceps cysticerci peritoneal infection induced a slight cellular immune depression evaluated by FACS, thus T cell proliferation response to total antigens of T.crassiceps cysticerci was depressed in Zn– mice (III); however mice infected with the parasite and supplemented with Zn (II) showed a normal response to specific cysticercal antigens. In addition spleen cells from group IV showed a significant decrease

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of CD3+, CD4+CD8– and CD4–CD8+ cells both “in vivo” and when kept in culture with RPMI or with specific cysticercal antigens added “in vitro”. However, this effect was not observed in infected mice Zn+(II). These results suggest the importance of zinc supplementation to increase the mice resistance to murine cysticercosis.

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THE DOGGED PATH TO ACCEPTANCE OF BORON AS A NUTRITIONALLY IMPORTANT MINERAL ELEMENT

Forrest H. Nielsen United States Department of Agriculture Agricultural Research Service Grand Forks Human Nutrition Research Center Grand Forks, North Dakota 58202-9034

U.S. Department of Agriculture, Agricultural Research Service, Northern Plains Area is an equal opportunity/affirmative action employer and all agency services are available without discrimination.

1. BACKGROUND BORON ESSENTIALITY STUDIES In 1923, Warington described signs of boron deficiency for several species of leguminous plants. This was followed by a report in 1926 by Sommer and Lipman showing that boron was essential for the completion of the life cycle of a number of monocotyledonous and dicotyledonous plants. These findings probably stimulated several early eminent nutrition scientists to make attempts to show that boron was essential for higher animals; these attempts failed (Follis, 1947; Hove et al., 1939; Orent-Keiles, 1941; Skinner and McHargue, 1945; Teresi et al., 1944). Thus, by 1950, the dogma had developed that boron was a unique element in that it was essential for plants but not for animals; a dogma so ingrained in nutrition that it has taken almost 20 years to overcome it. A happening in 1980 probably can be considered the seminal point in the long and vexatious path to boron being accepted as nutritionally important in higher animals including humans. After examining some growing chickens in an arsenic experiment being conducted by a graduate student, Eric Uthus, in my laboratory, I asked a quite pointed question that was preceded by a statement phrased something like: “Eric, your control chickens have abnormal looking legs and they are not growing well. Could you have left something out of the diet?” This led to the disclosure that he had made changes in the diet in an attempt to make it lower in arsenic; the changes were the Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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omission of boron, fluorine and nickel from the mineral mix, and a change in the source of vitamin D. Shortly thereafter, I asked Dr. Curtiss Hunt, a post doctorate who was working on vanadium in my laboratory at that time, to perform a simple experiment that had a profound effect on our subsequent research careers. Day-old chicks were divided into four groups and fed the modified arsenic diet supplemented with fluorine, boron, nickel, or all three elements. After three weeks, the chicks supplemented with boron grew better than those not receiving supplemental boron; neither nickel nor fluoride supplementation affected growth. The boron supplemented chicks also exhibited a more normal (but not completely normal) leg structure than the chicks supplemented with just nickel or fluoride. This led to the discovery that the new vitamin D source used in the modified arsenic diet was impotent, and to the decision to ascertain whether an interaction between boron and vitamin D affected growth and bone development in chickens. At the Trace Elements In Man and Animals (TEMA-4) symposium in 1981, I presented some of the first findings that the collaboration between Dr. Hunt and me had produced (Hunt and Nielsen, 1981). The reception of those findings could be best described as “remarkable indifference.” Attendance at the session was extremely light and questions and comments about the findings were minimal. Interestingly, in this same session, Rex Newnham gave a presentation in which he claimed boron could prevent or cure arthritis (Newnham, 1981). One of the findings I described at TEMA-4 was that rachitic long bones were found in 17 of 21 boron-deprived chicks, but only in 9 of 22 boron-supplemented chicks, fed a vitamin D deficient diet. Moreover, the lack of calcification was more severe in the boron-deprived chicks. The collaboration between Dr. Hunt and I subsequently confirmed the interaction between boron and vitamin D, and also showed that the dietary intake of calcium and magnesium affected the response to boron deprivation (Hunt, Shuler and Nielsen, 1983). In 1985, the senior scientist/post doctorate relationship between Dr. Hunt and I ended. Each of us continued to study boron independently with Dr. Hunt utilizing chicks several years before moving to rats as experimental animals, whereas I used only rats. Still, we apparently were the only scientists studying the possible essentiality of boron at this time; the reception to our findings could not be categorized as enthusiastic.

2. THE SIGNAL BORON ESSENTIALITY STUDIES BETWEEN 1987 AND 1991 Between 1987 and 1991, interest in boron essentiality was piqued beyond the Grand Forks Human Nutrition Research Center in North Dakota. In 1987, it was reported that a boron supplement of 3 mg/day markedly affected several indices of mineral metabolism of seven women consuming a magnesium low diet and five women consuming a magnesium adequate diet; the post menopausal women had consumed a conventional diet supplying about 0.25 mg of boron/day for 119 days (Nielsen et al., 1987). One finding in this experiment was that boron supplementation elevated serum concentrations of estradiol and testosterone with the elevation apparently more marked in the magnesium low women. Subsequent experiments confirmed that dietary boron can affect sex steroid status in humans (Nielsen et al., 1992; Samman et al., 1998). In 1987 and 1990, two additional studies performed at the Grand Forks Human Nutrition Research Center produced the most compelling findings to date (Nielsen et al., 1992; Nielsen, 1989; Nielsen et al., 1990; Nielsen et al., 1991) indicating that boron is of

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nutritional importance for humans. In these experiments men over the age of 45, post menopausal women, and postmenopausal women on estrogen therapy were fed a low boron diet or about 0.25 mg/2,000 kcal for 63 days, and then fed the same diet supplemented with 3.0 mg of boron for 49 days. Boron affected biochemical indicators related to bone turnover, physiological indicators of psychomotor and cognitive function, and blood cellular composition. For example, in both experiments, estrogen ingestion elevated serum 17 this elevation was higher during boron repletion than during boron depletion. This finding indicates that boron can enhance the effects of estrogen therapy which is used to prevent bone loss in postmenopausal women. Penland (1998) found that the boron supplementation after depletion altered electroencephalograms to suggest improved behavioral activation, or less drowsiness, and mental alertness, and improved psychomotor skills and cognitive processes of attention and memory. The suggestion that boron is a nutritionally important element based on these human findings drew mixed reactions. As bluntly stated in one review of the research: “The wisdom behind studying an element for which there is no credible evidence to indicate that it is nutritionally important can be questioned, especially when there is such a shortage of funds and opportunities to study nutritionally important elements such as zinc and iron in humans.” However, these experiments expanded the interest in the possible nutritional importance of boron beyond the Grand Forks Human Nutrition Research Center. For example, Hegsted et al. (1991) showed that boron deprivation decreased the apparent absorption and balance of calcium, magnesium and phosphorus in the vitamin D deficient rat. King, Odom et al. (1991) showed that in ovo injections of boron reduced the abnormal height of long bone growth plate in chicks hatched from vitamin D deficient chickens. In 1991, the U.S. Borax company began showing an interest in supporting boron essentiality research. Their “no strings attached” support accelerated the production of conclusive findings showing that boron is not only nutritionally important but essential.

3. BORON ESSENTIALITY VALIDATION STUDIES SUBSEQUENT TO 1991 Findings brought forth since 1991 has solidified the acceptance of boron as an essential nutrient with likely practical nutritional importance. Among the more important findings has been the demonstration that the lack of dietary boron interrupts the life cycle of the frog by interfering with normal development during organogenesis and markedly impairing normal reproductive function (Fort et al., 1998). Boron deficiency has been shown to have pathological consequences during two different stages of the life cycle of the zebrafish; these were membrane blebbing with cytoplasmic extrusion during the zygote and cleavage periods of embryogenesis which resulted in embryo death, and cone dystrophy in the adult stage (Eckhert and Rowe, 1999). Hunt and Idso (1999) have found that boron deprivation impairs immune function and exacerbates adjuvantinduced arthritis in rats. The finding that the lack of boron interrupts the life cycle of some animals is adequate evidence for establishing boron as an essential nutrient for higher animals. However, significant progress is being made in establishing another criterion for essentiality; that is, a defined biochemical function. In 1991, the basis for my hypothesis that boron has a biochemical function that influences hormone action, transmembrane signaling, and/or membrane function or stability was published (Nielsen, 1991). Substantiation for this

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hypothesis was findings indicating that boron deprivation affected the transport of calcium in and out of the cell (Nielsen, 1994). A fluorescent marker was used to measure cellular ionized calcium concentrations in platelets before and after activation with thrombin in the presence of external calcium; in potassium adequate rats the ionized calcium concentration upon activation was higher in platelets from boron deprived than supplemented rats. Potassium deficiency markedly reduced the ionized calcium concentration in boron deprived platelets activated with thrombin, but did not affect the concentration in platelets from boron supplemented rats. The frog and zebrafish findings described above also support the hypothesis that boron has a biochemical role at the membrane level. For example, membrane blebbing with cytoplasmic extrusion during the zygote and cleavage periods of embryogenesis, and cone dystrophy in the adult stage of zebrafish are changes occurring in cells that produce prodigious quantities of membrane (Eckhert and Rowe, 1999).

4. ACCEPTANCE OF BORON NUTRITIONALLY IMPORTANT An analysis of both human and animal data resulted in the suggestion in a WHO/FAO/IAEA (1996) publication that an acceptable safe range of population mean intakes of boron for adults could well be 1 to 13 mg/day. Many people apparently consistently consume less than 1 mg/day, the lower value for the safe range of intakes. For example, in a group of 43 peri-menopausal women studied in the eastern North Dakota area of the United States, two women apparently consumed an average of less than 0.5 mg of boron per day, and 14 women consumed between 0.5 and 1.0 mg of boron per day (Nielsen and Penland, 1999). Rainey and Nyquist (1998) also reported that many people consistently consume less than 1 mg of boron daily. The recent findings on boron essentiality, combined with the earlier human experiments, probably were instrumental in the decision by the Food and Nutrition Board of the National Academy of Sciences in the United States to consider establishing a Dietary Reference Intake (DRI) for boron. Based on findings to date, a DRI for boron should make people realize that consuming inadequate boron could possibly have detrimental consequences to good bone, brain, eye, immune, psychomotor, and reproductive function. In other words, all signs indicate that 20 years after boron was first suggested to be an essential nutrient for higher animals, boron will be accepted as a nutritionally important trace element in humans. At TEMA-9 in 1996, I ended my talk, which was the last one of the meeting, with the words “by the year 2000, boron most likely will be recognized as an element of clinical and nutritional importance” (Nielsen, 1997). The scientists that have labored in the dogged path to make this prediction come true should have a feeling of satisfaction for accomplishing a feat that should promote health and wellbeing throughout the world.

REFERENCES Eckhert, C.D. and R.I. Rowe, 1999, Embryonic dysplasia and adult retinal dystrophy in boron deficient zebrafish. J. Trace Elem. Exp. Med. 12: In press. Follis, R.H. Jr., 1947, The effect of adding boron to a potassium-deficient diet in the rat. Am. J. Physiol. 150:520–522. Fort, D.J., T.L. Propst, E.L. Stover, P.L. Strong, and F.J. Murray, 1998, Adverse reproductive and developmental effects in Xenopus from insufficient boron. Biol Trace Elem. Res. 66:237–259.

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Hegsted, M., M.J. Keenan, F. Siver, and P. Wozniak, 1991, Effect of boron on vitamin D deficient rats. Biol. Trace. Elem. Res. 26:243–255. Hove, E., C.A. Elvehjem, and E.B. Hart, 1939, Boron in animal nutrition. Am. J. Physiol. 127:689–701. Hunt, C.D. and J.P. Idso, 1999, Dietary boron as a physiological regulator of the normal inflammatory response: a review and current research program. J. Trace Elem. Exp. Med. 12: In press. Hunt, C.D., T.R. Shuler, and F.H. Nielsen, 1983, Effect of boron on growth and mineral metabolism, in: 4: Spurenelement Symposium (M. Anke, W. Baumann, H.E. Bräunlich, and C. Brückner, eds.), pp. 149–155, Friedrich-Schiller-Univ., Jena. Hunt, C.D. and F.H. Nielsen, 1981, Interaction between boron and cholecalciferol in the chick, in: Trace Element Metabolism in Man and Animals (TEMA-4) (J.McC. Howell, J.M. Gawthorne, and C.L. White, eds.), pp. 597–600, Australian Acad. Sci., Canberra. Newnham, R.E., 1981, Mineral imbalance and boron deficiency, in: Trace Element Metabolism in Man and Animals, (TEMA-4) (J.McC. Howell, J.M. Gawthorne, C.L. White, eds.), pp. 400–402, Australian Acad. Sci., Canberra. Nielsen, F.H. and J.G. Penland, 1999, Boron supplementation of peri-menopausal women affects boron metabolism, and indices associated with macromineral metabolism, hormonal status and immune function. J. Trace Elem. Exp. Med. 12: in press. Nielsen, F.H., 1997, Beyond copper, iodine, selenium and zinc: other elements that will be found important in human nutrition by the year 2000, in Ninth International Symposium on Trace Elements in Man and Animals (TEMA-9) (Fischer, P.W.F., M.R. L’Abbé, K.A. Cockell, and R.S. Gibson, eds.), pp. 653–655, NRC Research Press, Ottawa. Nielsen, F.H., 1994, Biochemical and physiologic consequences of boron deprivation in humans. Environ. Health Perspect. 102 (Suppl 7), 59–63. Nielsen, F.H., S.K. Gallagher, L.K. Johnson, and E.J. Nielsen, 1992, Boron enhances and mimics some effects of estrogen therapy in postmenopausal women. J. Trace Elem. Exp. Med. 5:237–246. Nielsen, F.H., 1991, Nutritional requirements for boron, silicon, vanadium, nickel and arsenic: current knowledge and speculation. FASEB J. 5:2661–2667. Nielsen, F.H., L.M. Mullen, and E.J. Nielsen, 1991, Dietary boron affects blood cell counts and hemoglobin concentrations in humans. J. Trace Elem. Exp. Med. 4:211–223. Nielsen, F.H., L.M. Mullen, and S.K. Gallagher, 1990, Effect of boron depletion and repletion on blood indicators of calcium status in humans fed a magnesium-low diet. J. Trace Elem. Exp. Med. 3:45–54. Nielsen, F.H., 1989, Dietary boron affects variables associated with copper metabolism in humans, in: 6th International Trace Element Symposium 1989, Vol 4 (M. Anke, W. Baumann, H. Bräunlich, C. Brückner, B. Groppel, and M. Grün, eds.), pp. 1106–1111, Friedrich-Schiller-Univ., Jena. Nielsen, F.H., C.D. Hunt, L.M. Mullen, and J.R. Hunt, 1987, Effect of dietary boron on mineral, estrogen and testosterone metabolism in postmenopausal women. FASEB J. 1:394–397. Orent-Keiles, E., 1941, The role of boron in the diet of the rat. Proc. Soc. Exp. Biol. Med. 44:199–202. Penland, J.G., 1998, The importance of boron nutrition for brain and psychological function. Biol. Trace Elem. Res. 66:299–317. Rainey, C. and L. Nyquist, 1998, Multicountry estimation of dietary boron intake. Biol. Trace Elem. Res. 66:79–86. Samman, S., M.R. Naghii, P.M. Lyons Wall, and A.P. Verus, 1998, The nutritional and metabolic effects of boron in humans and animals. Biol. Trace Elem. Res. 66:227–235. Skinner, J.T. and J.S. McHargue, 1945, Response of rats to boron supplements when fed rations low in potassium. Am. J. Physiol. 143:385–390. Sommer, A.L. and C.B. Lipman, 1926, Evidence of the indispensable nature of zinc and boron for higher green plants. Plant Physiol. 1:231–249. Terresi, J.D., E. Hove, C.A. Elvehjem, and E.B. Hart, 1944, Further study of boron in the nutrition of the rat. Am. J. Physiol. 140:513–518. Warington, K., 1923, The effect of boric acid and borax on the broad bean and certain other plants. Ann. Bot. 37:629–672. WHO/FAO/IAEA, 1996, Trace Elements in Human Nutrition and Health, pp. 175–199, World Health Organization, Geneva.

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HOMING IN ON THE MOLECULAR BASIS OF BORON ESSENTIALITY USING DIFFERENTIAL DISPLAY, GENE ARRAYS, AND NORTHERN BLOTS

C. D. Eckhert, A. Bennett, K. Becker, and D. Luo Department of Environmental Health Sciences University of California Los Angeles, California, USA 90095-1772

1. INTRODUCTION The first unambiguous demonstration of boron essentiality in animals was the discovery that it stimulated embryonic growth in trout (Ooncorhynchus mykiss) (Eckhert, 1998). In subsequent studies using zebrafish (Danio rerio) we found that boron was essential for both completing the cleavage stage of embryonic development (Rowe and Eckhert, 1998, 1999) and preventing dystrophy of mature photoreceptors (Eckhert, 1999). Based on these encouraging observations we initiated a search for boron responsive eukaryote genes. For this we prepared boron deficient yeast (Saccharromyces cerevisae) for genetic screening.

2. MATERIALS AND METHODS Yeast were grown at 30° to early log phase (9 h) in boron depleted media B) (Bennett, Rowe and Eckhert, 1999). The culture was then divided into two flasks. One flask was supplemented with boron using ultrapure boric acid and the other with an equivalent volume of ultrapure water. The yeast cultures were allowed to grow for an additional thirty minutes and then pelleted by centrifugation and frozen at –70°. RNA was prepared from the pellets and evaluated using differential display, gene arrays and Northern blots.

[email protected] Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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3. RESULTS All three techniques demonstrated the existence of a set of boron responsive genes. Boron supplementation resulted in mRNAs that increased and decreased (Table 1). The following figures give the results from the differential display (Fig. 1), gene array (Fig. 2) and Northern blots (Fig. 3). Boron responsive genes included those that were fundamental to cell growth and maintenance. The mRNAs that were increased in response to boron encoded for: two elongation factors (TEF2 and YEF2) that function in peptide elongation on the ribosome; a putative nuclear protein (MAK16); a putative cell surface glycoprotein (SED1) and a metallothionein-like protein (CRS5). The mRNAs that decreased in response to boron encoded for: a beta-transducin-like protein necessary for the maintenance of killer Ml double-stranded RNA (MAK11); an arginine specific carbamoyl phosphate synthetase (CPA1); saccharopine reductase (LYS9); ribosomal protein SIB (RPS1B); a calcium and phospholipid binding protein homologous to EF-1 gamma (CAM1) and methionine amino peptidase (MAP1).

4. DISCUSSION Boron is an essential element for vascular plants (Warington, 1923) and for fish (Eckhert, 1998, Rowe et al., 1998 and Rowe and Eckhert, 1999). There is strong evidence

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that frogs and rodents also require the element for development. Fort fed Xenopus, the African frog, a rat diet containing low boron rat diet mated them and cultured their eggs in a low boron salt solution The low boron conditions resulted in a high incidence of embryonic mortality and larval malformations (Fort et al., 1998). Laposata and Dunson obtained eggs from several frog species foraging in the wild and incubated them in graded concentrations of boron. They did not observe a negative impact on either the hatch or malformation rate when the boron concentration was

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below the limit of detection (Laposata and Dunson, 1998). This suggests that boron is needed during the formation of germ cells. Lanoue et al. fed the same lots of low boron diet used by Fort in his Xenopus study to rats in two studies. She observed a slightly lower number of implantation sites in her first, but not second study. When the same diet was fed to mice, which had their embryos removed and incubated in a low boron culture medium, a high rate of embryonic degeneration and death was observed (Lanoue et al., 1998). The molecular basis for boron essentiality has not been determined for either. Knowledge of the molecular trafficking of copper, zinc and iron in eukaryotes has been greatly advanced by studies using Saccharomyces cerevesiae (yeast) (Pufahl et al., 1997; Eide, 1998). This unicellular eukaryotic organism shares significant genetic and protein homologies with mammals and to a lesser extent plants. The liquid culture system allows for rapid growth and since the entire yeast genome has been sequenced the biological role of boron at the genetic level can be evaluated. Our use in this report, of Saccharomyces cerevesiae to study boron, has provided the first demonstration of the existence of boron responsive genes.

ACKNOWLEDGMENTS This study was performed with financial support from the UC Water Resources Center (Project W-846) and a gift from U. S. Borax.

REFERENCES Bennett, A., Rowe, R.I., and Eckhert, Boron Stimulates Yeast (Saccharomyces cerevisiae) Growth, J. Nutr. 1999 IN PRESS. Eckhert, C.D., 1998, Boron stimulates embryonic trout growth, J. Nutr. 128:2488–2493. Eide, D.J., 1998, The molecular biology of metal ion transport in Saccharomyces cerevisias, Annual Review of Nutrition 18:441–469. Fort, D.J., Propst, T.L., Stover, E.L., Strong, P.L., and Murray, F.J., 1998, Adverse reproductive and developmental effects in Xenopus from insufficient boron, Biol. Trace Element Res. 66:237–259. Lanoue, L., Taubeneck, M.W., Muniz, J., Hanna, L.A., Strong, P.L., Murray, F.J., Nielsen, F.H., Hunt, C.D., and Keen, C.L., 1998, Assessing the effects of low boron diets on embryonic and fetal development in rodents using in vitro and in vivo model systems, Biol. Trace Element Res. 66:271–298. Laposata, M.M. and Dunson, W.A., 1998, Effects of boron on nitrate on hatching success of amphibian eggs, Arch. Environ. Contam. Toxicol 35:615–619. Rowe, R.I. and Eckhert, C.D., 1999, Boron is required for zebrafish embryogenesis, J. Exp. Biol. 202:1649–1654. Rowe, R.I., Bouzan, C., Nabili, S., and Eckhert, C.D., 1998, The response of trout and zebraflsh embryos to low and high born concentrations is U-shaped, Biol. Trace Element Res. 66:262–270. Warington, K., 1923, The effect of boric acid and borax on the broad bean and certain other plants, Ann. Bot. 37:629–672.

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ESTIMATION OF DIETARY BORON INTAKE IN SIX COUNTRIES Egypt, Germany, Great Britain, Kenya, Mexico, and the United States

1

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Charlene Rainey , Leslie Nyquist , Jennifer Casterline , 3 and Dena Herman 1

Nutrition Research Group 4199 Campus Drive Suite 550, Irvine California 92612 2 Program in International Nutrition One Shields Avenue University of California Davis, California 95616 3 Department of Public Health 640 Charles Young Drive South University of California Los Angeles, California 90095

1. INTRODUCTION Dietary Reference Intakes for boron are currently under consideration by the Food and Nutrition Board panel for trace elements. Boron is a naturally-occurring trace element in the human diet. Estimated dietary boron intakes provide useful information for planning and assessing diets in healthy populations. Egypt, Germany, Great Britain, Kenya, Mexico and the United States were selected for this study because they represent a wide variety of dietary patterns and have adequate food consumption data.

2. MATERIALS AND METHODS 2.1. Food Consumption Survey Data Large-scale nationwide survey data were provided by the U.S., Germany and Great Britain. U.S. boron intake estimates are based on the U.S. Department of Agriculture’s Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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(USDA) Continuing Survey of Food Intakes by Individuals (CSFII) 1994–96 (USDA, 1996) and CSFII 1989–91 (USDA, 1998). German estimates are based on the German National Consumption Study (NVS) 1985–89 (Institute for Nutritional Research, 1992), and estimates for Great Britain utilize the Dietary and Nutritional Survey of British Adults 1986–87 (Office of Population Census Surveys, 1991). Survey data from rural agricultural communities of Mexico, Kenya and Egypt were provided by the Human Nutrition Collaborative Research Support Program (NCRSP) 1983–86 (Murphy and Bunch, 1994).

2.2. Boron Nutrient Database A boron nutrient database was created to include boron concentrations for the foods consumed in each country. Version 1.0 of the database incorporates boron analytical data from various sources in the U.S., Finland, U.K., Italy, Japan, and China. Version 2.0 of the Boron Nutrient Database was recently developed for the purpose for the purpose of updating and expanding the analytical data on the boron content of foods. The methods by which version 2.0 was developed are similar to those used for version 1.0 (Rainey et al., 1999). This included searching the literature for credible analytical data on the boron of foods. The data were evaluated using a quality index system to assess analytical methodology, analytical quality control, number of samples, sample handling and sampling plan. Data for like foods were aggregated to determine the median and range of boron concentrations for each food. The median boron values from the analytical database were then used to calculate boron for other foods. Both versions 1.0 and 2.0 of the boron nutrient database represent at least 95% (by weight) of foods consumed by survey respondents age 4 and older in the six countries.

2.3. Estimating Dietary Boron Intake Each person’s average daily boron intake was then estimated by linking the boron database with the survey food records. Version 1.0 of the database was used in estimating boron intakes for the U.S. (1989–91 estimates), Germany, Great Britain, Mexico, Kenya, and Egypt. Version 2.0 was used in estimating boron intakes from the new U.S. CSFII 1994–96. Dietary boron intake estimates were then generated for various age and sex groups. Estimates for the U.S. were weighted according the sample weights provided with the survey data for CSFII 1989–91 and CSFII 1994–96. In addition, the CSFII 1994–96 data were adjusted using C-SIDE version 1.02, courtesy of Iowa State University Statistical Laboratory (Dodd, 1996, Nusser, Carriquiry, Dodd, and Fuller, 1996).

3. RESULTS AND DISCUSSION Figures 1 and 2 illustrate the mean, percentile and percentile estimates for adult males and females (age 19 and older). The U.S., Great Britain, and and Egypt have lowest boron intakes. The highest intakes were found for Germany, Kenya, and Mexico. New adult intake estimates for the U.S. (CSFII 1994–96) are slightly higher than previous estimates based on CSFII 1989–91. Table 1 compares the mean, percentile and percentile estimates from the two surveys.

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REFERENCES Dodd, K.W., 1996, A technical guide to C-SIDE, software for intake distribution estimation, Technical Report 96-TR 32, Dietary Assessment Research Series Report 9 (September 1996), Department of Statistics and Center for Agricultural and Rural Development (CARD), Iowa State University. Institute for Nutritional Research (Institute für Ernährungswissenschaft, IfE), 1992, National Consumption Study (Nationale Verzehrsstudie, NVS) and Nutrition Survey and Risk Factor Analysis Study (Verbundstudie Ernährungserhebung und Risikofaktorenanalytik, VERA), Machine-readable data set, Public use files no. 029, Institute für Ernährungswissenschaft, Giessen, Germany. Murphy, S.P. and Bunch, S., 1994, Human Nutrition Collaborative Research Support Program (CRSP), Machine-readable data sets, University of California, Berkeley, Berkeley, CA. Nusser, S.M., Carriquiry, A.L., Dodd, K.W., and Fuller, W.A., 1996, A semi-parametric transformation approach to estimating usual daily intake distributions, Journal of the American Statistical Association 91(436):1440–1449. Office of Population Census Surveys, Social Survey Division, 1991, Dietary and Nutritional Survey of British Adults, 1986–1987, Machine-readable data set, Study No. 2836, The Data Archive (distributor), Colchester, Essex, UK. Rainey, C.J., Nyquist, L.A., Christensen, R.E., Strong, PL., Culver, B.D., and Coughlin, J.R., 1999, Daily boron intake from the American diet, J Am Diet Assoc 99:335-340. U.S. Department of Agriculture (USDA), Agricultural Research Service (ARS), 1995, 1989–91 Continuing Survey of Food Intakes by Individuals and 1989–91 Health Knowledge Survey, (CSFII/CHKS), CDROM, Available from National Technical Information Service, Springfield, VA. USDA, ARS, 1998, 1994–96 Continuing Survey of Food Intakes by Individuals and 1994–96 Health Knowledge Survey, (CSFII/CHKS), CD-ROM, Available from National Technical Information Service, Springfield, VA.

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NUTRITIONAL ESSENTIALITY OF BORON FOR DEVELOPMENT, MATURATION, AND REPRODUCTION IN FROGS A Review 1,2

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D. J. Fort , R. L. Rogers , E. L. Stover , P. L. Strong , 4 and F. J. Murray 1

The Stover Group, Stillwater, OK, USA Department of Zoology Oklahoma State University Stillwater, OK, USA 3 U.S. Borax, Valencia, California, USA 4 Murray and Associates Inc., San Jose, California, USA 2

1. INTRODUCTION In contrast to its essentiality in vascular plants, most research to date has focused on the toxicity of boron to lower vertebrates as well as mammals. Although investigators (Nielsen, 1992) have evaluated the nutritional significance of boron in mammals, including humans, it has been only recently demonstrated that boron likely plays an important role in the development of fish (Eckhert, 1998; Rowe et al., 1998; Rowe and Eckhert, 1999, in press) and frogs (Fort et al., 1998a; Fort et al., 1998b; Fort et al., 1999a, b and 1999c, in press).

2. DEVELOPMENTAL EFFECTS AND BORON DEFICIENCY In two separate depletion studies conducted previously (Fort et al. 1998a, b, 1999a, b, and 1999c, in press), frogs fed a low boron diet (–B; 45 (gB/kg) for either 28 d or 120 d produced a greater proportion of necrotic eggs and fertilized embryos which abnormally gastrulated at a greater rate. The affected embryos were substantially less viable at 96-h of development when compared to embryos from adults administered a diet supplemented with boron (+B; 1,850 (gB/kg). These studies showed that 28-d or 120-d of insufficient boron markedly impaired normal Xenopus laevis embryo development, such that no embryos from –B administered adults survive and develop normally, suggesting Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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that boron is nutritionally essential in Xenopus. These studies also demonstrated that both the incidence and severity of adverse effects are greatly increased after 120-d on the –B diet, in comparison to observations after 28-d on the –B diet. Complete concentration-response curves were developed for boron in Xenopus using a 4-d embryo-larval developmental model (Frog Embryo Teratogenesis Assay: Xenopus [FETAX]) (Dumont et al., 1983) documenting nutritionally essential, as well as toxic, concentration ranges (Fort, 1999a, b, and 1999c, in press). Deleterious developmental effects were induced at culture media concentrations of <0.3 (M B (essential) and >4980 (M (toxic). Four-d development in Xenopus embryos administered boron in the culture media at concentrations ranging from 0.5 to 3,320 (M was normal. An evaluation of the impact of –B diet administration on Xenopus maturation indicated that when compared to their +B counterparts, larvae administered the –B diet showed markedly slower rates of metamorphosis, specifically tail resorption and skin maturation. Because maturation, including tail resorption, is controlled by the thyroid axis, these studies suggest that low boron administration may be interfering with the thyroid axis. Reversal of the inhibitory effects of –B administration on tail resorption by both thyroxin and iodine further suggests that the effects observed were due to disruption of the thyroid axis. Initial measurements of triiodothreonine (T3), thyroid hormone in Xenopus, indicate that T3 levels are at least 2.5fold lower in –B larvae than detected in either the BLL or +B-reared larvae. The reversal of tail fin resorption inhibition by the addition of 100 (gB/L to the culture media indicates that boron is likely playing a role in the thyroid axis and may be important in the synthesis of T3. Thus, these studies represent the first indication of a biochemical or physiological role of boron in the vertebrate body without the addition of a physiological stressor.

3. BORON DEFICIENCY AND REPRODUCTION Adults administered the –B diet for 120d had distinctly atrophied ovaries and testes compared to adults administered the +B diet (Fort et al., 1999b, in press). Females administered the –B diet had a much greater proportion of immature oocytes in stages 1 and 2 [ca. –B = 87% vs. +B = 23%] than females fed the +B diet, which had a greater proportion of mature oocytes in stages 5 and 6 (mature) [ca. –B = 3% vs. +B = 45%]. Coculture of progesterone with normal stage 1 and 2 oocytes to stage 5 and 6 oocytes in vitro allowed for the successful maturation of stage 1 and 2 oocytes from the +B females but not from the –B females, which did not respond to hormone treatment. The adverse effects of low dietary boron in the female Xenopus were noted following both depletion periods; however, the effects were more dramatic following 120-d of boron depletion. Reductions in sperm counts and increases in sperm dysmorphology rates were noted in males fed the –B diet when compared to males fed the +B diet. Reproductive effects associated with boron deficiency in the female included ovary atrophy, oocyte necrosis, and incomplete oocyte maturation. Of these findings, the latter was the most intriguing. In the present study, incomplete maturation of “normal appearing” –B oocytes was marked by size and incomplete breakdown of the germinal vesicle. Normal germinal vesicle breakdown was observed in oocytes from the +B dietmaintained females. Unlike the oocytes from +B diet-maintained females, addition of progesterone in vitro did not stimulate germinal vesicle breakdown of oocytes from –B females. The inability of the –B oocytes which were “normal appearing” to mature in the presence of progesterone in vitro suggested that boron deficiency may disrupt the

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maturation process at either the progesterone receptor level, or possibly in the cascade of secondary and tertiary messenger events responsible for inducing the maturation of the oocyte. A statistically significant (P > 0.05) decrease in testis weight and sperm count were noted in the –B males when compared to the +B diet-maintained adults. Decreased sperm counts in the –B diet-fed males appeared to be simply due to a general decrease in testis health. An increase in sperm dysmorphology was also noted in the –B males. The primary site of dysmorphogenesis was identified in the base of the neck region of the sperm and characterized by a kinking of the tail. Aside from the flexure, sperm morphology appeared normal. Motility of the abnormal sperm was somewhat affected. Because the rate of fertilization in –B diet-administered Xenopus recorded in previous studies (Fort et al., 1998a, b, 1999a, b, and 1999c, in press) has only been somewhat reduced, it appears that the biological significance of these effects are not as substantial as observed in the females administered the –B diet. However, these results indicate that the impact of boron deficiency on male reproductive performance in higher vertebrates should not be overlooked. It is also interesting that a boron-enriched diet might decrease the rate of abnormal sperm cells in Xenopus and increase testis weight slightly. Similar to these enhancement effects in the male, the boron-enriched diet in the female appeared to increase oviduct size and ovary weight. Results of boron tissue analysis indicated that the –B diet reduced ovary, oviduct, oocyte, and testis boron levels and that the +B diet tissues contained the greatest levels of boron. These results were not necessarily surprising, since the +B diet contained the greatest quantity of boron. The length of boron depletion or supplementation dictated the magnitude of the tissue boron changes, with longer depletion (120d) reducing boron levels more appreciably than the shorter depletion period (28 d). Conversely, the longer supplementation period (120d) increased tissue boron concentrations when compared to the shorter dietary administration period. Boron levels in maturing oocytes generally increased from the immature (stages 1 and 2) to the mature (stages 5 and 6). Oocytes from the +B diet-administered females contained appreciably greater levels of boron than oocytes from the –B diet-fed group.

4. CONCLUSIONS These studies demonstrate that although the adverse effects of boron deficiency on early embryonic development have been documented in Xenopus (Fort et al., 1998a, b, 1999a, b, and 1999c, in press), responses to boron deficiency in Xenopus may be observed significantly earlier during the development and maturation of the oocytes and production of mature sperm cells. Although the biochemical link between these early reproductive effects and the embryo-larval developmental effects observed in previous studies (Fort et al., 1998a, b, 1999a, b, and 1999c, in press) has not yet been made, results from these studies clearly indicate the nutritional essentiality of boron for normal reproduction, development, and maturation in Xenopus.

REFERENCES Dumont, J.N., Schultz, T.W., Buchanan, M., and Kai, G., 1983, Frog embryo teratogenesis assay: Xenopus— A short-term assay applicable to complex mixtures, in: Symposium on the Application of Short-Term

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Bioassays in the Analysis of Complex Environmental Mixtures II (M.D. Waters, S.S. Sandhu, J. Lewtas, L. Claxton, N. Chernoff, and S. Nesnow, eds.), pp. 393–405, Plenum Press, New York. Eckhert, C.D., 1998, Boron stimulates embryonic trout growth, J. Nutr. 128:2488–2493. Fort, D.J., Propst, T.L., Schetter, T., Stover, E.L., Strong, P.L., and Murray, F.J., 1998a, Adverse developmental and reproductive effects of insufficient boron in Xenopus: Building a case for nutritional essentiality, FASEB J. 12(4):A207. Fort, D.J., Propst, T.L., Stover, E.L., Strong, P.L., Murray, F.J., 1998b, Adverse effects of insufficient boron in Xenopus, Biol. Trace Elem. Res. 66:237–259. Fort, D.L., Propst, T.L., Stover, E.L., Murray, F.J., and Strong, P.L., 1999a, Adverse effects from low dietary and environmental boron exposure on reproduction, development, and maturation in Xenopus laevis, J. Trace Elem. Exp. Med. 12(3):175-185, 1999. Fort, D.J., Stover, E.L., Strong, PL., and Murray, F.J., 1999b, The effect of boron deprivation on reproductive parameters in Xenopus laevis, J. Trace Elem. Exp. Med. 12(3): 187–204, 1999. Fort, D.J., Stover, E.L., Strong, P.L., Murray, F.J., and Keen, C.L., 1999c, Chronic Feeding of a Low Boron Diet Results in Adverse Reproductive and Developmental Effects in Xenopus laevis. J. Nutrition, in press. Nielsen, F.H., 1992, Biochemical and physiological consequences of boron deprivation in humans, Environ Health Perspect. 102(Suppl 7):59–63. Rowe, R.I. and Eckhert, C.D., 1999, Boron is required for zebrafish embryogenesis, FASEB J. in press. Rowe, R.I., Bouzan, C., Nabili, S., and Eckhert, C.D., 1998, The response of trout and zebrafish embryos to low and high boron concentrations is U-shaped, Biol. Trace Elem. Res. 66:261–270.

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EFFECTS OF BORON IN WOUND HEALING Experiments on Nude Mice

Brigitte Dousseta, Mohamed Benderdoura, Ketsia Hessa, Rosine Mayap-Nzietchuenga, Francine Bellevillea, and André Duprezb a

Laboratory of Medical Biochemistry School of Medicine University Henri Poincare Nancy I PO Box 184, 54505 Vandoeuvre-Les-Nancy Cedex France b Laboratory of Anatomopathology School of Medicine University Henri Poincare Nancy I PO Box 184, 54505 Vandoeuvre-Les-Nancy Cedex France

1. INTRODUCTION Boric acid is known to improve wound healing (Blech, 1990). It occurs on the proliferative phase and particularly on angiogenesis. Angiogenesis is a complex process positively regulated by compounds of extracellular matrix and soluble factors. The migration of endothelial cells in the wound, their proliferation and the basement membrane degradation are involved in this process (Madri et Bell, 1992). The effect of boron is perhaps which stimudue to the growth factors release (Benderdour et al., 1997) such as lates protease activity particularly collagenase activity (Bettinger et al., 1994). The VEGF (vascular endothelium growth factor) which is also a potent-up regulator of angiogenesis, promotes the endothelial cell proliferation and migration, induces macrophage migration and modifies extracellular matrix composition leading to the angiogenesis (Frelin et al., 1997). In this work, we investigated the boric acid effect on the release of angiogenic factors in vivo using sponges implanted on the backs of nude mice as experimental model. Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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2. MATERIAL AND METHODS 2.1. Materials 10 nude mice, weighing between 25 and 30 g, were included in the experiment. Sponges of polyethylene (2cm x 3cm x 2mm) previously boiled in distilled water for 30min and rinsed several times, were implanted by micro surgery under the skin of mice backs. The mice received 1 ml sterile aqueous solution of HCl pH 5 in the sponge (4 mice, control group) or 1 ml 3% boric acid, sterile solution (6 mice, assay group), every two days for 21 days. Then the sponges were removed by surgery and cut in small pieces.

2.2. Histology Pieces from all sponges were fixed in 4% buffered formaldehyde, included in paraffine, cut at and coloured with hematoxyline-eosine-saffron. The slices were observed in optic microscopy.

2.3. Biochimical Analysis Sponge fragments of identical weight were ground in NaCl 0.15M with a potter, the mixture was centrifuged at l,000g for 15min. The supernatant was collected and analysed. Total proteins were determined by bicinchoninic acid procedure (Sigma). The results were expressed in proteins/mg sponge.

2.4. SDS-PAGE Electrophoresis and Immunoblotting They were carried out according to the method described by Benderdour et al. (1997) with of each supernatant and anti-hsp 70 monoclonal antibody (Sigman H5147), diluted 1/5,000 in PBS-Tween 20 or rabbit-polyclonal anti-human TNFα antibody diluted 1/250 (Genzyme IP 130) or polyclonal antibody anti-human VEGF (calbiochem Ref. 676472) diluted 1/250. The membranes were washed three times in PBS-Tween and incubated with second antibody diluted 1/8,000 in PBS-Tween 20 (rabbit monoclonal antimouse IgG conjugated to peroxidase, Sigma A 9044) or with second antibody diluted 1/6,000 in PBS-Tween 20 (anti-rabbit IgG conjugated to peroxidase, Sigma A 0545) (Benderdour et al., 1997).

3. RESULTS The mice of assay group received 11 ml of 3% boric acid (330 mg of corresponding to 770 g for a man weighing 70 kg. No mouse died in control group, a mouse died at day 20 in assay group. No mouse had cutaneous reaction.

3.1. Histology In control group. The sponges were covered with normal mouse epidermis, densified dermis and few muscular fibrillis. The more external part of sponge contained fundamental substance, some fibromyoblasts and some new vessels. Accelular protein substance weakly coloured by eosinophile occupied the central zone of the sponge. In

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assay group, dermis in contact with sponge was greatly vascularised, numerous vessels were observed in the external zone of the sponge and the fibromyoblasts were more numerous than in control. Therefore, the histology showed that boric acid preferentially affected neoangiogenesis (Fig. 1).

3.2. Biochemistry Boric acid increased slightly but significantly the protein amonts in the sponge (4.6 proteins/mg sponge in control versus 5.7 ± 0.9 in assay). This result was confirmed by the electrophoresis. However, specific proteins such as hsp 70, VEGF were greatly increased by boric acid treatment (Fig. 2).

4. DISCUSSION The formation of granulation is a key phase in the wound healing. Clinically, boron acts on this phase, it potentiates the formation of new blood vessels; moreover, it is recommended to stop its application at this stage of healing.

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The morphology of fibroblasts changes during granulation tissue formation and they are transformed in fibromyoblasts (Walderf et Fewkes, 1995). We observed that colonization of sponges by fibromyoblasts is more important in assay than in control. Futhermore, the new vessels are more abundant in assay than in control confirming that boron stimulates angiogenesis. First, the angiogenesis induction affects the protease antiprotease activity of cells. In fact, proteolysis is necessary to the capillary development and maturation. Different factors regulate protease activity such as bFGF, TGFβ, TNFα (Madri et al., 1996). Boron stimulates the intracellular cell protease activity perhaps through its effect on the release of TNFα and VEGF (Benderdour et al., 1998). Moreover, stimulates the transcription of adhesion molecules as VCAM-1, molecule implicated in angiogenesis (Modur et al., 1996). VEGF is also more synthetized in assay than in control. VEGF is synthetized by different cells particularly by smooth muscular cells and it is highly specific for endothelial cells. VEGF is expressed in spatial and temporal association with events of angiogenesis in vivo (Shweiki et al., 1993). Futhermore isoforms of VEGF can bind to cell surface and extracellular matrix associated with heparan sulfate proteoglycans, and can release angiogenic factor such as bFGF (Neufeld et al., 1999). Boron also potentiates the synthesis of hsp 70 (heat shock protein 70). This protein protects the cells again the stress. Different hsp have been found in wound (Boehncke

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et al., 1994). Oberringer et al., (1995) found a strong correlation between wound healing and production of hsp 70. In conclusion, boron seems improve the wound healing in promoting the synthesis and the release of angiogenic factors.

AKNOWLEDGMENTS This study was performed with the financial support of region lorraine and ARIZE.

REFERENCES Benderdour, M., Hess, K., Dzondo-Gadet, M., Dousset, B., Nabet, P., and Belleville, F., 1997, Effects of boric acid solution on cartilage metabolism, Biochem. Biophys. Res. Comm., 234:263–268. Benderdour, M., Hess, K., Dzondo-Gadet, M., Nabet, P., Belleville-Nabet, F., and Dousset, B. 1998, Boron modulates extracellular matrix and TNFα synthesis in human fibroblasts, Biochem. Biophys. Res. Commun., 246:746–751. Bettinger, D.A., Pellicane, Y.V., Tary, W.C., Yager, D.R., Diegelman, R.F., Lee, R., Cohen, I.K., and Demarie, E.J., 1994, The role of inflammatory cytokines in wound healing: accelerated healing in endotoxinresistant mice, J. Trauma, 36:810–814. Blech, M.F., Martin, C., Pichon, M., Borelly, J., and Hartemann, P., 1990, The clinical and bacteriologic out come of wounds using different local antiseptics, J. Orthop. Surgery, 4:123–129. Boehncke, W.H., Dahle, A., Zollner, T.M., and Sterry, W., 1994, Differential expression of shock protein 70 (hsp 70) and heat shock cognate protein 70 (HSC 70) in human epidermis, Arch. Dermatol. Res., 287:68–71. Frelin, C., Ladoux, A., and Bauters, C., 1997, VEGF: mediateur de l’angiogénèse hypoxique, Med. Sci., 13:886–891. Madri, J.A. and Bell, L., 1992, Vascular-cell response to injury: modulation by extracellular matrix and soluble factors, in: Ultrastructure membrane and cell interactions in atherosclerosis, (H. Robenek and N. Severs, eds.), pp. 167–181, CRC press, Boca Raton F1. Madri, J.A., Sankar, S., and Romanie, A.M., 1996, Angiogenesis, in: The molecular and cellular biology of wound repair (2nd edition), (R.A.F. Clark, eds.), pp. 355–371, Plenum Press, New-York. Modur, V., Zimmerman, G.A., Prescott, S.M., and McIntyrc, T.M., 1996, Endothelial cell inflammatory response to tumor necrosis factor alpha, J. Biol. Chem., 271:13094–13102. Neufeld, G., Cohen, T., Gengrinovitch, S., and Poltorak, Z., 1999, Vascular endothelial growth factor and its receptor, FASEB J., 13:9–22. Oberringer, M., Baum, H.P., Jung, V, Welter, C., Frank, J., Kuhlmann, M., Mutschler, W., and Hanselmann, R.G., 1995, Differential expression of heat shock protein 70 in well healing and chronic human wound tissue, Biochem. Biophys. Res. Comm., 214:1009–1014. Shweiki, D., Itin, A., Neufeld, G., Gitay-Goren, H., and Keshet, E., 1993, Patterns of expression of vascular endothelial growth factor (VEGF) and VEGF receptors in mice suggest a role in hormonally regulated angiogenesis, J. Clin. Invest., 91:2235–2243. Walderf, H. and Fewkes, J., 1995, Wound healing, Adv. Dermatol, 10:77–96.

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EFFECT OF DIETARY BORON ON BONE CHARACTERISTICS AND PLASMA PARAMETERS IN YOUNG PIGS

T. A. Armstrong, J. W. Spears, T. E. Engle, and C. L. Wright Department of Animal Science and Interdepartmental Nutrition Program North Carolina State University Raleigh, North Carolina, 27695-7621, USA

1. INTRODUCTION Boron (B) has been receiving increased attention as a possible essential trace mineral. Boron appears to function in bone structure by inhibiting cartilage calcification in cholecalciferol and magnesium deficient chicks (Hunt, 1989). Boron supplementation increased bone mineralization and bone strength characteristics in pullets (Wilson and Ruszler, 1997) and broilers (Rossi et al., 1993). In addition to effects on bone, boron appears to be involved in lipid metabolism. Boron supplementation increased plasma triglyceride concentrations in chicks (Hunt, 1996). Consequently, the effect of dietary boron on bone and lipid metabolism in weanling pigs was investigated.

2. MATERIALS AND METHODS In experiment one, 48 weanling pigs (18 to 22 d of age) received a corn-soybean meal basal diet (6.67mgB/kg basal diet). The animals were randomly assigned to one of three dietary treatments. Treatments consisted of: 1) control, 2) 5mgB/kg diet, or 3) 15 mg B/kg diet. Boron was supplemented as sodium borate. In experiment two, 48 weanling pigs were treated identically to the animals in experiment one; however, they received a semi-purified basal diet (0.98mgB/kg basal diet). Blood samples were obtained from 8 animals per treatment on d 40 of the study. In addition, 8 animals per treatment were killed on d 40, and right and left femurs were removed. Plasma calcium, phosphorus, magnesium, triglycerides, and cholesterol concentrations and alkaline phosphatase activity were determined. Left and right femurs were removed of connective tissue and muscle and were frozen at –20 °C until analysis. Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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Bone ash and bone lipid percentages were determined on cross-sections obtained from the right femur. Bending moment was determined using an Instron Materials Testing Machine on whole left femurs. Total area, moment of inertia, and stress were determined using crosssections obtained at the point of fracture along the shaft of the left femur. Moment of inertia was determined via digitization of the cross-sections with video analysis using Optimas software. The moment of inertia parallel to the direction of the applied force was calculated from the digitized image using the SLICE program (Nagurka and Hayes, 1980). The moment of inertia is a measure of the area distribution around the axis of center load and accounts for geometrical differences among bones of different sizes (Vidal, 1995). Bone stress is a measure of force per unit of area. Stress accounts not only for the load applied to the bone, but also for the area and shape at the site of loading.

3. RESULTS AND DISCUSSION Supplementation of B to a corn-soybean meal diet had no effect (P > 0.10) on any of the variables measured in experiment 1. In experiment 2, daily gain and daily feed intake were not affected (P > 0.10) by B supplementation. However, feed efficiency was improved (P < 0.05) by the addition of 5mgB/kg diet to the semi-purified basal diet (Table 1). Plasma mineral concentrations and plasma alkaline phosphatase activity were not different (P > 0.10) between treatments. Plasma cholesterol concentrations were increased (P < 0.05) by supplementation of 5mgB/kg diet (Table 2). In addition, plasma triglyceride concentrations were increased (P < 0.05) with the supplementation of 15mgB/kg diet. Bone ash percentage was not

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different (P > 0.10) between treatments, and bone lipid percentage was decreased (P < 0.10) with B supplementation. Bending moment was increased (P < 0.05) by 5mgB/kg diet in pigs consuming the semi-purified diet. Total area, moment of inertia, or bone stress were not different (P > 0.10) between treatments. These data support previous research that indicates some possible biological functions for B in animals. However, these data are the first data to demonstrate that B may have an essential role in pigs receiving diets low in B. The lack of a response to B supplementation in experiment 1 may be related to the B content of the corn-soybean meal basal diet. The basal diet in experiment 1 contained 6.67mgB/kg diet. This concentration of B was equivalent to the B concentration of the semi-purified basal supplemented with 5mgB/kg diet as sodium borate.

REFERENCES Hunt, C.D., 1989, Dietary boron modified the effects of magnesium and molybdenum on mineral metabolism in the cholecalciferol-deficient chick, Biol. Trace Elem. Res. 22:201-220. Hunt, C.D., 1996, Biochemical effects of physiological amounts of dietary boron, J. Trace Elem. Exp. Med. 9:185–213. Nagurka, M.L. and Hayes, W.C., 1980, An interactive graphics package for calculating the cross-sectional properties of complex shapes, J. Biomech. 13:59–64. Rossi, A.F., Miles, R.D., Damron, B.L., and Flunker, L.K., 1993, Effects of dietary boronsupplementation of broilers, Poult. Sci. 72:2124–2130. Wilson, J.H. and Ruszler, P.L., 1997, Effects of boron on growing pullets, Biol. TraceElem. Res. 56:287–294. Vidal, M.A., 1995, Age-related changes in mechanical and geometrical properties of bone measured with invasive and noninvasive methods, Masters of Science Thesis, University of Wisconsin-Madison, Madison, WI.

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326

DIETARY BORON IS A PHYSIOLOGICAL REGULATOR OF THE NORMAL INFLAMMATORY RESPONSE

Curtiss D. Hunt United State Department of Agriculture Agricultural Research Service 1 U.S. Department of Agriculture Agricultural Research Service

1. BORON AND INFLAMMATORY DISEASE He normal inflammatory response serves to focus host defenses at a site of tissue injury or infection. In fact, absence of inflammation after introduction of an injurious agent leads to a compromised host (Gallin et al., 1988). However, excessive inflammation leads to inflammatory disease. There is emerging evidence that physiological amounts of dietary boron act to regulate the normal inflammatory process. The Unani traditional medical system in India uses sodium tetraborate or borax as an ingredient of some prescriptions for treatment of inflammatory diseases (Shah and Vohora, 1990). In the only reported controlled human study of the interaction between dietary boron and inflammation (Fracp et al., 1990), 20 patients presenting radiographically confirmed osteoarthritis received either daily 6mg of boron (as sodium tetraborate decahydrate [borax]) or a placebo as oral supplements for 8 weeks in a double-blind trial. The arthritic individuals who received boron supplements self-reported substantial improvement in subjective measures of their arthritic condition. Animal models of rheumatoid arthritis respond positively to boron. For example, boron (l0mg/kg body weight) as borax was reported to have anti-arthritic and Northern Plains Area is an equal opportunity/affirmative action employer and all agency services are available without discrimination, Grand Forks Human Nutrition Research Center, Grand Forks, ND, USA 582029034 Address all correspondence to: Dr. Curtiss D. Hunt; USDA, ARS, GFHNRC; Grand Forks, ND 58202-9034 USA; telephone: 701-795-8423; fax: 701-795-8230; email: [email protected] Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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anti-pyretic activities because it reduced paw volume and fever in albino rats with formaldehyde-induced arthritis (Shah and Vohora, 1990). A preliminary report (Bai and Hunt, 1995). Suggested that ample (but probably not pharmacologic) amounts of dietary boron compared to very low amounts significantly delayed the onset of adjuvant-induced arthritis in rats (incidence of arthritis at 12 d postinjection with M. tuberculosis [expressed as % of animals in each treatment]: 41%; B/g, 0%). Rodent chow typically contains (Hunt, Halas, and Eberhardt, 1988). A recent study (Hunt and Idso, In press) with an animal model shows that physiological amounts of boron help control the normal inflammatory process. Weanling rats (8/group) were fed either boron-low or boron-supplemented as diets and made arthritic on d 41 (postnatal d 63) by i.d. injection of M. butyricum in the subplantar region of the right hindpaw. All rats exhibited signs of inflammation after injection. Paw swelling, contralateral to the injection site, remained relatively constant the first 10 d after injection regardless of dietary treatment, but then increased to a much greater degree in rats fed the boron-low diet (boron × day, p < 0.01) (Fig. 1). Whereas the effective dosage for treatment of joint pain by the Unani system may exceed amounts normally consumed, the effect of boron on arthritis in this study (Hunt and Idso, In press) cannot be considered pharmacologic because the amount of boron in the boron supplemented diet was only one sixth of that typically present in rodent chow.

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2. BORON AND THE RESPIRATORY BURST MECHANISM Failure to end PMN leukocyte recruitment at the inflammation site creates a counterproductive cycle in which activated PMN leukocytes release chemoattractants that attracts other PMNs (Harris, 1988). Activated neutrophils and other phagocytes produce large quantities of superoxide, the precursor of a group of powerful reactive oxygen species (ROS) that are used as microbicidal agents during the normal inflammatory process, but destroy normal tissue during inflammatory disease. The primary electron donor for the reduction of oxygen during ROS generation is NADPH that comes mainly from reduction of in the pentose-phosphate (P-P) pathway (Klebanoff, 1988). In plants, a substrate in the P-P pathway, 6-phosphogluconate, complexes with boron, which thereby inhibits 6-phosphogluconate dehydrogenase (PGD) (Lovatt and Dugger, 1984). A serious problem in boron-deficient plants is increased substrate metabolism via the PP pathway that ultimately causes plant death (Lovatt and Dugger, 1984). Therefore, it is reasonable to hypothesize that proper boron nutriture may cause a simple reduction in leukocyte ROS generation through down-regulation of leukocyte PGD with subsequent alleviation of arthritic symptoms.

3. BORON AND POSSIBLE EFFECTS ON ROS METABOLISM There is emerging evidence that boron may hasten destruction of ROS that are scavenged and destroyed by defense mechanisms that employ glutathione (GSH), superoxide dismutase (SOD), and catalase. Serine-borate complex is a transition-state inhibitor of gamma glutamyl transpeptidase (GGT) (Tate and Meister, 1978) and by that mechanism, apparently elevates GSH concentrations in cultured fibroblasts taken from patients suffering from GSH synthase deficiency (Spielberg et al., 1979). Boron supplementation significantly increased erythrocyte SOD activity (2578 ± 74 vs. 2257 ± 99U/g hemoglobin; p < 0.03) in men and postmenopausal women with marginal copper status (Nielsen, 1989). It remains to be determined whether SOD activity increased because boron induced free radical formation, or whether boron improved antioxidative capacity.

4. BORON AND SERINE PROTEASES There is emerging evidence that boron facilitates the normal inflammatory process by dampening the activity of serine proteases. During a severe inflammatory response, various cells, including PMNs, release lysosomal proteases both extracellularly and into the circulation. These enzymes exacerbate the inflammatory response by degrading connective tissue structures (Fritz et al., 1986). Compromised status of natural protease inhibitors (Niehaus et al., 1990) can lead to diseases including rheumatoid arthritis which is essentially a problem of uncontrolled proteolysis (Kavanaugh and Kipsky, 1995). The serine proteases are major proteolytic enzymes released by activated leukocytes that, in addition to degrading structural proteins, have many essential regulatory roles in normal inflammation (Kettner, Mersinger, and Knabb, 1990). Boron compounds reversibly inhibit the activity of many serine proteases and are thought to do so by forming a tetrahedral boron adduct that mimics the tetrahedral adduct formed during normal substrate hydrolysis (Berry et al., 1988). The available experimental evidence (Bachovchin et al., 1988) suggests that formation of the boron

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adduct occurs when boron completes its octet by reacting with either the of the activesite serine residue 195 or the of the imidazole group of histidine residue 57 to form either a B-O or B-N bond respectively. Natural, simple unsubstituted boric acid compounds which contain a trigonal boron atom, bind to serine proteases to form a reversible tetrahedral transition state analogue complex. For example, borate reversibly inactivates the serine protease, alpha-chymotrypsin, by accepting the free electron pair of the nitrogen atom on the imidazole group of the histidine residue at the active site (Berezin et al., 1967). Various microbial subtilisin-type serine proteases bind to borates to form tetrahedral transition state analogues (Hausdorf et al., 1987). It is reasonable to hypothesize that boron helps regulate the normal inflammatory process by dampening the activity of leukocyte serine proteases and thereby reducing degradation of connective tissue structures.

5. BORON AND EICOSANOIDS The eicosanoids, a collective term for arachidonate and the prostaglandins, leukotrienes (LT), and thromboxanes, are important components of the inflammatory response. Lipoxygenase is required for the production of the LTs, cyclooxygenase for the prostaglandins and thromboxanes. Boron may play an important role in LT metabolism at two different sites. For example, in vitro studies demonstrate that boron inhibits (30% inhibition at under standard reaction conditions) the lipoxygenase enzyme in the sunflower, a plant very sensitive to boron deficient media (Belver and Donaire, 1983). Certain synthetic boron compounds inhibit human leukocyte 5'-lipoxygenase activity, protect against free-radicals generated by the Fenton reaction and inhibit prostaglandin cyclooxygenase and 5'-lipoxygenase in cultured mouse macrophages (Rajendran et al., 1994). Boron may affect the metabolism of the leukotrienes. The n-6 sulfidopeptide leukotrienes and together constitute the biological activity ascribed to slow reacting substance of anaphylaxis. The bioconversion of LTC4 to LTD4 by GGT is very strongly inhibited by serine-borate (Snyder et al., 1984). Because LTs are the major rate-limiting mediators of immune-inflammatory events, boron may be in a key position to modulate the inflammatory process.

6. BORON AND IMMUNE CELL FUNCTION The addition of boron in vitro over a range between 0 and inhibited proliferation of splenic cells isolated from boron-deprived rats and subsequently stimulated by 0, 5, or phytohemagglutinin/mL (Bai and Hunt, 1995). Another study showed that physiologic amounts of boron added to a boron-low diet more than doubled serum total antibody concentrations to injected antigen (human typhoid vaccine) in rats (Bai et al., 1997). Recently, we reported (Hunt and Idso, In press) that supplemental dietary boron had a beneficial immunomodulatory effect in the arthritic rat. The animals were fed either boron low or boron (as orthoboric acid supplemented diets and made arthritic on d 41 of the experiment by i.d. injection of M. butyricum. On d 14 after injection, rats fed the boron-supplemented diet compared to those fed the boron-low diet exhibited increased concentrations of natural killer cells (0.42 ± 0.13 vs.

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0.22 ± 0.10 million cells/mL whole blood; p < 0.008). On d 28, supplemental boron decreased neutrophil concentrations (0.89 ± 0.20 vs. 2.06 ± 1.13 million cells/mL whole blood; p < 0.02). The large changes in circulating concentrations of NK cells and neutrophils brought about by physiologic amounts of boron can be considered beneficial because supplemental boron also attenuated the rate of paw swelling (Fig. 1). There are several lines of evidence (Hellstrand et al., 1994) that the cytotoxic and proliferative activities of NK cells are inhibited by a simple ROS that is released during respiratory burst activity of leukocytes in general. Therefore, it is reasonable to conclude that boron beneficially dampens ROS production that in turn up-regulates cytotoxic and proliferative activities of NK cells. That is, physiologic amounts of boron may reduce the risk for inflammatory disease by helping hold in check a system that is constantly poised to attack, a balance that permits pathogen elimination but avoids autoimmunity.

SUMMARY Various diverse research findings have been summarized to develop the boroninflammation regulation hypothesis that boron reduces the risk for inflammatory disease by serving as a suppressive signal that down-regulates enzymatic activities typically elevated during the normal inflammatory process. There is in vivo evidence for an immunomodulatory effect of physiologic amounts of dietary boron. Specifically, boron helps control the normal inflammatory process by modulating the response of key immune cells to antigens by an undefined mechanism. Boron may act to regulate the normal inflammatory process by serving as a signal suppressor that down-regulates the activities of specific enzymes involved in the inflammatory process and thus may play a role in modulating the development of inflammatory disease. Suppression, but not elimination, of activities of these enzymes is hypothesized to reduce the incidence and severity of the symptoms of inflammatory disease.

REFERENCES Bachovchin, W.W., Wong, W.Y.L., Farr-Jones, S., Shenvi, A.B., and Kettner, C.A., 1988, Nitrogen-15 NMR spectroscopy of the catalytic-triad histidine of a serine protease in peptide boronic acid inhibitor complexes, Biochem. 27:7689–7697. Bai, Y. and Hunt, C.D., 1995, Dietary boron alleviates adjuvant-induced arthritis (AIA) in rats, FASEB J. 9:A576. Bai, Y., Hunt, C.D., and Newman Jr, S.M., 1997, Dietary boron increases serum antibody (IgG and IgM) concentrations in rats immunized with human typhoid vaccine, Proc. N. D. Acad. Sci. 51:181. Belver, A. and Donaire, J.P., 1983, Partial purification of soluble lipoxygenase of sunflower cotyledons: action of boron on the enzyme and lipid constituents, Z. Pflanzenphysiol Bd. 109:309-317. Berezin, I.V., Vill, K.H., Martinek, K., and Yatsimirshii, A.K., 1967, Reversible inactivation of alphachymotrypsin resulting from interaction of Cu++ ions with the imidazole group of a histidine residue, Molek. Biol. 1:719–728. Berry, S.C., Fink, A.L., Shenvi, A.B., and Kettner, C.A., 1988, Interaction of peptide boronic acids with elastase: circular dichroism studies, Proteins: Structure, Function, and Genetics 4:205–210. Fracp, R.L.T., Rennie, G.C., and Newnham, R.E., 1990, Boron and arthritis: the results of a double-blind pilot study, J. Nutr. Med. 1:127–132. Fritz, H., Jochum, M., Geiger, R., Duswald, H.H., Dittmer, H., Kortmann, H., Neumann, S., and Lang, H., 1986, Granulocyte proteinases as mediators of unspecific proteolysis in inflammation: a review, Folia Histochem. Cytobiol. 24:99–115.

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Gallin, J.I., Goldstein, I.M., and Snyderman, R., 1988, Inflammation: Basic Principles and Clinical Correlates, ed., Raven Press, New York. Harris Jr, E.D., 1988, Pathogenesis of rheumatoid arthritis: a disorder associated with dysfunctional immunoregulation, in: Inflammation: Basic Principles and Clinical Correlates, (J.I. Gallin, I.M. Goldstein, and R. Snyderman, eds.), pp. 751–773, Raven Press, New York. Hausdorf, G., Krüger, K., Küttner, G., Holzhütter, H.-G., Frömmel, C., and Höhne, W.E., 1987, Oxidation of a methionine residue in subtilisin-type proteinases by the hydrogen peroxide/borate system–an active site-directed reaction, Biochim. Biophys. Acta 952:20–26. Hellstrand, K., Asea, A., Dahlgren, C., and Hermodsson, S., 1994, Histaminergic regulation of NK cells. Role of monocyte-derived reactive oxygen metabolites, J. Immunol. 153:4940–4947. Hunt, C.D., Halas, E.S., and Eberhardt, M.J., 1988, Long-term effects of lactational zinc deficiency on bone mineral composition in rats fed a commercially modified Luecke diet, Biol. Trace Elem. Res. 16:97–113. Hunt, C.D. and Idso, J.P., In press, Dietary boron as a physiological regulator of the normal inflammatory response: a review and current research progress, J. Trace Elem. Exp. Med. Kavanaugh, A.F. and Kipsky, P.E., 1995, Rheumatoid arthritis, in: Inflammation: Basic Principles and Clinical Correlates, (R.R. Rich, T.A. Fleisher, B.D. Schwartz, W.T. Shearer, and W. Strober, eds.), pp. 1093–1116, Mosby, St. Louis. Kettner, C., Mersinger, L., and Knabb, R., 1990, The selective inhibition of thrombin by peptides of boroarginine, J. Biol. Chem. 265:18289–18297. Klebanoff, S.J., 1988, Phagocytic cells: products of oxygen metabolism, in: Inflammation: Basic Principles and Clinical Correlates, (J.K. Gallin, I.M. Goldstein, and R. Snyderman, eds.), pp. 391–444, Raven Press, New York. Lovatt, C.J. and Dugger, W.M., 1984, Boron, in: Biochemistry of the Essential Ultratrace Elements, (E. Frieden, eds.), pp. 389–421, Plenum Press, New York. Niehaus, G.D., Kimura, R., Traber, L.D., Herndon, D.N., Flynn, J.T., and Traber, D.L., 1990, Administration of a synthetic antiprotease reduces smoke-induced lung injury, J. Appl. Physiol. 69:694–699. Nielsen, F.H., 1989, Dietary boron affects variables associated with copper metabolism in humans, in: 6th International Trace Element Symposium 1989, (M. Anke, W. Baumann, H. Braünlich, C. Brückner, B. Groppel, and M. Grün, eds.), pp. 1106–1111, Karl-Marx-Universitat, Leipzig and Friedrich-SchillerUniversitat, Jena, DDR. Rajendran, K.G., Burnham, B.S., Sood, C.A., Spielvogel, B.F., Shaw, B.R., and Hall, I.H., 1994, Antiinflammatory and anti-osteoporotic activities of base-boronated nucleosides and phosphate-boronated nucleotides in rodents, J. Pharm. Sci. 83:1391-1395. Shah, S.A. and Vohora, S.B., 1990, Boron enhances anti-arthritic effects of garlic oil, Fitoterapia 61:121-126. Snyder, D.W., Aharony, D., Dobson, P., Tsai, B.S., and Krell, R.D., 1984, Pharmacological and biochemical evidence for metabolism of peptide leukotrienes by guinea-pig airway smooth muscle in vitro, J. Pharmacol. Exp. Ther. 231:224–229. Spielberg, S.P., Butler, J.D., MacDermot, K., and Schulman, J.D., 1979, Treatment of glutathione synthetase deficient fibroblasts by inhibiting gamma-glutamyl transpeptidase activity with serine and borate, Biochem. Biophys. Res. Commun. 89:504–511. Tate, S.S. and Meister, A., 1978, Serine-borate complex as a transition-state inhibitor of gamma-glutamyl transpeptidase, Proc. Natl. Acad. Sci. USA 75:4806–4809.

327

BORON STIMULATED YEAST DIFFERENTIAL GENE EXPRESSION

A. Bennett, V. Gilman, N. Soch, D. Luo, and C. D. Eckhert Department of Environmental Health Sciences University of California Los Angeles, California, USA

Although boron has been shown to be essential for plant and fish growth no molecular mechanism has been elucidated. Boron depleted growth media has been shown to retard the growth and limit the population size of Saccharomyces cerevisae (a budding yeast). By screening subsets of the mRNA populations of log-phase yeast cultures, with and without boron supplementation, we have developed a profile of genes that are differentially expressed relative to boron status. Total RNA was extracted from 4 groups of log-phase cultures of yeast grown in boron-depleted media and supplemented as follows: 1) NBNB: never supplemented with boron, 2) NBB: initially unsupplemented, later supplemented with 500ppb boron, 3) BNB: initially supplemented with 500ppb boron, 4) BB: initially supplemented with 500ppb boron, later supplemented with an additional 500ppb boron. Using differential display analysis, the total RNA, with anchored oligo dT primers, was used to synthesize first strand cDNA species. The cDNA was further amplified using the corresponding anchored primer with an arbitrary primer and labeled with alpha The cDNA fragments, representing specific subsets of the mRNA population, were separated on a conventional sequencing gel to the point that fragments of 300–1,000bp could be adequately resolved. Differentially expressed cDNA fragments were excised from the gel and sequenced by conventional protocols. A comparison the specific subsets (pairs of anchored and arbitrary primers) of cDNA fragments between the 4 treatments of yeast (NBNB, NBB, BNB and BB) showed that 5–6 genes fundamental in cell growth and maintenance are differentially expressed relative to boron status. For all of these genes the NBNB treatment showed significantly lower expression than the other treatments, indicating that the supplementation of boron is an essential element of key molecular events in yeast growth.

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IDENTIFICATION BY GENE ARRAYS OF BORON RESPONSIVE GENES IN YEAST

K. Becker, A. Bennett, and C. D. Eckhert Department of Environmental Health Sciences University of California

Los Angeles, California, USA

Boron is a ubiquitous element found in water, soil and rocks. It is known that boron is a required nutrient in vascular plants, diatoms, and some species of marine algal flagellates. However, the primary role has not yet been established. In boron deprivation studies, we have shown that there is a decline in yeast, Saccharomyces cerevisae, growth rate as well as population size. By analyzing log-phase mRNA of yeast that have been supplemented and/or depleted with boron, we have determined by differential gene analysis that several genes, fundamental in cell growth and maintenance are differentially expressed relative to boron status. The present study was undertaken to validate these results using gene filter arrays of the entire yeast genone. Two groups were evaluated: 1) NBNB yeast grown in boron depleted media into the log phase, 2) yeast grown in NB media during the lag phase and then transferred to a media supplemented with boric acid to a concentration of 500ppb B. Total RNA was extracted from the two groups of yeast during the log-phase. Genes of this extracted RNA were converted to cDNA using reverse transcription and labeled with . The labeled cDNAs were hybridized to high density GeneFilters (Research Genetics) containing all genes of the yeast genome. The results of the high density arrays hubridization validate our previous observation that several genes, fundamental in cell growth and maintenance, are differentially expressed relative to boron status.

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LIMB TERATOGENESIS INDUCED BY CHRONIC BORON OR COPPER DEFICIENCY IN XENOPUS D. J. Fort, E. L. Stover, F. J. Murray, and P. L. Strong The Stover Group Stillwater, Oklahoma, USA Murray and Associates San Jose, California, USA U.S. Borax, Valencia, California USA

As a part of separate large-scale studies evaluating the effects of boron or copper deficiency on reproduction and development, studies evaluating the effect of long-term boron or copper deficiency on Xenopus development were performed. Sets of adult male and female Xenopus were administered a boron deficient (–B) diet under low boron culture conditions, a boron supplemented (+B) diet under ambient boron culture conditions, a copper deficient (–Cu) diet under low copper culture conditions, or a copper supplemented (+Cu) diet under ambient copper culture conditions, for 120d. Adults from each group were subsequently bred, the progeny cultured, and subsequently bred. Although pronounced effects on adult reproduction and early embryo-larval development were noted in the generation, no effects on limb development were observed. No significant effects on reproduction, early embryogenesis, or limb development were noted in the +B group, irrespective of generation. However, highly specific fore- and hind limb defects, including axial flexures resulting in crossed limbs and reduction deficits, were observed in larvae. These effects were not observed in the larvae. More specifically, 12 of the larvae possessed mal-developed forelimbs only, 4 possessed maldeveloped hind limbs only, and 2 had both deformed fore- and hind limbs (n = 28). These specific limb defects were only observed in the generation. As was noted in the boron deficiency studies, significant effects on reproduction and early embryo development were observed in the generation, but not in the generation. However, unlike the effects associated with boron deficiency, mal-development of the hind limbs (32 responders, n = 40), characterized as incomplete connective tissue development resulting in various axial flexures, were found. These results further support the teratogenic effects associated with boron and copper deficiency in Xenopus.

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AN ASSESSMENT OF FERTILITY IN BORON-EXPOSED TURKISH SUBPOPULATIONS AN EPIDEMIOLOGICAL APPROACH

M. Çöl1, B. S. Sayli2, Y. Genç3, E. Erçevik1, A. H. Elhan3, and A. Keklik1 Ankara University Faculty of Medicine Departments of Public Health1 Genetics2 and Biostatistics3 Ankara, Turkey

Our knowledge is too limited in human reproductive data related with boron exposure. There are only a few studies reporting reduced sexual function in men occupationally exposed to boron. Some other studies, revealed that occupational or environmental exposures to inorganic borate did not lead to a reduction in birth rate. Data, about boric acid’s developmental toxicity on rats, mousses and rabbits are already available. Although not significant, some studies have shown that there has been a change in sex ratios of occupationally exposed populations (in favor of female offspring). Since Turkey is one of the countries which has large borate deposits and plants, the conduction of the study in this area is important. The purpose of this study was to investigate borate’s; Reproductive effects, Developmental effects, Effects on the sex ratio on environmentally and occupationally exposed male workers’ families in a crosssectional design. Study regions were planned as three areas in Turkey where Region I was Bigadiç (Bal.kesir), Region II was Emet-Hisarc.k (Kütahya), Region III was K.rka (Eski.ehir). 850 married-male workers from these plants were chosen by random sampling method but the number of workers who were contacted were 799. Data collection was made by personal interviews of workers at their working places. For data analysis and statistics SPSS for Windows package and Chi-Square, Student’s t test were used. 1080

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Infertility rates were 1.2% among 328 borate workers from Region I, 1.1% among 298 workers from Region II and 4.1% among 173 workers from Region III. Total infertility rate was 1.8% for all of the workers. These rates were similar to the results of studies made in the same region and in other parts of Turkey. Total male/female ratio was found to be 1.2% so no increase in the number of female offspring could be found when compared with previously reported data. No significant influence was observed in parameters used to define possible developmental effects. Stillbirths, abortions, prematuries or having low birth weights and early deaths of offspring were not more than the ones found in any part of the country. There were no differences in infertility rates, sex ratios and possible developmental effects between the production workers and office workers. As a conclusion, the level of boron that people from the three regions were exposed to seems not to affect the reproductive characteristics investigated.

331

PHOTORECEPTOR DYSTROPHY IN BORON DEPLETED ZEBRAFISH

S. Dohs and C. D. Eckhert Department of Environmental Health Sciences University of California Los Angeles, California, USA

Adult wildtype (WT) zebrafish (Danio rerio) were maintained in acrylic aquariums filled with ultrapure grade Milli-Q purified water containg Brine shrimp, reared in a low boron environment, were proveded as food. After six months WT fish were bred. About 5% of the zygotes (Fo) survived to adulthood. These boron depleted (B–) fish were reared to adulthood and used as stock to develop an F1 (B–) generation. The Fl fish were photophobic. Light sensitivity was not apparent in Fl (B+) fish reared in ultrapure water that had been supplemented with boric acid to achieve a concentration of the concentration of Los Angeles tap water. Retinas from both (B–) and (B+) adults were fixed and embedded for histological evaluation. Retinas from (B–) zebrafish exhibited cone dystrophy. This manifested as poorly developed outer segments with small myoid regions of the inner segment. In contrast, the outer sigments of (B+) retinas were well developed and their inner segments exhibited well developed myoid regions. Boron depletion is manifested in embryonic fish as retarded growth (trout) and death resulting from a failure to complete the cleavage stage (zebragish) of development. The present observation adds photoreceptor dystrophy in the adult as an additional symptom of boron deficiency.

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ADVERSE IN VITRO DEVELOPMENT OF PREIMPLANTATION MOUSE EMBRYOS EXPOSED TO LOW BORON DIET AND LOW BORON

L. Lanoue, F. J. Murray, P. L. Strong, and C. L. Keen Department of Nutrition University of California Davis, California Murray & Associates San Jose, California U.S. Borax, Valencia California, USA

Recent studies have shown that boron is critical for the early survival of frog and zebrafish embryon. In the present study we tested the hypothesis that boron is required to support the preimplantation development of mouse embryon using an in vitro model system. Female CD-1 mice (20–25g) were fed a low –B) or a supplemented +B) boron diet, or received a commercial stock diet In study I, two-cell embryos were collected from superovulated dams at 10, 12 and 16 weeks of dietary treatment and cultured for 72h in T6 media containing boron +T6). In study II, two-cell embryos were collected after a 16 week dietary period and cultured in boron-free media –T6). In both studies, liver and tibia boron concentrations were signigicantly lower in low boron fed-dams than in dams fed the supplemented boron or stock diets. In study I, exposure to low boron diet was associated with a moderate reduction in blastocyst formation (83.5%, –B vs 90.1%, +B) and an increased number of degenerate embryos (13.0%, –B vs 8.0%, +B) when two-cell embryos were grown in the presence of boron. In study II, exposure to a boron-free environment for 72h significantly compromised the in vitro development of the embryos as shown by the lower rate of blastocyst formation in embryos taken from dams fed a supplemented boron diet (54.5%, +B/–T6 vs 90.1%, +B/+T6), and from dams fed a low boron diet (39.7%, –B/–T6 vs 83.5%, –B/T6). Low dietary boron combined with low 1083

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media boron resulted in the highest rate of degerate embryos (57.0%), while stock diet with media-containing boron, the lowest (8.5%). These date reflect the sunergistic adverse effects of dietary and media boron depletion. Collectively, these date support the concept that boron deficiency impairs early embryonic development in rodents.

333

THE TEMPORAL EFFECTS OF LOW DIETARY BORON (B) ON TISSUE B CONCENTRATIONS IN RATS

J. Y. Uriu-Adams, B. J. Horwath, F. J. Murray, G. Downing, P. Strong, and C. L. Keen Nutrition Department University of California Davis, California Murray & Associates San Jose, California R. G. Research, Niskayuna, New York U.S. Borax Inc., Valencia, California, USA

Recent reports have demonstrated the essentiality of B for frogs and fish. We are involved in several ongoing studies determining whether B is essential for mammals. A key question with regard to essentiality, is how quickly and to what extent do tissue concentrations of B change when an animal is introduced to a diet low in B. Female rats (50–60 g) were fed Purina chow for three days and a subset killed (Day 0) for baseline B concentrations. Rats were then divided into one of 5 dietary groups. Three of the diet groups were fed corn meal based diets containing either (LOW), (CON). A fourth diet group was fed a low B sucrose/dextrose based diet (DYET), and the fifth group received Purina chow (CHOW). Rats were killed on Days 3, 7, 10, 14, 21, 28, 42, 56, 84 and 112, and tissues analysed for B concentrations using ICP-MS. Preliminary results from Days 0–84 show that B concentrations in tibia and liver were relatively constant over time for the CHOW fed rats. In contrast, within 3 days of introduction to the diets, the CON rats had a 20% decrease, while the DYET, LOW, and INT B groups had a 75% reduction in tibia and liver B levels compared to baseline. These group differences were maintained over the next 84 days. The kidney B levels in CHOW fed rats declined slightly over time such that by Day 56, kidney B was 65% of baseline. In contrast, Day 3 kidney B levels decreased markedly in all other groups (56%–74%); from then on, the kinetic profiles were similar to CHOW. Uterus B concentrations decreased in all groups by Day 3 of the study, with varying degree of severity. Overall, 1085

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our data show a clear difference in tissue B concentrations among the CHOW, CON, INT groups; whether the LOW and DYET groups also follow a dose response is unclear. These data suggest that there is a labile pool of B in a diverse group of tissues that is quickly and easily mobilized during conditions of low B. The functional consequences of low tissue B remains to be determined.

334

BORON STIMULATES YEAST GROWTH

A. Bennett, N. Soch, R. Rowe, and C. D. Eckhert Department of Environmental Health Sciences University of California Los Angeles, California, USA

Sacchromyces cerevisiae (yeast) is a single-celled eukaryote that has been successfully applied to elucidate molecular mechanisms involving elements, such as copper and iron. This has been possible because they are essential nutrients for growth and metabolism and their intracellular trafficking is similar in yeast and mammals. We report here that a boron deficient growth media slows the growth and limits the stationary population size of yeast in liquid culture. YPD growth media was prepared and passed through an Amberlite column to remove the boron. The deficient media (NB) was prepared by the addition of ultrapure water (<0.2ppb B) to achieve its final concentration. The supplemented media (B) was prepared by the addition of boric acid to achieve a final concentration of 500ppb B. As early as the lag phase, we observed a significant difference in the rate of growth between yeast grown in boron-depleted growth media and boron-supplemented media. At the point where the growth difference became statistically significant each culture was split into two groups. One was maintained in media of the same concentration and the other group was transferred to the other level of boron. The four groups were as follows: 1) NBNB: unsupplemented throughout 2) NBB: unsupplemented and then supplemented with 500 ppb of boron, 3) BNB: intially supplemented with 500 ppb of boron and then transferred to unsupplemented media, BB: supplemented with 500 ppb of boron throughout the experiment. Repletion of yeast grown in the boron deficient media with 500 ppb boron stimulated their growth during the log phase of growth. In the stationary phase, three of the groups (NBB; BNB & BB) leveled-off at the same population size while the fourth group, the yeast grown in boron-deficient YPD throughout the experiment (NBNB) leveled-off at a significantly lower population size. These results demonstrate that boron is required for optimal S. cerevisiae growth. In tandem with previous reports in fish, these data argue that boron is essential for eukaryotic organisms.

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DOES BORON ACT ON TRANSCRIPTION AND TRANSLATION OF PROTEINS?

M. Dzondo-Gadet*, R. Mayap Nzietchueng*, K. Hess*, P. Nabet*, F. Belleville*, and B. Dousset Laboratoqire de Biochimie Médicale Facutlé de Médecine NANCY, France

Boron was found to modulate the formation of extracellular matrix by increasing the release of its components such as proteoglycans, collagene and proteins. Furthermore, boron stimutales synthesis and release of No receptor to boron has been yet described, but boron might generate colmplexes with cis-diol groups of ribose and subsequently by this mechanism it might modify the protein synthesis. We investigated this protein in vitro system.

MATERIAL AND METHOD The transcription assays were carried out with nuclei extracted from human placenta. The transcription was performed in presence of and boric acid solution from 0.063 to 0.5% (w/v) or HCl solution pH 5 (control). The synthesis of total RNA was evaluated by incorporated radioactivity and by measurement of absorbance at 260 nm after phenol/chloroforme extraction. The translation assays were performed in acelllular system of wheat germ extract? The translation mixture (final volume was supplemented with placenta RNA, and boric acid solultion 0.063 to 0.5% (w/v) or HCl pH 5. The pH of translation medium was adjusted to 7.1. Proteins synthetised were precipited by adding TCA, the radioactivity was counted and the proteins analysed by electrophoresis and immunoblot.

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RESULTS Boric acid enhanced the synthesis of total RNA measured either by incorporation (×–11) or by absorbance DO at 260 nm (×6). Boric acid inscreased also the protein synthesis (×2). Autoradiography showed that synthesis of various proteins was enhanced and among these proteins, angiogenic factors (VEGF, bFGF, will be searched by immunoblot.

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336

NEW SENSITIVE METHODS IN THE DETERMINATION OF TRACE ELEMENTS

P. Schramel GSF-National Research Center for Environment and Health Institute of Ecological Chemistry D-85758 Neuherberg

INTRODUCTION Discussing modern analytical techniques for trace element analysis in the biomedical field, firstly one has to define the problems which should be solved. It is likely to stress out that there doesnít exist any analytical technique which is able to analyze all the elements of interest in all naturally given concentration ranges and in all the matrices of interest. That means all the methods donít compete but they are complementary to each other or to another. The best method for a given problem should be used and this is in most the cases not one method which is available in a laboratory per chance. So it is better to speak about a modern analytical laboratory which should be equipped with different modern analytical techniques and therefore would be able to react adequately to the different tasks. The preanalytical steps, which are very important and significantly influences the reliability of the results should not be discussed here. Only some aspects of the final analysis in the laboratory, that means sample digestion and final methods of determination should be shortly discussed. The elements of interest in this field are shown in Table 1 (Zumkley, 1983). The main or mineral elements will not be mentioned here because these show normally no analytical problems. At the one hand there are the known essential trace elements for investigating their biochemical behaviour in the living organism and on the other hand all the others which may play a role in environmental and/or occupational health medicine. We know something about well known heavy metals like Pb, Cd, Hg and others but nearly nothing about

Address all correspondence to: Prof. Dr. Peter Schramel; GSF-National Research Center for Environment and Health, Institute for Ecological Chemistry; telephone 0049/89/3187-4062; fax: 0049/89/3187-3348; email: [email protected] Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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the rest of the periodic table. All the elements show an ambivalent behaviour but we know nearly nothing about the area of health because of their very often low concentrations. Our knowledge is only a question of concentration and in this connection of the analytical coverage. Generally we have the following analytical techniques for the determination of trace elements (Table 2). Not mentioned here are highly sophistacated methods, which are up to now not used for Ñroutineì analysis, like for example all the different laser induced techniques. Commonly used are atomic spectroscopy (AAS and ICP-OES), ICP-mass spectroscopy and the electrochemical techniques. XRF, especially TXRF does not play any important role in this field and NAA looses more and more the importance because of the lack of sufficient nuclear reactors for these purposes. But especially for quality assurance NAA as a completely independent technique is still an important tool—if available.

METHODS Sample digestion—Firstly modern digestion procedures, which are an important step in the whole analytical procedure should be briefly discussed. Two techniques, namely pressurized (Schramel et al., 1980) and UV-digestion (Begerow et al., 1997), can be used nearly universally for atomic and mass spectroscopy. No high demands on the quality of the digested samples are necessary for these techniques because of the additionally high thermal energy available in flames, graphite

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furnaces and especially in an ICP. This thermal energy destroys the not digested organic matrix during the analyzing procedure. So, quality of the digested sample solution is defined as the rest of organic carbon in the solution. Obviously no solid residues should be present in the solution. Microwave assisted and conventionally heating pressurized digestion procedures fulfill normally these demands for organic materials. Higher demands have analytical techniques like stripping voltammetry (Hasse et al., 1983) and/or a chemical step which is connected to the final determination like matrix separation, preconcentration and hydride generation (Schramel et al., 1993). The past is a typical example for high demands especially for the As determination using hydride generation in urine samples. At least six different As-species are present, which show very different answers in hydride generation. In this case and/or for voltammetric techniques like DPASV or DPCSV a complete destruction of the organic matrix (TOC lower than 1%) is necessary for reliable results. A high potential for atomic and mass spectroscopy has also UV-digestion, but which is limited to urine and serum samples. It reduces significantly the difficulties with the organic matrix, especially in ICP-MS, due to residues on the interface.

FINAL METHODS OF DETERMINATION AAS-techniques are still widely used for the determination of mineral elements (flame), some essential trace elements like Cu, Cr, Se, Mn and/or heavy elements like Pb, Cd or others (graphite furnace) and for Hg (CVAAS), As and Se (hydride generation). Very often matrix separation and/or preconcentration steps are necessary to overcome the lack in detection power, at least for heavy metal determination in body fluids. Well known matrix interferences influence the reliability of the results, therefore very often the use of standard addition method for evaluation is necessary, especially using graphite furnace techniques. ICP-OES has very similar possibilities as flame AAS, that means, especially in body fluids only Cu, Fe and Zn from the known essential trace elements and of course all mineral elements can be analyzed without any difficulties in body fluids (Schramel et al., 1982). But the use of an internal standard is strongly recommended because of interferences on the sample introduction system due to different viscosity and density of individual samples. The advantage of ICP-OES compared to AAS-techniques are the multielement possibilities and the significantly reduced chemical matrix interferences (like molecular interferences) because of the high temperature of the ICP available for the excitation of the atoms or ions. The voltammetric techniques like DPASV or DPCSV are very powerful analytical techniques, with absolut detection limits in the pg—range, but less commonly used. One reason is the high demand on the quality of the sample solution. The electrochemical techniques are species specific and not element specific, that means any change in the bounding state of an element interferes the results. The other reason is that this technique mostly asks for evaluation by standard addition method and therefore it is more or less very time cosumable. The third reason is that one needs a really qualified (from the chemical point of view) and experienced person for running this technique

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to get reliable results. Thatís not a technique to press a buttom and to expect accurate results. The practical detection limits are for below those of GFAAS and in the range of ICP-MS. Today ICP-MS can be regarded as one of the most powerful techniques for trace and ultratrace analysis of elements. Detection limits in the ng/L range or below are quite normal especially when using SF-ICP-MS. The ICP-MS detection limits, related to the different matrices and their necessary dilution or digestion, for all given elements are better, in some cases up to three orders of magnitude, than those achieved by AAS— techniques (Schramel et al., 1999). The use of ICP-MS for biological samples and the choice of the appropriate instrument (Q-ICP-MS or SF-ICP-MS) will of course depend to a large extent on the task facing a particular laboratory. From the economic point of view, the investment and the operational costs are not justified for the investigation of only a few samples for example one element only. In this case AAS, particulary GF-AAS, CV-AAS and HG-AAS, will continue to be used. If available, stripping voltammetry, as mentioned before, should not be forgotten in this context. However, ICP-MS will be indispensable for the analysis of a large number of samples, in which several elements are to be determined simultaneously or for frequently changing the analytes. This implies also the so-called Ñnon-targetì screening and research in the biomedical field. Very little is known about most of the elements, especially of those present in low concentrations, ICP-MS enables their detection and hence the better investigation of these elements. As mentioned in the beginning, it should be emphasized that also ICP-MS is not a universally applicable method. Serious systematic errors can occur when potential sources of interferences are disregarded. Today mass spectrometers with quadrupole filters with a resolution of 300 or expensive double focusing sector field instruments with resolutions between 7,500 to 15,000 are commercially available. In some cases high-resolution mass spectrometers enables separation of the analyte peak from the interferring peak. An overview of the minimum resolution required for an interference-free determination of some trace elements is given in Table 3 (Begerow et al., 1996; Williams, 1992; Vanhoe et al., 1993). In this Table some of the essential trace elements are included. One can see, that strong interferences in case of Q-ICP-MS may occur. By no means all spectral interferences can be resolved with SF-ICP-MS, dependent on the achievable resolution. However, it must be pointed out that adjustment to a higher resolution level causes a distinct decrease in the ion yield, that means in sensitivity. But this is normally not combined with the same decrease in the detection limit. Different sample introduction systems—only mentioned here—can be used:

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Nebulizer (pneumatic and ultrasonic) ETV (electrothermal vaporization) FIAS (flow injection, hydride generation) These often permit the user to better react to the given problem and to create the best possible conditions for detection. For a better understanding of the interferences in an ICP-MS it is important that in a mass spectrometer mass to charge ratios (m/z) and not masses are analyzed. Normally z = 1 (single charged ions), but dependent on the ionization energy, ions bearing double or even multiple charges can occur. These interferences have different origins. They can be divided into spectral interferences with the masses to be determined, and non-spectral interferences, for example influences of the sample on the sample introduction system, interferences on the interface because of high concentrations of salts or organic matrix constituents, effects on the plasma conditions caused by matrix variations—some more seriously than in ICP-OES—and by variations in the isotopic composition. The degree of interference and thus its influence on the analytical result naturally depends to a great extent on the concentration ratio between the element causing the interference and the analyte element, which is, however, often very unfavourable for the analyte in biological materials. The possible spectral interferences (Schramel et al., 1999) are: polyatomic interferences with the formation of cluster ions and oxides isobaric interferences multiple charged ions contribution from neighbouring masses The polyatomic interferences certainly pose the main problem and represent the most limiting factor in ICP-MS, at least when the quadrupole technique is applied. The cluster ions are formed from the plasma gas, auxiliary gases, the constituents of the sample, the reagents and the solvent. A great variety of atomic combinations is possible and they hamper the analysis of elements, especially in the mass range between 40 and 80. The Table 4 shows some examples for such interferences. When using Q-ICP-MS, spectral interferences, which depend on the instrumental parameters, plasma conditions and the matix, are very difficult to control. As these

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influences cannot be reproduced exactly, an mathematic correction in the strict sense, as ever highly sophisticated they are, is imposssible, as the correction is valid only for the solution used for determination of this correction factor, that means for the ratio of the analyte element to the interferent. The second and generally more serious reason is that a large number must be subtracted from another large very similar number in such cases (Ñ 100—99.9ì ). As both numbers are statistically uncertain, no reliable result can be obtained. In general it can be stated that the smaller the analyte/interferent ratio, the less effective these corrections are. Thus elimination of the interference is naturally the main objective. This can be sometimes achieved with the aid of SF-ICP-MS or to some extent also with the aid of other sample introduction systems like ETV (electrothermal vaporization) or HG (hydride generation). The spectral interferences should be mentioned. For the correction of these, normally the use of an internal standard is the method of choice. An approriate internal standard is one which a) is originally present in the sample at a negligible concentration b) ideally has a mass ans ionization energy which is as close as possible to those of the analyte c) does not adversely affect subsequent analysis of this element As a consequence of these r1equirements the choice of an—or better more—internal standards—dependent on the elements to be determined—is often difficult. One important application of ICP-MS should not be forgotten here: it presents the most preferred detector for species-analysis, due to the capabilities for on-line coupling with species separation techniques and to the low detection limits available with this technique. In the field of human biomonitoring ICP-MS will be the preferred method in future for routine analysis of many samples which are to be analyzed for several elements and for very low concentrations, for non-target screening and for research. All the other techniques have still their place in a modern trace element analytical laboratory. The application of these methods depends on the problem.

REFERENCES Begerow, J., Turfeld, M., and Dunemann, L., 1997, Determination of physiological platinum levels in human urine using magnetic sector field inductively coupled plasma mass spectrometry in combination with ultraviolet photolysis, J. Anal. At. Spectrom. 11:913–916. Begerow, J. and Dunemann, L., 1996, ICP-MS bei biologischen Proben, Nachr. Chem. Tech. Lab. 44(7/8):739–743. Hasse, S. and Schramel, P., 1983, Voltammetric determination of Cd, Cu, Co, Ni and Pb in milkpowder and other biological materials with special regard to the ashing method, Mikrochim. Acta III:449–455. Schramel, P., Wolf, A., Seif, R., and Klose, B.-J., 1980, Eine neue Apparatur zur Druckveraschung von biologischem Material, Fresenius Z. Anal. Chem. 302:62–64. Schramel, P. and Hasse, S., 1993, Destruction of organic materials by pressurized microwave digestion, Fresenius J. Anal. Chem. 346:794–799. Schramel, P., Klose, B.-J., and Hasse, S., 1982, Die Leistungsf %ohigkeit der ICP-Emissionsspektroskopie zur Bestimmung von Spurenelementen in biologisch-medizinischen und in Umweltproben, Fresenius Z. Anal Chem. 310:2098–216.

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Schramel, P., Begerow, J., and Emons, H., 1999, ICP-MS for the human biomonitoring, in: Analysis of Hazardous Substances in Biological Materials (Angerer, J. and Schaller, K.-H., Eds.) Vol. 6, 1–50, Wiley—VCH, Weinheim, New York. Williams, J.G., 1992, Instrument Options. In: Handbook of Inductively Coupled MassSpectrometry (Eds. Jarvis, K.E., Gray, A.L., and Houk, R.S.), Blackie, Glasgow, 58–80. Vanhoe, H., Dams, R., and Versieck, J., 1994, Use of ICP-MS for the Determination of Ultra-Trace Elements in Human Serum, J. Anal. Atom Spectrom. 9:23–31. Zumkley, H. (Ed.), 1983: Spurenelemente, Georg Thieme Verlag Stuttgart, New York.

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DISTRIBUTION OF SELENIUM, ARSENIC, IRON, AND SCANDIUM IN SELENIUM-DEFICIENT RATS USING THE MULTITRACER TECHNIQUE

Rieko Hirunuma, Shuichi Enomoto, Shizuko Ambe, and Fumitoshi Ambe The Institute of Physical and Chemical Research (RIKEN) Wako, Saitama 351-0198 Japan

1. INTRODUCTION Selenium is an essential and a poisonous element to mammals, which means administration of Se compounds has a narrow margin of safety between therapeutic and toxic doses (Berry et al., 1995). A number of studies on the role of Se in biochemistry are found in literature: selenium is known as the central elements glutathione peroxidase (Burk, 1994). Thirteen Se-containing proteins (molecular weights from 12,100 to 75,400) have been found in organs of rats (Behne et al., 1988). The binding of selenite to plasma proteins and the role of erythrocytes have been studied (Mas et al., 1988, 1989). In rats and human, at least two Se-binding proteins, in which selenite is not incorporated, are present in plasma. It is well known that fish with high concentration of Hg in their bodies have high concentration of Se also, and that Hg poisoning is inactivated by some effect of Se (Urano et al., 1997). Other than this, it is reported that Se is in a competitive or synergetic relationship with several elements such as Mn, Fe, Co, Zn, and As. Therefore, it is expected that distributions of other trace elements in various organs are also influenced by the amount of Se fed to the mammals. However, no systematic study has been reported on the behavior of trace elements in Se-deficient rats. This paper describes the uptake and distribution of trace elements in two types of Se-deficient rats examined by the multitracer technique. This technique is useful to evaluate the behavior of many elements under the identical experimental condition (Ambe et al., 1995). In this investigation, rats bred on Se-deficient diet from the fetal period were used as Se-deficient rats (I). Se-deficient rats (II) were fed on Se-deficient diet from the weanling period. Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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2. MATERIAL ET METHOD Preparation of a multitracer solution: Multitracer solutions containing various kinds of radionuclides were prepared from an Ag target irradiated with or beam of 135 MeV/nucleon from RIKEN Ring Cyclotron. The irradiated Ag target was dissolved in (1:1) Then Ag was precipitated as AgCl with cone. HCl and the AgCl was filtered out. The solution was evaporated to dryness under reduced pressure. The residue was dissolved in a physiological saline solution. Animals: Wistar male rats of 12-week-old were used in the present study. Sedeficient rats (I): Wistar dam rats had been fed with Se-deficient diet (produced by Oriental Yeast Co., LTD., Japan) since their 14th day of pregnancy until the weanling period of their baby rats. The weanling male rats were isolated from their dams, and were fed with Se-deficient diet until the age of 12 weeks. These male rats were used as Se-deficient rats (I). Se-deficient rats (II): Weanling male rats (4 weeks old) were fed with Sedeficient diet for 8 weeks to make them Se-deficient rats (II). Control rats: Four-week-old male rats were fed with Se-deficient diet added with 0.2 ppm of Se (adequate level of Se for normal growth) for 8 weeks and used as control rats. Administration of the multitracer and g-ray measurement: A tenth ml of saline solution containing the multitracer was injected intravenously into each rat. The Se-deficient (I), (II), and control rats were sacrificed at 72 hours after injection, and the radioactivity of their organs, tissues and blood was measured using highpurity Ge detectors. The observed g-rays were assigned to each nuclide on the basis of its energy and half-life. The behavior of Be, Na, Ca, Sc, V, Cr, Mn, Fe, Co, Zn, Ga, As, Se, Rb, Sr, Y, Zr, Tc, Ru, and Rh was examined.

3. RESULTS AND DISCUSSION Influences exerted by Se-deficiency were observed on the behavior of Se, As, Fe, and Sc in various organs. Figure 1 shows the uptake of Se in the brain, the testicles, and the liver of the Se-deficient (I), (II), and control rats. The uptake of Se was higher in the brain of the Se-deficient rats (I) and (II) than in that of the control ones. On the other hand, the uptake of Se in the testicles was higher for the Se-deficient rats (II) only com-

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pared with that for the control ones. And, there was almost no influence on the uptake of Se in the testicles of the Se-deficient rats (I). In the liver, the uptake of Se in the control rats was the highest among the three types, and that in the Se-deficient rats (I) was the lowest. For control rats, the uptake of Se in the liver was much larger than that in the brain and the testicles (Fig. 1). For Se-deficient rats (II), the uptake in the brain and the testicles was larger than that in the liver. It is reported that Se fed to Se-deficient rats is absorbed preferentially in organs such as the brain and the testicles. It is considered that the results described above are attributed to this reason. Against this idea, however, the uptake in the testicles of Se-deficient rats (I) was smaller than that of Se-deficient rats (II), while almost comparative uptake was observed for the brain. Only a limited number of investigations, in which rats in so severe Se-deficient state as Se-deficient rats (I) were used, has been reported. The spermatozoa formation is the only one function, to our knowledge, which is so sensitive that Se-deficient state from the early stage of pregnancy makes the development of the function impossible. The decrease of the uptake of Se in the testicle of rats (I) is, therefore, considered to be related to this fact. Larger uptake was observed for As, Fe, and Sc in Se-deficient rats (I) compared with that in Se-deficient rats (II) and control rats (Fig. 2). Selenium is known to enhance As excretion into the bile in rats (Glattre et al., 1995; Levander and Baumann, 1966a, b). The observed increase in As uptake suggests that the bile excretion of As was decreased by the severe Se-deficiency. The increase of Fe uptake in the liver suggests an increase of Fe-binding proteins, such as ferritin or catalase in the liver. One of the reasons for the similar behavior between Sc and Fe is presumably the similarity of their ionic valence and of their ionic radii (0.73Å for and 0.64Å for (Weast, 1989–1990)). On the other hand, the behavior of other trace elements except Se in the Se-deficient rats (II) was not influenced by Se-deficiency. From this it is suggested that the metabolism of these elements is affected by Se-deficiency for fetus.

REFERENCES Ambe, S., Chen, S.Y., Ohkubo, Y., Kobayashi, Y., Maeda, H., Iwamoto, M., Yanokura, M., Takematsu, N., and Ambe, F, 1995, "Multitracer" a new tracer technique—Its principle, features, and application., J. Radioanal. Nucl. Chem. 195:297–303. Behne, D., Hilmert, H., Scheid, S., Gessner, H., and Elger, W., 1988, Evidence for specific selenium target tissues and new biologically important selenoproteins., Biochim. Biophys. Acta 966:12–21. Berry, J.P., Zhang, L., and Galle, P., 1995, Interaction of selenium with copper, silver, and gold salts. Electron microprobe study., J. Submicrosc. Cytol. Pathol. 27:21–28. Burk, R.F. (ed.), 1994, Selenium in biology and human health, Springer-Verlag, New York.

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Glattre, E., Mravcova, A., Lener, J., Vobecky, M., Egertova, E., and Mysliveckova, M., 1995, Study of distribution and interaction of arsenic and selenium in rat thyroid., Biol. Trace Elem. Res. 49:177–185. Mas, A., Jiang, J.Y., and Sarkar, B., 1988, Selenite metabolism in rat and human blood., Biol. Trace Elem. Res. 15:97-110. Mas, A. and Sarkar, B., 1989, Role of glutathione in selenite binding by human plasma, Biol. Trace Elem. Res. 20:95-104. Levander, O.A. and Baumann, C.A., 1966a, Selenium metabolism V. Studies on the distribution of selenium in rats given arsenic., Toxicol. Appl. Pharmacol. 9:98–105. Levander, O.A. and Baumann, C.A., 1966b, Selenium metabolism VI. Effect of arsenic on the excretion of selenium in the bile., Toxicol. Appl. Pharmacol. 9:106–115. Urano, T., Imura, N., and Naganuma, A., 1997, Inhibitory effect of selenium on biliary secretion of methyl mercury in rats., Biochem. Biophys. Res. Commun. 239:862–867. Weast, R.C. (ed.), 1989-1990, CRC Handbook of Chemistry and Physics, 70th ed., pp. F-187, CRC Press, Inc., Boca Raton, Florida.

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SEPARATION OF METALLOTHIONEIN ISOFORMS AND IDENTIFICATION OF COMPLEXED METALS BY CAPILLARY ZONE ELECTROPHORESIS USING DIODE ARRAY DETECTION

V. Virtanen, G. Bordin, and A.-R. Rodriguez European Commission Joint Research Centre Institute for Reference Materials and Measurements Retieseweg, B-2440 Geel Belgium

1. INTRODUCTION Metallothioneins (MTs) constitute a class of metalloproteins with low molecular mass (6–7 kDa) and are characterized by a high cysteine content (~30%), by lack of aromatic amino acids and by their resistance to thermocoagulation and acid precipitation. MT is a protein induced by various stimuli such as metals, stress and some drugs. MT has the ability to bind with high affinity and stoichiometry metals such as zinc, cadmium, copper and mercury through the formation of thiolates. The MTs seem to play an important role in number of metabolic mechanisms including the homeostasis of some essential metals (Zn, Cu) and detoxification of harmful (Cd, Pb) and excessive essential metals. Most MTs have several isoforms which are product of genetic polymorphism characteristic of MT genes in animals and humans. Mammalian MTs have often 2 major isoforms (MT-1 and MT-2) which have a pI value between 3.9 and 4.6 and differences of charge due to certain amino acid substitutions. Furthermore, many animal species generate various sub-isoforms of MT-1 and MT-2, therefore displaying significant microheterogeneity. Investigation of the functional significance of the individual MT isoforms and subisoforms requires techniques that offer a high degree of resolution. We used capillary zone electrophoresis (CZE) with diode array detector for the separation of metallothionein isoforms and for the identification of complexed metals. Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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2. EXPERIMENTAL CZE was performed on a P/ACE System 5000 capillary electrophoresis instrument (Beckman, Fullerton, CA, USA) equipped with a P/ACE Station software. Uncoated fused-silica capillaries of and containing polyimide cladding were obtained from Beckman. Overall capillary length was 57cm with on-line detection at 50cm. Pressure injection mode was used (0.5psi). Separated components were detected by using a diode array detector. All buffers (tris-borate, tricine, tricine-MeOH) were prepared with ultrapure water obtained from a Milli-Q Plus 185 water purification system (Millipore S.A., Molsheim, France) and degassed in an ultrasonic bath. The buffers were filtered using a filter (Gelman Sciences, Ann Arbor, MI, USA).

3. RESULTS AND DISCUSSION In capillary zone electrophoresis the separation of analytes is based on the differences in the electrophoretic mobilities resulting in different velocities of migration of ionic species in the electrolyte solution in the capillary. Separation is mainly based on the differences of the charge to mass ratios of the analytes. In fused-silica capillaries cations with highest charge/mass ratio migrate first, followed by cations with reduced ratios. Neutral components are then transported with the electroosmotic flow. Finally the anions migrate; first anions with lower charge/mass ratio and last anions with greatest charge/mass ratio. Also the type of interaction between the analyte and both the capillary inner surface and running electrolyte play an important role. A critical point for analysis of metalloproteins is the pH of the running electrolyte also called running buffer. If the analytes are expected to preserve their state of origin then pH’ s similar to those found in the original tissues should be used. This means that the running electrolyte should have a pH value where the metals are still bound to the organic part in the complex and not dissociated. For metallothioneins neutral or nearly neutral conditions are well suited for CZE separations. Horse kidney MT has two isoforms marked MT-1 A and MT-1 B. Their molecular masses for apoforms are quite similar 6,040 and 6,067 Daltons, respectively. In total they have six differences in their amino acid sequences. However, variations are known to exist within horse kidney MT-1 A. Namely in position 39 glysine can be replaced by arginine, called MT-1R39, and in position 54 serine can be replaced by lysine, called MT1L54. Using tris-borate buffer we found that horse kidney MT sample (Sigma, St. Louis, MO, USA) exhibited 5 peaks (Virtanen, V. et al., 1996). We observed batch to batch variations so that two of the peaks could multiply their abundance in one sample and they could be nearly absent from another sample. However, by using diode array detection we could observe that three of these 5 peaks did not show typical UV spectra for metallothionein (Virtanen, V. and Bordin, G., 1998). These seemed to lack almost totally metals binding to thiol groups. Namely, Zn-thiol and Cd-thiol bonds showing absorbance at 225 nm and 250 nm were missing. We could also see from the electropherogram that these three species have either higher molecular mass or lower charge than horse kidney MT-1. Using liquid chromatography with electrospray mass spectrometric detection, LC-ES-MS, Chassaigne and Lobinski (1998) studied a horse kidney sample in apoform applying acid conditions. They also found, in agreement with us, three other proteins than horse kidney metallothionein-1. These proteins have higher molecular mass than horse kidney metallothionein isoforms.

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Identification of impurities is possible using diode array detection. MT-1 sample from rats administrated with zinc (Ciemat, Madrid, Spain) showed three peaks, one of them being clearly minor compared to the two others. UV spectra of each of them were obtained. We knew from gel electrophoresis results previously done by the supplier that there are impurities present in the sample. However, the RP-HPLC method used by the supplier could not separate these impurities. By using the UV spectra we could identify the impurity peaks. Furthermore, by adding cadmium to the sample we could see the appearance of an absorbance shoulder at 250 nm in the UV spectra of one of the peaks, due to complexation of cadmium to the thiol groups of the metallothionein isoform. However, with the impurities no change in the UV spectra was observed indicating that the impurities did not form cation-thiol bonds and confirming that these compounds are not metallothioneins. Organic solvents can be added to electrolyte solution as modifiers. They increase the viscosity of the electrolyte solution and induce variations in the dielectric constant of the buffer. This causes changes in the double-layer of the capillary and results in decreasing electroosmotic mobility. It also changes the type of interaction between analytes, capillary surface and electrolyte solution. We tested several different organic modifiers, both protic and aprotic (Virtanen, V. et al., 1998). With addition of methanol to the electrolyte solution, we observed improvement of the separation of rabbit liver MT isoforms (Sigma, St. Louis, MO, USA). Very interestingly we observed splitting of the horse kidney MT isoform peaks. Using higher methanol content we could find eight MTlike species together with one non-MT-like species in horse kidney sample. These eight species can be either metalloforms of the horse kidney MT isoforms or isoform variants. Further investigations are still needed to know the true character of these peaks. However, Chassaigne et al., (1998) have found in their LC-ES-MS studies that in acidic conditions eight isoform variants are present in total. At neutral conditions they could find four metal variants for both horse kidney isoforms. From all these studies, we can conclude that capillary zone electrophoresis is a powerful tool for MT isoform separation. When separation is carried out at neutral or slightly alkaline pH, the original MT is preserved since No dissociation of metals occurs. Information of cation-thiol bonds can be obtained by using diode array detection giving more information about the species and this can be used as an identification method for MTs. Similarly, further metal complexation with thiol groups of the analyte can be investigated by assessing the changes in the UV spectra.

REFERENCES Chassaigne, H. and Lobinski, R., 1998, Characterization of horse kidney metallothionein isoforms by electrospray MS and reversed-phase HPLC-electrospray MS, Analyst, 123, 2125–2130. Virtanen, V. and Bordin, G., 1998, Characterization of mammalian metallothionein isoforms by capillary zone electrophoresis with diode array detection using tris-borate buffer, J. Liq. Chrom. & Rel. Technol., 21(20), 3087–3098. Virtanen, V., Bordin, G., and Rodriguez, A.-R., 1996, Separation of metallothionein isoforms with capillary zone electrophoresis using an uncoated capillary column: Effects of pH, temperature, voltage, buffer concentration and buffer composition, J. Chromatogr. A, 734, 391–400. Virtanen, V., Bordin, G., and Rodriguez, A.-R., 1998, The influence of experimental conditions and of organic solvents as modifiers on the separation of metallothionein isoforms by capillary zone electrophoresis in an uncoated capillary column, Chromatographia, 48, 637–642.

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INDUCTIVELY COUPLED PLASMA TIME OF FLIGHT MASS SPECTROMETRY FOR TRACE ELEMENT AND SPECIATION ANALYSIS

Håkan Emteborg, Xiaodan Tian, Monika Heisterkamp, and Freddy C. Adams Department of Chemistry Micro- and Trace Analysis Center MiTAC, University of Antwerp UIA, Universiteitsplein 1 B-2610 Antwerp Belgium

1. INTRODUCTION Analytical instrumentation based on inductively coupled plasma mass spectrometry was introduced in 1983. With a highly energetic argon plasma as a continuous ion source, these instruments incorporate quadrupole mass filters for obtaining unit mass resolution of the elements in the periodic table. A very sensitive and truly multielemental technique was from that point available to users in many fields. A restriction to very rapid multi-elemental determinations is the quadrupole mass filter itself since the mass range from to is swept one mass unit at a time. The user must define integration times on each isotope and on top of this there is a settling time for the rods in the quadropole. These features make the detection mode sequential and the overall time for analysis is proportional to the number of isotopes determined. It would apparently be a considerable advantage with a much faster mass spectrometer coupled to ICP, for example, time of flight mass spectrometry, as outlined by Hieftje et al.1 During 1998, an axial Inductively Coupled Plasma Time of Flight Mass Spectrometer was commercialised by LECO, St. Joseph, MI, USA. Following an outline of the principles of operation for the ICP-TOF-MS, figures of merit will be reported along with results for coupling capillary GC to ICP-TOF-MS.

1.1. Principle of Operation for ICP-TOF-MS The ions are formed in the plasma and extracted into the mass spectrometer through the sampler- and skimmer cones. All ions have roughly the same kinetic energy Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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once they have been accelerated into the flight tube by a high voltage pulse. This means that ions of different mass have different velocity and the arrival time at the detector is thus proportional to m/z. In the system used here, travels the flight path in approximately whereas needs to travel the same distance. This also means that the ions can not be extracted continuously into the mass spectrometer, therefore the ion extraction from the plasma has to be modulated and the detector must also be able to respond to very rapid changes. To increase resolution, to double the flight path and as a photon stop a reflectron or ion mirror is also implemented. The present system can measure up to 65 isotopes quasi-simultaneously. The overall measurement-time of one or 65 isotopes is thus the same and the time required is determined by the selected integration time and the number of replicates desired. As an example, five replicate measurements of 13 isotopes with a 10-second integration time takes 50s using ICP-TOF-MS, for a quadrupole ICP-MS, almost eleven minutes are necessary for the same measurement. Since ions of all masses are extracted into the mass spectrometer, there is a need for removing highly abundant matrix ions online not to over-saturate or destroy the detector. For this purpose a Transverse Rejection Ion Pulse, TRIP is implemented to deflect matrix ions such as and The narrrowest deflection window affects roughly 5 amu. The main advantage of ICP-TOF-MS is the high speed, which allows full elemental coverage in minute volumes of sample and very rapid sample transients from ETV, chromatographic or laser ablation systems. An example of this will be given below for capillary gas chromatography coupled to ICP-TOF-MS. The high speed should also enhance the precision for isotope ratio measurements.

2. RESULTS In Table 1 typical figures of merit are given. The values reported are not highly different for a quadrupole ICP-MS system with the important exception that the time

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spent for collecting the information is much shorter using the ICP-TOF-MS system. To illustrate the high potential for this instrumentation when used for speciation- or transient sample analysis, Figure 1 has been included. The benefit of the high speed of the mass spectrometer clearly becomes evident when comparing the spectral skew that can be observed for the four lead isotopes in a chromatographic peak of organolead using a quadrupole ICP-MS for detection as a contrast to cGC-ICP-TOF-MS.

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3. CONCLUSIONS The main advantage of ICP-TOF-MS to previous ICP-MS instrumentation is the high speed and full elemental coverage in or rapid transients. For trace element determinations in samples from various biological systems the levels of several essential trace elements may interact and depend on each other in an intricate way. ICPTOF-MS should be the method of choice if such inter-relationships are to be characterised since it incorporates unsurpassed speed and isotopic information allowing the use of stable isotope tracers. Moreover, coupling to chromatographic or laser ablation systems may lead to a better understanding of the trace element content in specific bands from a gel or a chromatographic peak adding even more selectivity to the analysis. The need for the TRIP is one of the weakest points of the present system and this feature is likely to be refined further in the future. ACKNOWLEDGMENT We thank the Fund for Scientific Research (FWO, Brussels, Belgium) for financial support.

REFERENCE Hieftje, G.M., Myers, D.P., Li, G., Mahoney, P.P., Burgoyne, S.J., Ray, S.J., and Guzowski, J.P., 1997, Toward the Next Generation of Atomic Mass Spectrometers, J. Anal. At. Spectrom., 12, 287.

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SILICON ABSORPTION FROM STABILIZED ORTHOSILICIC ACID AND OTHER SUPPLEMENTS IN HEALTHY SUBJECTS M. Calomme1,*, P. Cos1, P. D’Haese2, R. Vingerhoets1, L. Lamberts2, M. De Broe2, C. Van Hoorebeke1, and D. Vanden Berghe1 1

Department of Pharmaceutical Sciences Department of Nephrology-Hypertension University of Antwerp (U.I.A.) Universiteitsplein 1, B-2610 Antwerp Belgium

2

1. INTRODUCTION Silicon (Si) is, next to oxygen, the most abundant element in the earth’ s crust. The distribution of Si in vertebral tissue and physiological changes in bone caused by dietary Si deficiency indicate that Si influences bone formation by affecting cartilage composition and ultimately cartilage calcification (Carlisle, 1997). The dietary intake of Si was estimated for US citizens to be between 20–50 mg with the lower intake for animal-based diets and the higher intake for plant-based diets (Pennington, 1991). However, studies on the minimum Si requirement and supplementation experiments, comparing the bioavailability of different Si compounds, are lacking. Orthosilicic acid was suggested to have an important function in Si metabolism (Berlyne et al., 1986; Carlisle, 1997) and is found in both fresh water and sea water. This monomeric form of silicic acid is stable in dilute concentrations of about but condenses into silica gels at higher concentrations and low pH. The absorption and urinary excretion of Si from (a) stabilized orthosilicic acid (OSA), (b) standardized herbal silica extract from the Si-accumulator plant Equisetum arvense, and (c) colloidal silicic acid were compared in a double-blind study with healthy subjects.

Fax 32.3.820.25.44, email: [email protected] * Author for correspondence Trace Elements in Man and Animals 10, edited by Roussel et al., Kluwer Academic / Plenum Publishers, New York, 2000.

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2. MATERIALS AND METHODS Fourteen healthy subjects (8 females, 6 males, aged 22–34 y) were included after informed, written consent. None had taken Si supplements within 3 months before the start of the study. Each fasting subject received in a cross-over protocol an equimolar dose of Si p.o. (20 mg Si) in the form of stabilized orthosilicic acid (OSA, 1 ml of BioSil containing 20 g Si/l as stabilized monomeric silicic acid, Bio Minerals NV, Belgium [Calomme and Vanden Berghe, 1997]), herbal silica (533 mg of a dry Equisetum arvense extract containing 8% (w/w) colloidal silicic acid (2ml of or a placebo (10ml mineral water) with 1 week wash-out period between each supplement or the placebo. Blood samples were collected in Si free polypropylene tubes prior to supplementation and after 1,2, 4, 6, and 8 hours post partem. Urine was collected between 0 and 24 hours post partem in polypropylene containers. Identical meals were consumed during the experiment after 2 and 6 hours supplementation. The Si concentration in serum and urine was determined for each subject in one batch with AAS (Huang et al., 1993). A Zeeman/3030 Atomic Absorption Spectrometer equipped with a HGA-600 graphite furnace was used in combination with an AS-60 autosampler (Perkin-Elmer Corp. Norwalk CT). Pyrolytic coated graphite tubes were used. The hollow cathode lamp settings were respectively 30 mA lamp current, 251.6 nm spectral line and 0.7 nm band width. The injected sample volume was and signals were measured in the peak-area mode. Serum and urine samples were diluted 1/10 in a matrix-modifier solution containing 0.50 g/1 and 0.1% (v/v) HNO3. Samples were measured against a calibration curve prepared in the matrix-modifier solution, using as standards 125 and Si, respectively. The sensitivity determined as the amount of silicon yielding a 0.0044 Abs.s signal was 82 pg. The precision determined as the inter-assay c.v. was 13.3%, 6.2%, and 3.1% for Si concentrations of (n = 6), (n = 6), and (n = 6), respectively. The area under the time concentration curve (A.U.C.) was calculated using the linear trapezoidal rule. Statistical significance was investigated with the Wilcoxon matched-pairs signed ranked-test. The relation between two parameters was investigated with the Spearman correlation procedure.

3. RESULTS The mean baseline silicon concentration in serum was 112 ± 42 (mean ± S.E.). A significant increase in serum Si concentration from the baseline value was observed after respectively 1 h for OSA (mean ± S.E.: p < 0.005), 4h for the placebo (23 ± p < 0.025) and herbal silica p < 0.025), and 8 h for colloidal Si (36 p = 0.01). The mean A.U.C. (table) was significantly higher after OSA supplementation (p < 0.005), but was not significantly different for respectively herbal silica and colloidal Si compared to the placebo. A significant correlation was found between the individual A.U.C. and the individual urinary Si concentration (r = 0.43, p = 0.0011, n = 56, figure). The urinary silicon excretion (table) was significantly higher after OSA supplementation (p < 0.005) but was not significantly different for respectively herbal silica and colloidal Si compared to the placebo.

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4. DISCUSSION The serum Si concentration of subjects receiving a placebo increased 2 hours after consumption of a meal, indicating a significant variation of the serum silicon concentration during the day. The present study demonstrates that the bioavailability of silicon is largely dependent on its chemical form. The absorption of Si is faster, higher and less subject dependent for stabilized orthosilicic acid compared to herbal silica and colloidal silicon which are polymerized forms of orthosilicic acid. Consequently, the chemical form of Si in the diet will influence the minimal Si requirement for humans. The difference in absorption between Si supplements is confirmed by a higher urinary Si excretion for OSA and is in agreement with the dose-dependent Si absorption from stabilized orthosilicic acid which was demonstrated in an animal supplementation study (Calomme and Vanden Berghe, 1997). The significant correlation between the

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absorption measured by the A.U.C. and the urinary excretion confirms earlier reports suggesting renal clearance as a major route for silicon excretion.

REFERENCES Berlyne G.M., Adler A.J., Ferran N., Bennett S., and Holt J., 1986, Some aspects of renal silicon handling in normal man, Nephron 43:5–9. Calomme M.R. and Vanden Berghe D.A., 1997, Supplementation of calves with stabilized orthosilicic acid: Effect on the Si, Ca, Mg, and P concentrations in serum and the collagen concentration in skin and cartilage, Biol. Trace Elem. Res. 56:153–165. Carlisle E.M., 1997, Silicon. In: O'Dell B.L. and Sunde R.A., eds. Handbook of Nutritionally Essential Mineral Elements. New York: Marcel Dekker, Inc., 603–618. Huang Z., D'Haese P.C., Lamberts L.V., Van Landeghem G.F., and De Broe M.E., 1993, Silicon determination in biological fluids by Zeeman atomic absorption spectrometry. In: Anke M., Meissner D., and Mills C.F., eds. Trace Elements in Man and Animals—TEMA 8. Gersdorf: Verlag Media Touristik, 98-99. Pennington J.A., 1991, Silicon in foods and diets. Food Additives and Contaminants 8:97–118.

341

PREPARATION AND ICP-MS MEASUREMENTS OF MAGNESIUM STABLE ISOTOPES IN HUMAN SAMPLES M. Sabatier1, W. R. Keyes2, M. J. Arnaud1, and J. R. Turnlund2 1

Perrier Vittel Water Institute Vittel, France and 2 USDA, Western Human Nutrition Center San Francisco, USA

Thermal Ionization Mass Spectrometry (TIMS) is the reference method for the analyses of stable isotopes. However, the high precision and accuracy obtained with TIMS need time-consuming tasks for the preparation of the samples and for their quantitative measurements. In this study, the optimal conditions have been determined for Mg isotopic ratios in human samples by Inductively Coupled Argon Mass Spectrometry (ICP-MS). From standard solutions and diluted and ashed urine samples analyzed at 10 and 100ppb magnesium (Mg) concentrations it was shown that both matrix and Mg concentrations had no effect on and ratio measurements. No difference as well was observed between 1.5 and 5 minutes run time analyses. A precision of the and ratios of 0.39% RSD (range 0.18–0.53%) and 0.38% (range 0.26–0.50%) respectively was obtained at 100 ppb Mg concentration and for 1.5 minute running time. With standard solutions and urine samples spiked with and to levels of 5 to 30% isotope excess, similar isotope recoveries were obtained at either 10 or 100ppb Mg concentrations and for 1.5 and 5 minutes running time. Accuracy of the measurements were shown by the agreement between measured and expected ratios found in natural and enriched samples. These results demonstrated that stable isotope Mg measurements by ICP-MS of untreated urine samples are rapidly obtained and are in agreement with standards values. Analytical results on fecal and blood samples will be also presented.

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342

CHARACTERIZATION OF ZINC METALLOTHIONEINS USING ELECTROANALYTICAL METHODS

M. Dabrio and A. R. Rodríguez Commission of the European Communities Joint Research Centre (JRC) Institute for Reference Materials and Measurements (IRMM) Retieseweg, B-2440 Geel, Belgium

Metallothioneins (MTs) are proteins with a relatively low molecular weight and a high content of thiol groups (~30mol%), giving them a high affinity to metals. This property enables them to be involved in some major biological processes such as the detoxification of heavy metals, metabolism of essential metals or controlling cell growth. Sub-isoform MT-2 of two zinc metallothioneins have been investigated using electrochemical techniques such as Differential Pulse Polarography (DPP) and Square Wave Voltammetry (SWV), in order to have a better understanding of their ion exchange properties when the addition of cadmium and/or zinc were carried out to the molecule. These MT, containing almost only zinc in their structure, were isolated and purificated from liver tissue of two different origins, rat and human foetal, provided by the CIEMAT (Spain) and the Catholic University of Leuven (Belgium), respectively. Three different responses due to the Zn(II) reduction were distinguished, indicating different forms of complexation of zinc with the thionein. The additions of cadmium, which is quickly incorporated into the molecule, provoke a clear reorganization of their structure affecting not only peaks of the Cd(II) reduction attributed to two forms of complexation of cadmium-thionein but also changes in the co-ordination of zinc complexes. The influence of the pH in the complexation equilibrium was also studied in a narrow range of pH due to the low stability of the samples. The pH plays an important role in the stability of the different sorts of cadmium and zinc complexes. Voltammetric peaks attributed to different species are interrelated.

1116

343

HEPATIC METALLOTHIONEIN ISOFORMS INDUCTION BY CADMIUM AND ITS DETECTION USING CAPILLARY ELECTROPHORESIS

B. Ribas Ozonas, O. García Arribas, and M. Pérez Calvo

Instituto de Salud Carlos III CNSA, Majadahonda 28220 Madrid

Cadmium is a heavy metal widely distributed in the environment arising from industrial emissions and wastes. Cadmium has been reported to produce several toxic effects in animals and man. The liver and kidney are the critical organs. One of the biochemical mechanism of cadmium toxicity is the induction and accumulation of Metallothionein (MT), a peptide of 7,000D (well known as low molecular weight protein), cysteine-rich, heavy metal binding protein. The MT has a great capacity to bind in its molecule metal ions forming stable complexes.1 Although the physiological role of metallothionein and posibly its isoforms are still unclear, it is known that these proteins participate in the detoxication of some heavy metals such as cadmium and in the homeostatic regulation of some essential metals such as zinc and copper. In this work we propose to use the MT as bioindicator of cadmium exposure and toxicity. We used two groups of male Wistar rats, a control group and the treated one with cadmium with twelve intraperitoneal doses from 0.1 to 1mg Cd/Kg rat/day. After we extract blood to analyze differents blood parameters, the rats are sacrificed with ether anesthesia and the liver is removed. The MT is isolated from this tissue after homogenization, and applying the classical technique of purification, the differents MT isoforms are determinated by Capillary Electrophoresis, using as electrolyte, phosphate buffer at pH 5, 7, and 8. The results show that with treated rats the levels and the number of MT isoforms are higher than control rats. In the cadmium exposure, the animals defend themselves synthesizing “de novo” MT, but this protein is again blocked and loses its biological function. The effect of cadmium as endocrine disruptor was showed before in rat testes, growth, weight, libido, reproduction and bone deposition, with body, morphological and molecular inhibitions.2 With capillary electrophoresis it is shown that, when the pH is becoming basic the peaks gain in hight, more separation and in resolution. At pH 8 there is obtained more numbers of 1117

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peaks, better separated and with higher resolution, increasing the retention time (Rt). The results of blood analysis show that hemoglobin and hematocrit are below the control values because of the anemia produced by cadmium treatment. The transaminase GOT and LDH are increased concerning the controls, due to the enzyme release by the hepatic damage and also the competition between both ions. The proteins in blood are decreased because cadmium exposure induces proteinemia.

REFERENCES 1. Bremner, I. (1991) Nutritional and physiological singificance of metallothionein. Methods Enzimol. 205, 25–35. 2. Bondía, S., Jaudi, A., Ribas, B., Santos Ruiz, A., and Sánchez Reus, M.I. (1980) Some physiological data of rats exposed to cadmium. Trace Element Anal. Chem. Med. Biol. vol 2, Walter de Gruyter and Co. Edt., Berlin-New York (P. Brätter and P. Schramel, Eds.) 37–46.

344

RECENT DEVELOPMENTS IN HPLC-ICP-MS FOR THE “DIFFICULT” ELEMENTS

F. Abou-Shakra and P. Booker Micromass UK Limited Floats Road, Manchester UK

Speciation studies have tended to concentrate on elements such as Sn, Hg, Pb, Cd, Se and As. The growing interest in speciation analysis includes the study of trace element speciation in human and animal health. The importance of trace elements in optimal health is largely due to the protein chemistry of the element. The simultaneous coupling of an HPLC system to a UV/VIS detector and an ICPMS supplies the analyst with information regarding the type of metals bound to various proteins. Speciation studies traditionally suffer from a number of problems when coupling chromatographic systems to ICP-MS. Firstly, a number of important elements such as Se and As determinations are subject to severe spectral interferences in conventional ICP-MS from argide-based molecular ions. In addition, As and Se have relatively high first ionisation energies and are only ionised to ~30% in an argon ICP, which in turn leads to relatively poor sensitivity for these elements. Thirdly, the concentrations of these elements after dilution and fractionation of the sample is normally in the sub-ng/ml range. Finally, the on-line coupling of chromatography systems has traditionally led to problems with software control of the third party hardware and the inability to undertake simultaneous multi-element analyses. The use of a hexapole collision cell in the ICP-MS and specialised integrated software has eliminated these problems. Data will be presented to illustrate the benefits of this system when using it for the coupling of HPLC to ICP-MS and the analysis of some of the traditionally “difficult” elements in biological fluids.

1119

345

DETERMINATION OF HEAVY METALS IN CALCIUM AND HERBAL SUPPLEMENTS UTILIZING INDUCTIVELY COUPLED PLASMA MASS SPECTROMETRY (ICP-MS)

Elzbieta (Ela) Bakowska Hewlett-Packard Company, 2850 Centerville Road, Wilmington, DE 19808, USA

Recent studies have prompted concerns regarding the composition of mineral and herbal supplements. These utimately have resulted in mandated heavy metals testing of calcium carbonate based supplements in the United States. Proposals for similar actions are now being considered for regulating the contents of herbal supplements. The analytical techniques most commonly used for metals determination in these types of samples are graphite furnace atomic absorption spectroscopy (GFAAS) and inductively coupled plasma opticial emission spectroscopy (ICP-OES). The requirements for increased detection capability, improved selectivity and enhanced productivity has stimulated interest in the use of ICP-MS as an alternative to these methods of analysis. ICP-MS is a rapide multi-element technique with superb detection capability that can provide both qualitative and quantitative information for up to 70 elements. The determination of trace metals in a variety of over-the-counter calcium supplements, calcium based antacids, and herbal supplements by ICP-MS was conducted and analytical figures merit established.

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OVERVIEW OF BIOMEDICAL APPLICATIONS OF INDUCTIVELY COUPLED PLASMA MASS SPECTROMETRY (ICP-MS)

Elzbieta (Ela) Bakowska

Hewlett-Packard Company, 2850 Centerville Road, Wilmington, DE 19808, USA

The importance of monitoring trace metals concentrations in biological systems for use as a research, diagnostic, and toxicological indicator has been well established. Analytical challenges associates with these analysis include limited sample availability, low analyse concentrations, and complex samples matrices. Many of these concerns can be addressed through use of an appropriate sample preparation protocol followed by analysis using inductively coupled plasma mass spectrometry (ICP-MS). ICP-MS is a powerful analytical technique allowing simultaneous elements including aluminium, boron, cadmium, lead, selenium and zinc by ICP-MS in matrices such as whole blood tissue, serum and urine will be discussed.

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347

DETERMINATION OF SERUM COBALT AT THE NANOMOLAR LEVEL BY DIRECT ELECTROTHERMAL ATOMIC ABSORPTION SPECTROMETRY J. Poupon1, V. Gleizes2, G. Saillant2, and M. Galliot-Guilley1 1

Laboratoire de Biochimie-Toxicologie Hôpital Fernand Widal Paris, France 2 Service de Chirurgie Orthopédique Traumatologique et Réparatrice de l’Appareil Locomoteur CHU Pitié-Salpêtrière, Paris France

Cobalt is an essential trace element present at very low level in blood: 0.5 to 9.0nmol/l, mean 5.5nmol/l, according to the most reliable authors. At this level, contaminations of the sample is an important drawback, but the sensitivity of analytical methods is a worrying matter too. RNAA, although very sensitive, is not very useful. To date, almost all techniques dealing with physiological levels include a pre-concentration step followed by electrothermal absorption spectrometry (ETAAS), leading, at the best, to a detection limit of 1.7nmol/l. However, these techniques are time consuming, delicate and increase the risk of contaminations. Cobalt and others elements such as chromium and molybdenum, are common components of orthopedic implants. Their release from implants and their potential toxicity is at present a question of debate. To compare serum cobalt of patients with metal hip arthroplasty to control subjects, we needed a simple and sensitive method for cobalt determination. We have developed a direct method by ETAAS in which of serum diluted 1:1.1 with a Pd-Triton modifier are injected into the furnace of a 5,100 PC Zeeman AAS (Perkin Elmer). Preliminary attempts showed that, although more sensitive, wall atomization was not suitable and we chose to use an intregrated platform that allows the deposit of Drying steps and, above all, pyrolysis steps are critical to minimize the accumulation of carbon residues. The standard addition technique was used for calibration. The detection limit (3 sd) of our technique is less than 1 nmol/l of serum (0.7nmol/l) and CV’s are satisfactory. 1122

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1123

Nineteen controls subjects were sampled with polyurethane catheter (Insyte Becton Dickinson) and blood was drawn in two 7.5ml syringes Sarsted). Great care was taken to avoid any further contamination. Accuracy was checked by analysis of the “Second-generation” Biological Reference Material (Freeze-Dried Human Serum) from Pr. J. Versieck. Mean value of controls was 4.51 nmol/l, sd = 3.31, range: 1.59–12.55. Our technique is fast, requires a minimum of handling and is sensitive enough to evidence moderate elevations of serum cobalt. Results obtained in patients with metalmetal total hip arthroplasty are reported elsewhere.

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LIST OF PARTICIPANTS

AASEN Ivar AGRICULTURAL UNIVERSITY OF NORWAY Department of soil and Water Sciences N-1432 AAS NORWAY Tel: ++47 64948212 Fax: ++47 64948211 ABOU SHAKRA Fadi MICROMASS UK LTD Floats Rd Wythenshawe M23 9LZ MANCHESTER ENGLAND Tel: ++44 161 945 4170 Fax: ++44 161 998 8915 [email protected] ABURTO Enrique UNIVERSITY OF PRINCE EDWARD ISLA 550 University Avenue CIA 4P3 CHARLOTTE TOWN CANADA Tel: ++1 902 566 0779 Fax: ++1 902 566 0851 AFANAS’EV Igor VITAMIN RESEARCH INSTITUTE Russian State Medical University 117513 MOSCOU RUSSIA Tel: ++7 95 4347187 Fax: ++7 95 4347187 [email protected] AGUILAR Ana Esther UNIVERSITY OF MEXICO SCHOOL Cd. Universitaria S/N Fac Quimica 04510 MEXICO, D.F MEXICO Tel: ++52 622 37 40 Fax: 52 622 37 40 AKESSON Bjorn LUND UNIVERSITY P.O. Box 124 Biomedical Nutrition S-22100 LUND SWEDEN Tel: ++46 462 22 45 23 [email protected] ALCARAZ Arlette CHU A. MICHALLON Laboratoire de Biochimie C 38043 GRENOBLE Cedex 9 FRANCE Tel: ++4 76 76 57 54 Fax: ++4 76 76 56 64 [email protected] ALCOCK Nancy UNIVERSITY OF TEXAS MEDICAL BRANCH 700 Harborside Drive 77555 1109 GALVESTON, Texas USA Tel: ++1 409 772 4661 Fax: ++1 409 772 6287 ALI Simon UNIVERSITY OF LIVERPOOL Dept of Medicine L69 3GA LIVERPOOL ENGLAND Tel: ++44 151 794 6233 Fax: ++44 151 706 3802 [email protected] ALIMONTI Alessandro ISTITUTO SUPERIORE DI SANITA Viale Regina Elena 299 00161 ROME ITALY Tel: ++ 39 06 4990 2080 Fax: ++39 06 4990 2366 [email protected] ALLEN Lindsay UNIVERSITY OF CALIFORNIA, DAVIS Dept Nutrition CA 95616 DAVIS USA Tel: ++1 530 752 5920 Fax: ++1 530 752 3973 AMANCIO Olga Maria S. UNIVERSIDADE FEDERAL DE SAO PAULO Escola Paulista de Medicina 04023-062 SAO PAULO—SP BRAZIL Tel: ++55 115498993 Fax: ++55 115498993 [email protected] AMMERMAN Clarence UNIVERSITY OF FLORIDA Dept of Animal Science FL 32611 0900 GAINESVILLE USA Tel: ++1 3523929635 Fax: ++1 3523927652 [email protected] 1125

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List of Participants

ANDERSON Richard A. HUMAN NUTRITION RESEARCH CENTER Bldg 307 Rm 224 MD 20705-2350 BELSTVILLE USA Tel: ++1 301504 80 91 Fax: ++1 301 504 90 62 [email protected] ANDRIOLLO Maud UJF—FACULTE DE PHARMACIE Laboratoire de Biologie du Stress Oxydant 38700 LA TRONCHE FRANCE Tel: ++4 76 63 71 16 Fax: ++4 76 51 86 67 ANKE Sabine UNIVERSITY OF JENA Institut fur Ernahrung & Umwelt 07743 JENA GERMANY Tel: ++49/365 1949 675 [email protected] ANKE Manfred FRIEDRICH SCHILLER UNIVERSITAT Institut fur Ernahrung & Umweittoxicologie 07743 JENA GERMANY Tel: ++49 3641949671 Fax: ++49 364194902 [email protected] ARMSTRONG Todd NORTH CAROLINA STATE UNIVERSITY Department of Animal Sciences 27695 7621 NORTH CAROLINA USA Tel: ++1 919 5154049 Fax: ++1 919 5154463 [email protected] ARNAUD Josiane CHU A. MICHALLON Laboratoire de Biochimie C 38043 GRENOBLE Cedex 9 FRANCE Tel: ++4 76 76 56 40 Fax: ++4 76 76 58 21 [email protected] ARNAUD Maurice INSTITUT DE L’EAU PERRIER VITTEL M.T B.P 101 88804 VITTEL FRANCE Tel: ++3 29 08 70 41 Fax: ++3 29 08 70 49 AROLA Maria UNIVERSITY OF OULU Faculty of Medicine 90210 OULU FINLAND ARTHUR John R. ROWETT RESEARCH INSTITUTE Division of Biochemical Sciences AB21 9SB ABERDEEN SCOTLAND Tel: ++ 44 1224 716630 Fax: ++ 44 1224 7 16622 [email protected] ARTURO Leone Dipartimento di Scienze Farmaceut 84080 FISCIANO ITALY ASHMEAD De Wayne ALBION LABORATORIES 101 North Main Street CLEARFIELD, UTAH USA Tel: +1 801 773 4631 Fax: ++1 801 773 4633 [email protected] ATHERTON Caroline INSTITUTE OF FOOD RESEARCH Dept of Nutrition, Diet & Health NR4 7UA NORWICH ENGLAND Tel: ++44 1603 255 190 Fax: ++44 1603 452 578 [email protected] AUDETTE Robert J. UNIVERISTY OF ALBERTA HOSPITAL Dept of Laboratory Medicine T6G 2B7 EDMONTON, Alberta CANADA Tel: ++1 403 492 6648 Fax: ++1 403 492 6267 [email protected] BAJ Arturo OSPEDALE DI DESIO Medicina del Lavoro 20033 DESIO ITALY Tel: ++39 362 383 211 Fax: ++39 362 383 299 [email protected] BAKOWSKA Elzbieta HEWLETT PACKARD COMPANY 2850 Centerville Road DE 19808 WILMINGTON USA Tel: ++1 302 633 7317 Fax: ++1 302 993 5955 [email protected] BARCLAY Denis NESTLE RESEARCH CENTER Lausanne P.O. Box 44 CH-1000 LAUSANNE 26 SWITZERLAND Tel: ++41 21 785 8826 Fax: ++41 21 785 8925 [email protected] BAREAU Xavier 6 rue Francis Poulenc 19100 BRIVE FRANCE Tel: ++5 55 74 33 63 BARNES Ramon UNIVERSITY OF MSSACHUSETTS Chemistry LGRC Tower MA 01003 4510 AMHERST USA Tel: ++1 413 545 2294 Fax: ++1 413 545 3757 BARTON Henryk JAGIELLONIAN UNIVERSITY School of medicine 30-688 KRAKOW POLAND Tel: ++48 12 659 00 20 Fax: ++48 12 657 02 62

List of Participants

1127

BEATTIE John H. ROWETT RESEARCH INSTITUT Greenburn Road AB21 9SB ABERDEEN SCOTLAND Tel: ++44 1224716631 Fax: ++44 1224716629 [email protected] BECKER Kristine UNIVERSITY OF CALIFORNIA Dept of Environmental Health Sc. 90095 1772 LOS ANGELES USA Tel: ++1 310 825 8429 Fax: ++1 310 825 8429 BEHN Dietrich Hahn Meitner Institut Dept of Trace elements in Health and Nutrition D-14109 BERLIN GERMANY Tel: ++30 8062 2784 Fax: ++30 8062 2781 BEHN Claus UNIVERISTY OF CHILE Faculty of Medicine—Physiology & Biophysics SANTIAGO 7 CHILE Tel: ++56 2 7776916 [email protected] BELL Alice RESEARCH CENTRE FOR FOOD & NUTRITION P.O. Box 6163 YAOUNDE CAMEROUN Tel: ++237 221001 Fax: ++237 226262 BELLISOLA Guiseppe UNISERSITY OF VERONA Institute of immunology & Infectious Disease I-37134 VERONA ITALY Tel: ++39 45 8074257 Fax: ++39 45 580900 [email protected] BENNETT Aaron UNIVERSITY OF CALIFORNIA Dept of Environmental Health Sc. 90095 1772 LOS ANGELES USA Tel: ++1 310 825 8429 Fax: ++1 310 825 8429 BERNTSSEN Marc H.G DIRECTORATE OF FISHERIES Institute of nutrition N-5002 BERGEN NORWAY Tel: ++47 5523 8138 Fax: ++47 5523 8095 [email protected] BERR Claudine INSERM U360 Hôpital Salpetrière 75654 PARIS Cedex 13 France Tel: ++1 42 16 25 47 Fax: ++1 42 16 25 41 BERTOUZE Marjorie HOPITAL ALBERT MICHALLON Service de Diabetologie 38043 GRENOBLE FRANCE Tel: ++4 76 76 55 09 Fax: ++4 76 76 50 42 BERTRANDT Jerzy MILITARY INSTITUTE OF HYGIENE & EPIDEMIOLOGY 4 Kozielska St. 01-163 WARSAW POLAND Tel: ++48 22 8381069 [email protected] BLAZI Poljak R. BOSKOVIC INSTITUTE Bijenicka 54 10000 ZAGREB CROATIA BOGDEN John D. UMDNJ-NJ MEDICAL SCHOOL Dept of Preventive medicine NJ 07103-2714 NEWARK USA Tel: ++1 9739725432 Fax: ++1 9739727625 [email protected] BONHAM Maxine UNIVERSITY OF ULSTER Cromdre Road BT5 ZISA COLERAINE NORTHERN IRELAND BORSCHEL Marlene ROSS PRODUCTS DIVISION Abbott Laboratories 43215 COLUMBUS, OH USA Tel: ++1 6146247578 Fax: ++1 6146243453 BOST Muriel TRACE ELEMENT—INSTITUT POUR L’UNESCO 1, Place de l’Ecole 69342 LYON Cedex 07 FRANCE Tel: ++4 72 80 82 90 Fax: ++4 78 58 86 71 BOURDONNAIS Alain ALBION LABORATORIES 22000 SAINT BRIEUC FRANCE Tel: ++2 96 50 80 90 Fax: ++2 96 61 05 51 [email protected] BOUVARD Sophie UNIVERSITE JOSEPH FOURIER UFR de Pharmacie 38700 LA TRONCHE FRANCE Tel: ++4 76 63 71 16 Fax: ++4 76 51 86 67 BRATTER Peter HAHN MEITNER INSTITUT BERLIN Gmbh Dept Trace Element in Health & Nutrition D-14109 BERLIN GERMANY Tel: ++49 3080622262 Fax: ++49 3080622781 [email protected] BRAZZOLOTTO Xavier CEA Direction des Sciences du Vivant Dept biologie moleculaire et structurale 38054 GRENOBLE Cedex 9 FRANCE Tel: ++4 76 88 56 23 Fax: ++4 76 88 58 72 BREMNER Ian ROWETT RESEARCH INSTITUT Greenburn Road, Bucksburn AB21 9SB ABERDEEN ENGLAND Tel: ++44 1224716602 Fax: ++44 1224715349 [email protected]

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List of Participants

BROKKEN Kyle QUALI TECH, INC 318 Lake Hazeltine Drive 55318 CHASKA, MN USA Tel: ++1 612 448 5151 Fax: ++1 612 448 3603 BUNDY Rafe BRITISH NUTRITION FOUNDATION 52-54 High Holborn WCIV 6RQ LONDON ENGLAND Tel: ++44 171 404 6504 Fax: ++44 171 404 6747 BUREAU Isabelle UNIVERSITE JOSEPH FOURIER Laboratoire de Biologie du Stress Oxydant 38700 LA TRONCHE FRANCE Tel: ++4 76 63 71 16 Fax: ++4 76 51 86 67 BURK Raymond VANDERBITT UNIVERSITY G-2104 Medical Center North 372322279 NASHVILLE, TN USA Tel: ++1 6153434747 Fax: ++1 6153436229 BUTTRISS Judith BRITISH NUTRITION FOUNDATION 52-54 High Holborn WUV 6QR LONDON ENGLAND Tel: ++44 171 404 6504 Fax: ++44 171 404 6747 CALOMME Mario UNIVERSITY OF ANTWERP Dep Pharmaceutical Sciences Lab Microbiology B-2610 ANTWERP (WILRIJK) BELGIUM Tel: ++32 38202552 Fax: ++32 38202544 [email protected] CANTONE Marie Claire UNIVERSITY OF MILANO Dipartimento di Fisica 20133 MILANO ITALY Tel: ++39/2 2392212 Fax: ++39 2 2392 630 CARLSON Marcia UNIVERSITY OF MISSOURI COLUMBIA S-133 animal Sciences Center 65211 COLUMBIA USA Tel: ++1 573 882 7589 Fax: ++1 573 884 4545 CATTY Patrice C.E.A Laboratoire DBMS/BMC 38054 GRENOBLE FRANCE Tel: ++4 76 88 96 51 Fax: ++4 76 88 54 87 CEBALLOS-PICOT Irène HOPITAL NECKER ENFANTS MALADES Laboratoire Biochimie Medicale 75743 PARIS Cedex 15 FRANCE Tel: ++1 44 49 51 34 Fax: ++1 44 49 51 30 CEFALU William T. UNIVERSITY OF VERMONT Department of Medicine VT— 5405 BURLINGTON USA [email protected] CESARINI Jean Pierre INSERM FOR FOR 25, rue Manin 75019 PARIS FRANCE Tel: ++1 48 03 69 48 Fax: ++1 48 03 65 10 CHACON CASTRO Pilar GENERAL VALL D’HEBRON Passeig Vall d’Hebron 08035 BARCELONE SPAIN Tel: ++34 93 2746100 Fax: ++34 93 2746188 CHAKRABORTI Dipankar JADAVPUR UNIVERSITY School of Envrironmental Sutides 700 032 CALCUTTA INDIA Tel: ++91 33 473 5233 Fax: ++91 33 473 4266 [email protected] CHAPPUIS Philippe HOPITAL LARIBOISIERE Laboratoire Central de Biochimie 75475 PARIS Cedex 10 FRANCE Tel: ++1 49 95 64 31 Fax: ++1 49 95 84 77 [email protected] CHARLTON C J WALTHAM Centre for Pet Nutrition Waltham-on-the-wolds LE14 4RT LEICESTERSHIRE ENGLAND Tel: ++44 1664 415308 Fax: ++44 1664 415440 [email protected] CHAUD Daniela, Maria, Alves FEDERAL UNIVERSITY OF SAO PAULO Paulista Medicine School 02926 000 SAO PAULO BRAZIL Tel: ++55 118779680 Fax: ++55 118578162 CHAUDHARI Ram FORTITECH INC Riverside Technology Park NY 12308 SCHENECTADY USA Tel: ++1 518 372 5155 Fax: ++1 518 372 5599 CHAUDHARI Panna FORTITECH INC Riverside Technology ark NY 12308 SCHENECTADY USA Tel: ++1 518 372 5155 Fax: ++1 518 372 5599 CHAZOT Guy TRACE ELEMENT—INSTITUT POUR L’UNESCO 1, Place de 1’Ecole 69342 LYON Cedex 07 FRANCE

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1129

CHENG Behling FACULTY OF MEDICINE KUWAIT UNIVERSITY Biochemistry Safat 13110 KUWAIT KUWAIT Tel: ++965 531 9489 Fax: ++965 533 8908 [email protected] CHIMIENTI Fabrice LABORATOIRE DE BIOLOGIE DU STRESS OXYDANT Faculte de Pharmacie 38700 LA TRONCHE FRANCE Tel: ++4 76 63 71 16 CHIPLONKAR Assem S. AGHARKAR RESEARCH INSTITUTE Agarkar Road 411 004 PUNE INDIA Tel: ++91 212 354357 Fax: ++91 212 351542 [email protected] CHOAY Patrick LABORATOIRE C.C.D. 60, rue Pierre Charron 75008 PARIS FRANCE CHURCHMAN David MICROMASS UK Ltd Tudor Road WA14 5RZ CHESHIRE ENGLAND Tel: ++44 161 282 4315 Fax: ++44 161 282 4400 david. [email protected] CLARK Larry ARIZONA CANCER CENTER 2504 East Elm Street AZ 857A6 3417 TUCSON USA COLLI Celia UNIVERSITY OF SAO PAULO Food and Nutrition Department 05508-900 SAO PAULO BRAZIL Tel: ++55 11 8183656 Fax: ++55 11 8154410 [email protected] COMBAZ/MOUCHET Sylvie/Nadège LABORATOIRES ROCHE NICHOLAS SA 33, rue de 1’Industrie 74240 GAILLARD FRANCE Tel: ++4 50 87 70 89 Fax: ++4 50 87 77 50 CONSTANTINESCU Bogdan INSTITUT OF ATOMIC PHYSICS Cyclotron Lab MG-6 BUCHAREST ROUMANIE Tel: ++4014231650 COOPER Lesley INTERNATIONAL MANGANESE INSTITUTE 17 Av. Hoche 75008 PARIS FRANCE Tel: 01 42 89 42 92 Fax: 01 45 63 06 34 CORNELIS Rita UNIVERSITY OF GENT Laboratory for Analytical Chemistry B-9000 GENT BELGIUM Tel: ++32 9 264 6626 Fax: ++32 9 264 6699 COUDRAY Charles INRA Unité Maladies Metaboliques & Micronutriments 63122 SAINT GENES CHAMPANILLE FRANCE Tel: ++4 73 62 46 38 Fax: ++4 73 62 42 38 [email protected] COUGHLIN James R. COUGHLIN & ASSOCIATES 118 Park Crest 92657 NEWPORT COAST USA Tel: ++1 7146404080 Fax: ++1 7146401037 [email protected] COUSINS Robert J. UNIVERSITY OF FLORIDA Center for Nutritional Sciences FL 32611 GAINESVILLE USA Tel: ++1 352 392 2133 Fax: ++1 352 392 1008 COYLE Peter INSTITUTE OF MEDICAL AND VETERINARY SCIENCE Division of clinical biochemistry 5000 ADELAIDE AUSTRALIA Tel: ++8 82223223 Fax: ++8 82223538 [email protected] COZZOLINO Silva M.F UNIVERSIDADE DE SAO PAULO Ave. Lineu Prestes 580, Bl 1405508 900 SAO PAULO BRAZIL Tel: ++55 11 8183625 Fax: ++55 11 8183509 [email protected] CROMWELL Gary UNIVERSITY OF KENTUCKY Animal Sciences Dept 40564 LEXINGTON, Kentucky USA Tel: ++1 606 257 7534 Fax: ++1 606 323 1027 [email protected] CZARNECKI MAULDEN Gail FRISKIES R&D 3916 Pettis Road MO 64504 ST JOSEPH USA Tel: ++1 816 387 4122 Fax: ++1 816 387 4115 [email protected] CÖL Meltem ANKARA UNIV. MED Faculty Med. Dept Public Health ANKARA 06460 TURKEY Tel: ++90 312 482 4407 Fax: ++90 312 4192120

1130

List of Participants

DABRIO RAMOS Marta IRMM-JRC Retieseweg B-2440 GEEL BELGIUM Tel: ++32 14 571 273 Fax: ++32 14 584 273 [email protected] DANHIER Philippe LABORATOIRE UNDA 118 Avenue J. Bordet B 1140 BRUSSELS BELGIUM Tel: ++32 2 7269972 Fax: ++32 2 726 74 58 DANIELS Lynne FLINDIARS MEDICAL CENTRE Dept Public Health SOUTH AUSTRALIA AUSTRALIA Tel: ++61 882045693 [email protected] DARBY Christopher MINISTRY OF AGRICULTURE, FISHERIES & FOOD Str Christopher house SE1 OUD LONDON ENGLAND Tel: ++44 171 921 2393 Fax: ++44 171 921 1121 DARMON Nicole CNAM/ISTA Groupe Nutrition—Santé 75003 PARIS FRANCE Tel: ++1 40 27 23 82 Fax: ++1 40 27 24 75 [email protected] DAVIDSSON Lena LAB. HUMAN NUTRITION ETH Zurich CH-8803 RUSCHLIKON SWITZERLAND Tel: ++41 1 724 2144 Fax: ++41 1 7240183 [email protected] DE CREMER Koen INSTITUT VAN DE NUCLEAIRE WETENSCHAPPEN Proeftuinstraat, 86 B-9000 GENT BELGIUM Tel: ++32 92646603 Fax: ++32 92646699 [email protected] DE LORGERIL Michel CHU DU NORD DE ST ETIENNE Laboratoire de Physiologie 42055 ST ETIENNE FRANCE Tel: ++4 77 82 83 00 Fax: ++4 77 82 80 04 [email protected] DE PORTELA Maria Luz Pita UNIVERSITY OF BUENOS AIRES School of Pharmacy and Biochemistry 1113 BUENOS AIRES ARGENTINE Tel: ++54 1 964 8242 Fax: ++54 1 964 8243 [email protected] DECLERCQ Lieve ESTEE LAUDER COMPANIES Nijverherdsstraat 15 B-2260 OEVEL BELGIUM Tel: ++32 14 25 88 52 Fax: ++32 14 25 88 60 [email protected] DEFLERS Jean CHEMGAS 31, Bis Avenue Robert Schuman 92100 BOULOGNE FRANCE Tel: ++1 48 25 33 37 Fax: ++1 48 25 92 40 DEMAEGDT Gérard Ste INOBIO 761, Avenue de la Gare 27610 ROMILLY SUR ANDELLE FRANCE Tel: ++2 32 49 98 95 Fax: ++2 32 49 16 75 DEUTCH Bente INST. OF ENVIRON. & OCCUPATIONAL MED. University of Aarhus, building 180 DK-8000 AARHUS DANEMARK Tel: ++45 8942 2958 Fax: ++45 8942 2970 DIDIER Christine L B S O CHU A. Michallon 38043 GRENOBLE Cedex 9 FRANCE Tel: ++4 76 76 54 84 Fax: ++4 76 76 56 64 DIETER Hermann H. FEDERAL ENVIRONMENTAL AGENCY P.O. BOX 330022 DEPT V 2.7 D-14191 BERLIN ALLEMAGE Tel: ++49 89 031400 Fax: ++49 89 031830 [email protected] DOREA Jose Garrofe UNIVERSIDADE DE BRASILIA Caixa Postal 04322 70919970 BRASILIA-DF BRAZIL Tel: ++51 613685853 [email protected] DOSS Sudhuen UNIVERSITY OF CALIFORNIA Dept of Environmental Health Sciences 90095 1772 LOS ANGELES, CA USA Tel: ++1 310 825 8429 Fax: ++1 310 825 8429 [email protected] DOUSSET Brigitte HOPITAL CENTRAL Laboratoire Central de Biochimie 54035 NANCY Cedex FRANCE Tel: ++3 83 85 13 57 Fax: ++3 83 85 27 43 [email protected] DOVAL Alain STE NICOMED Pox Box 24 N 1375 BILLINGSTAD NORWAY Tel: ++47 66 84 65 60 Fax: ++47 66 98 22 01

List of Participants

1131

DUCROS Véronique CHU A. MICHALLON Laboratoire de Biochimie C 38043 GRENOBLE Cedex 9 FRANCE Tel: ++4 76 76 56 40 Fax: ++4 76 76 58 21 [email protected] De VECCHY Hélène CABINET DE VECCHY 3, rue Niepce 75014 PARIS FRANCE Tel: ++1 43 20 66 59 Fax: ++1 40 47 80 84 ECKHERT Curtis D. UNIVERSITY OF CALIFORNIA School of Public Health—Dept Environmental HS LOS ANGELES USA Tel: ++1 310 825 8429 [email protected] EDER Klaus INSTITUT FOR TIERERNAHRUNG & VORRASTSHATTUNG Emil Abderhaldeustrasse 25b D-06108 HALLE (Saale) GERMANY Tel: ++345 5522702 Fax: ++345 5527124 [email protected] EIDE David UNIVERSITY OF MINNESOTA-DULUTH Department of Biochemistry & Molecular Biology MN 55812 DULUTH USA Tel: ++1 218 726 6508 Fax: ++1 2187268014 [email protected] EISENSTEN Rick UNIVERSITY OF WISCONSIN Dept of Nutritional Sciences WI—MADISON USA Tel: ++1/6082625830 Fax: ++1/6082625860 ELMES Margaret RETIRED Dawros house, CF64 4HB DINAS POWYS PAYS DE GALLES, ROYAUME UNIE Tel: ++44 1222512102 Fax: ++44 1222515975 EMTEBORG Hakan UNIVERSITY OF ANTWERPEN, UIA Department of chemistry B-2610 ANTWERPEN BELGIUM Tel: ++32 3 820 23 70 Fax: ++32 3 820 23 76 [email protected] ENGLE Terry NORTH CAROLINA STATE UNIVERSITY Department of Animal Science 27695 7621 NORTH CAROLINA USA Tel: ++1 919 515 4049 Fax: ++1 919 515 4463 [email protected] ENOMOTO Shuichi THE INSITUTE OF PHYSICAL & CHEMICAL RESEARCH Wako 351-01 SAITAMA JAPAN Tel: ++81 484679421 Fax: ++81 48 467 9423 [email protected] ESCHMANN Klaus URSAPHARM ARZNEIMITTEL GMBH Industriestrasse D66129 SAARBRUECKEN GERMANY Tel: ++49 6805929230 Fax: ++49 6805929287 FAES Jean Marc ROBAPHARM AG Gewerbestrasse 18 CH-4123 ALLSCHWIL SWITZERLAND Tel: ++41 61 487 88 88 Fax: ++41 61 487 88 99 FAIRWEATHER-TAIT Sue INSTITUT OF FOOD RESEARCH Norwich Research Park NR4 7VA NORWICH ENGLAND Tel: ++44 1603255306 Fax: ++44 1603452578 [email protected] FAKLER Timothy ZINPRO CORPORATION 6500 City West Parkway #300 55344 EDEN PRAIRIE, MN USA Tel: ++1 612 944 2136 Fax: ++1 612 944 5824 [email protected] FALNOGA Ingrid JOSEF STEFAN INSTITUTE Dept of Environmental Sciences 1000 LJUBLJANA CROATIA Tel: ++386 61 1885 354 [email protected] FARRE ROVIRA Rosaura UNIVERISITY OF VALENCIA Faculty of pharmacy 46100 BURJASSOT (VALENCIA) SPAIN Tel: ++34 963864950 Fax: ++34 963864954 [email protected] FAURE Patrice CHU A. MICHALLON Laboratoire de Biochimie C 38043 GRENOBLE Cedex 9 FRANCE Tel: ++4 76 76 52 77 Fax: ++4 76 76 56 64 [email protected] FAVIER Max CHU DE GRENOBLE Hopital Sud 38043 GRENOBLE FRANCE Tel: ++4 76 76 54 06 Fax: ++4 76 76 51 95

1132

List of Participants

FAVIER Alain CHU A. MICHALLON Laboratoire de Biochimie C 38043 GRENOBLE Cedex 9 FRANCE Tel: ++4 76 76 54 84 Fax: ++4 76 76 56 64 [email protected] FEILLET COUDRAY Christine INRA—CENTRE DE RECHERCHE Laboratoire des maladies metabolliques 63122 ST GENES CHAMPANELLES FRANCE Tel: ++4 73 62 45 79. Fax: ++4 73 62 46 38 [email protected] FELDMANN Jorg UNIVERSITY OF ABERDEEN Dept of Chemistry AB 24 3UE ABERDEEN ENGLAND Tel: ++44 1224 272911 Fax: ++44 1224 272921 [email protected] FENYOHAZI Jeno ST ISTVAN HOSPITAL BUDAPEST Nagyvarad Tes 1 1091 BUDAPEST HUNGARY Tel: ++36 2 160 350 Fax: ++36 2 62 9322 FERGUSON Elaine L. UNIVERSITY OF OTAGO Dept Human Nutrition DUNEDIN NEW ZEALAND Tel: ++643 479 8374 Fax: ++643 479 7958 [email protected] FERRY Monique CENTRE HOSPITALYR 64, Avenue du Dr Santy 26953 VALENCE FRANCE Tel: 04 75 75 75 53 Fax: 04 75 75 77 98 FIELDS Meira USDA. ARS. BHNDO Human Nutrition Research Center MD 20705 BELTSVILLE, MD USA Tel: ++1 301 5049412 Fax: ++1 301 5049456 [email protected] FISCHER Peter NUTRITION RESEARCH DIVISION Health Protection Branch KIA OL2 OTTAWA, Ontario CANADA Tel: ++1 6139570919 Fax: ++1 6139416182 [email protected] FLEITES P. CENTRO NACIONAL DE TOXICOLOGIA (CENATOX) Ave 31 y calle 114, 11500 LA HAVANA CUBA Tel: ++53 7 336075 FONTECAVE Marc DBMS/CB—CEA GRENOBLE 17 Avenue des Martyrs 38054 GRENOBLE Cedex 9 FRANCE Tel: ++4 76 88 91 03 Fax: ++4 76 88 91 24 [email protected] FORCEVILLE Xavier CH MEAUX Service de réanimation 77104 MEAUX FRANCE Tel: ++1 64 35 38 38 Fax: ++1 64 33 08 54 FORD Rodney COMMUNITY PAEDIATRIC UNIT P.O. Box 25-265 8001 CHRISTCHURCH NEW ZEALAND Tel: ++64 3 3777196 Fax: ++64 3 3770596 FORT Douglas F. THE STOVER GROUP P.O. Box 2056 OK 74076 STILLWATER USA Tel: ++1 405 743 1435 Fax: ++1 405 743 1489 FOX Tom INSTITUTE OF FOOD RESEARCH Norwich Research Park Colney NR4 7UA NORWICH ENGLAND Tel: ++44 1603 255191 Fax: ++44 1603 452578 [email protected] FREELAND-GRAVES Jean UNIVERISTY OF TEXAS Nutrition A 2700 78712 AUSTIN, Texas USA Tel: 0015124710657 Fax: 0015124710657 FRIEL James K. MEMORIAL UNIVERSITY OF NEWFOUNDLAND Department of Biochemistry AIB 3X9 ST JOHN’S CANADA Tel: ++17097377954 Fax: ++17097372427 [email protected] FUCHSWANS Werner INSTITUT FUR MEDIZINISCHE PHYSIOLOGIE Gusenleithnergasse 30/12 A-l140 WIEN AUTRICHE Tel: ++43 1 4277 62511 Fax: ++43 1 2572 32 884 FUENTEALBA Carmen UNIVERSITY OF PRINCE EDWARD ISLAND C1A 4P3 PRINCE EDWARD ISLAND CANADA Tel: ++1 902 566 0868 Fax: ++1 902 566 0851 [email protected] GALLAHER Daniel UNIVERSITE OF MINNESOTA dept of Food Science and Nutrition 55108 MINNESOTA USA Tel: ++1 612 624 0746 Fax: ++1 612 625 5272 [email protected]

List of Participants

1133

GARCIA ARRIBAS Olga INSTITUTO DE SALUD CARLOS III Dept of Toxicology E-28220 MADRID SPAIN Tel: ++34 915097981 Fax: ++34 915097991 [email protected] GARCIA BELTRAN Lidia GENERAL VALL D’HEBRON Passeig Vall d’hebron 08035 BARCELONA SPAIN Tel: ++34 93 2746100 Fax: ++34 93 4906725 GARREL Catherine CHU A. Michallon—Laboratoire de Biochimie C 38043 GRENOBLE FRANCE Tel: ++4 76 76 54 84 Fax: ++4 76 76 56 64 GARROW Timothy UNIVERSITY OF ILLINOIS AT URBANA Department of food Science and Human Nutrition IL 61801 URBANA USA Tel: ++1 217 333 8455 Fax: ++1 217 333 9368 [email protected] GIALA NELLA Giancarlo Dipartimento scienze fisiche 80126 NAPOLI ITALY Tel: ++39 081 676349 Fax: ++39 081 676346 GIBSON Rosalind Susan UNIVERSITY OF OTAGO Dept of Human Nutrition DUNEDIN NEW ZEALAND Tel: ++64 3 479 7955 Fax: ++64 3 479 7958 [email protected] GIMENEZ Maria Sofia UNIVERSIDAD NACIONAL DE SANLUIS Avenida Egercito de los andres 950 5700 SAN LUIS ARGENTINE Tel: ++54 2 692426780 Fax: ++54 2 652 430224 [email protected] GITLIN J.D WASHINGTON UNIVERSITY SCHOOL OF MEDICINE Department of Pediatrics MO 63110 ST LOUIS USA Tel: ++1 314 454 6124 Fax: ++1 314 454 4861 [email protected] GLEICHMANN Helga DIABETES RESEARCH INSTITUTE Auf’M Hennekamp 65 D-40225 DUSSELDORF GERMANY Tel: ++49 211 33820 Fax: ++49 211 3382603 GLEIZES Valéry HOPITAL RAYMOND POINCARE Service d’orthopédieTraumatologie 92380 GARCHES FRANCE Tel: ++1 47 10 77 02 GLUODENIS Tom HEWLETT PACKARD COMPANY 2850 Centerville Road DE 19808 WILMINGTON USA Tel: ++1 302 633 7317 Fax: ++1 302 993 5955 *[email protected] GOKMEN Inci MIDDLE EAST TECHNICAL UNIVERSITY Dept of Chemistry 06531 ANKARA TURKEY Tel: ++90 312 210 3194 Fax: ++90 312 210 1280 [email protected] GRALAK Mikolaj A. WARSAW AGRICULTURAL UNIVERSITY Dept animal Physiology 02-787 WARSZAWA POLAND Tel: ++48 22439041 Fax: ++48 472452 Gralak@alpha. sggw. waw.pl GREENBERG Danielle AMBI INC 4 Manhattanville Rd NY 10577 PURCHASE USA Tel: ++1 914 701 4500 Fax: ++1 914 696 0860 GRIZARD Damien FACULTE DE MEDICINE ET PHARMACIE Laboratoire de pharmacognosie et de biotechnologies 63001 CLERMONT FERRAND FRANCE Tel: ++4 73 60 80 49 Fax: ++4 73 27 24 15 genevieve.grizard@u. clermont1.fr GROMADZINSKA Jolanta INSTITUTE OF OCCUPATIONAL MEDICINE 8 Tevesy St 90-950 LODZ POLAND Tel: ++48 42314631 Fax: ++48 42538331 GUEGUEN Leon INRA Station de rehcerches nutrition 78350 JOUY EN JOSAS FRANCE GUEUX Elyett INRA—CENTRE DE RECHERCHE Unité des Maladies Métaboliques 63122 ST GENES CHAMPANELLES FRANCE Tel: ++4 73 62 42 35 GUILLARD Olivier HOPITAL JEAN BERNARD Laboratoire de BiochimieToxicologie 86021 POITIERS FRANCE Tel: ++5 49 44 45 44 10 Fax: ++5 49 44 38 34 [email protected]

1134

List of Participants

GUIRAUD Pascale CHU ALBERT MICHALLON Laboratoire de Biochimie C 38043 GRENOBLE Cedex 9 FRANCE Tel: ++4 76 76 54 84 Fax: ++4 76 76 56 64 GURBAY Aylin UNIVERSITY OF HACETTEPE Faculty of Pharmacy 06100 ANKARA TURKEY HADRZYNSKI Christian LABCATAL 7, rue Roger Salengro 92120 MONTROUGE FRANCE Tel: ++1 46 54 27 92 Fax: ++1 46 54 22 21 HAFLIGER Toni ROCHE PHARMA AG Schoenmattstr. 2 CH-4153 REINACH SWITZERLAND Tel: ++41 61 71542 19 Fax: ++41 61 915 42 44 [email protected] HALLFRISCH Judith BELTSVILLE HUMAN NUTRITION RESEARCH CENTER Agricultural Research Service 20705 BELTSVILLE, MA USA Tel: ++1 3015049061 Fax: ++1 3015049098 [email protected] HAMBIDGE Michael UNIVERSITY OF COLORADO Health Sciences Center CO 80262 DENVER USA ++1 303 315 5672 Tel: ++1 303 315 3273 [email protected] HANSEN Marianne THE AGRICULTURAL UNIVERSITY Research Department of Human Nutrition DK-1958 FREDERIKSBERG C DANEMARK Tel: ++ 45 35282483 Fax: ++45 35282483 HANSEN Gilbert LABORATOIRE NATIONAL DE SANTE 42, rue du laboratoire L1911 LUXEMBOURG LUXEMBOURG Tel: ++352 491191364 Fax: ++352 400745 HARIVEAU Elisabeth LABORATOIRE BOIRON 20, rue de la Libèration 69110 STE FOY LES LYON FRANCE Tel: ++4 72 16 40 78 Fax: ++4 78 59 69 16 HARVEY Linda INSTITUTE OF FOOD RESEARCH Norwich Research Park Colney NR4 7UA NORFOLK ENGLAND Tel: ++44 1603255308 Fax: ++44 1603452578 [email protected] HAYWOOD Susan UNIVERSITY OF LIVERPOOL Department of Veterinary Pathology L69 3BX LIVERPOOL ENGLAND Tel: ++44 1517944265 Fax: ++44 1517944268 HEALTH Anne Louise UNIVERSITY OF OTAGO Dept of human nutrition DUNEDIN NEW ZEALAND Tel: ++64 34710774 Fax: ++64 34797958 [email protected] HEDIGER Mathias A. WOMAN’S HOSPITAL AND HARVARD MEDICAL SCHOOL Renal Division Department of Medicine Brigham MA 02115 BOSTON USA [email protected] HEINS Ulrike INSTITUTE OF NUTRITIONAL SCIENCE Wilhelmstr. 20 D-35392 GIESSEN ALLEMANGE Tel: ++49 641 9939051 Fax: ++49 641 9939049 [email protected] HERBRETEAU Marie Christine LABORATOIRE RICHELET 15 Rue Lapérouse 75116 PARIS FRANCE Tel: ++1 40 73 82 50 Fax: ++1 40 73 82 69 HERCBERG Serge CONSERVATOIRE DES ARTS ET METIERS Centre de Recherche sur les Anémies Nutrition 75003 PARIS FRANCE Tel: ++1 40 27 24 75 Fax: ++1 40 27 01 53 [email protected] HINCAL Filiz UNIVERSITY OF HACETTEPE Faculty of pharmacy 06100 ANKARA TURKEY Tel: ++90 4 310 88 31 Fax: ++90 4 311 47 77 [email protected] HININGER-FAVIER Isabelle FACULTE DE PHARMACIE L.B.S.O 38700 LA TRONCHE FRANCE Tel: ++4 76 63 71 16 Fax: ++4 76 63 71 80 [email protected]

List of Participants

1135

HIRUNUMA Rieko THE INSTITUTE OF PHYSICAL AND CHEMICAL RESEARCH RI Beam Factory Project Office 351-0198 SAITAMA JAPAN Tel: ++81 48467 9421 Fax: ++81 48 467 9423 [email protected] HOFFMAN Eric ACTIVATION LABORATOIRE Ltd 1336 Sandhill Dr L96 4V5 ANCASTER, ON CANADA Tel: ++1 905 648 9611 Fax: ++1 905 648 9613 HOOGEWERFF Jurian ARSENAL RESEARCH Environmental Division A-1030 VIENNA AUTRICHE Tel: ++43 1 79747 411 Fax: ++43 1 79747 592 HOSOKAWA To Shiyuki HOKKAIDO UNIVERSITY CENTER FOR R&P IM HIGHER EDUCATION 060 0809 SAPPORO JAPAN Tel: ++81 11 7062194 Fax: ++81 117064922 [email protected] HUBBARD Susan BORAX EUROPE Gorsey Lane WA8 ORP WIDNES, CHESHIRE ENGLAND Tel: ++44 1514 205522 Fax: ++44 1514 208815 HUGHES Joyce BRITISH NUTRITION FOUNDATION 52 -54 High Holborn WUV 6RQ LONDON ENGLAND Tel: ++44 171 404 6504 Fax: ++44 171 404 6747 [email protected] HUGHES Sally Ann SMITHKLINE BEECHAM 11 Stoke Poges Lane SL1 3NW SLOUGH Berks ENGLAND Tel: ++44 1753 502137 Fax: ++44 1753 502007 [email protected] HUNT Curtiss D. USDA—ARS Grand Forks Human Nutrition Res Center ND 58202 7166 GRAND FORKS—North Dakota USA Tel: ++1 701 795 8423 Fax: ++1 701 795 8220 [email protected] HURREL R LABORATOIRE FUR HUMANERNAHRUNG eth Seestrasse 72 CH8803 RUSCHLIKON SWITZERLAND Tel: ++41 1 7045701 Fax: ++41 1 7045710 [email protected] ILLINGWORTH Doreen UNIVERSITY OF LEEDS Centre for Animal Sciences LS2 9JT LEEDS ENGLAND Tel: ++44 1132333068 Fax: ++44 1132333072 [email protected] IYENGAR Venkdatesh INTERNATIONAL ATOMIC ENERGY AGENCY P. Box 1000 A-1400 VIENNA AUTRICHE Tel: ++43 1 260021657 Fax: ++43 1 26007 [email protected] JACK Catherine HOSPITAL Department of medicine L69 3GA LIVERPOOL ENGLAND ++44/151 794 4089 Tel: ++44/151 706 5802 JACKSON Malcolm J. UNIVERSITY OF LIVERPOOL Department of Medicine L69 3GA LIVERPOOL ENGLAND Tel: ++44/151 706 4071 Fax: ++44/151 706 5802 [email protected] JACOB Claus HARVARD MEDICAL SCHOOL Lerchenweg 13 66459 KIRKEL 3 GERMANY Tel: ++1 617 731 32 86 Fax: ++1 617 566 3137 [email protected] JACOBS Richard M. U.S. Food & Drug Administration 1431 Harbor Bay 94502 SAN FRANCISCO USA Tel: ++1/5013376814 Fax: ++1/5103374704 [email protected] JAUDON Marie Chantal HOPITAL DE LA SALPETRIERE Laboratoire Central de Biochimie 75013 PARIS FRANCE Tel: ++1 42 16 20 44 Fax: ++1 42 16 20 33 JEEJEEBHOY K. ST MICHAEL’S HOSPITAL ON M58 1WB TORONTO CANADA Tel: ++1 416 864 5388 Fax: ++1 416 864 5882 [email protected] JOHANSSON Magnus INST. FOR REF. MATERIALS AND MEASUREMENTS Retieseweg B-2440 GEEL BELGIUM Tel: ++32 14571256 Fax: ++32 14584273 [email protected]

1136

List of Participants

JORHEM Lars NATIONAL FOOD ADMINISTRATION Box 622 S-75126 UPPSALA SWEDEN Tel: ++46 484 755 00 Fax: ++46 181 054 84 LA. [email protected] JOURDAN Eric LBSO CHU A. Michallon 38043 GRENOBLE Cedex 9 FRANCE Tel: ++4 76 76 54 84 Fax: ++4 76 76 56 64 JUDSON Geoffrey SOUTH AUSTRALIAN RESEARCH 33 Flemington Street SA 5065 GLENSIDE AUSTRALIA Tel: ++61 88 207 7979 Fax: ++61 88 07 7854 [email protected] JURASOVIC J INST FOR MEDICAL RES & OCCUPAT HEALTH Ksaverska cesta 2 10001 ZAGREB CROATIA Tel: ++385 1 4673 188 Fax: ++385 1 4673 303 [email protected] KAATS Gilbert HEALTH AND MEDICAL RESEARCH FOUNDATION 4900 Brodway, Suite 200 TX 78284 7762 SAN ANTONIO USA KAMIYAMA Masumi JISSEN WOMEN’S UNIVERSITY 36-11, Akabane 2-chome 115-0045 TOKYO JAPAN Tel: ++3 3901 6644 Fax: ++3 3901 5963 [email protected] KANTOLA Marjatta UNIVERISTY OF KUOPIO Dept of Chemistry 70211 KUOPIO FINLAND Tel: ++358 17163246 Fax: ++358 17163259 [email protected] KAPUSTA Yakov ACTIVATION LABORATOIRE Ltd 1336 Sandhill Drive L9G 4V5 ANCASTER, ON CANADA Tel: ++1 905 648 9611 Fax: ++1 905 648 9613 [email protected] KASTENMAYER Peter NESTLE RESEARCH CENTER P.O. Box 44 CH-1000 LAUSANNE 26 SWITZERLAND Tel: ++41 21785 5944 Fax: ++41 21785 8563 [email protected] KAUP Susan Marie WYETH NUTRITIONALS INTERNATIONAL 145 King of Prussia Road 19087 RADNOR, PA USA Tel: ++1 610 341 2336 Fax: ++1 610 989 4856 [email protected] KEMP Francis UNIVERSITY OF MEDICINE & DENTISTRY OF N. J. New Jersey Medical School MSB F507 07103 NEWARK USA Tel: ++1 973 972 4404 Fax: ++1 973 972 7625 [email protected] KENDALL Nigel CENTRE FOR ANIMAL SCIENCES L’BS school of Biology LS2 9JT LEEDS ENGLAND Tel: ++44 113 233 3068 Fax: ++44 113 233 3072 [email protected] KERKENI Abdelhamid FACULTE DE MEDECINE 5000 MONASTIR TUNISIE Tel: ++216 3462200 Fax: ++216 3460737 KESSLER Juerg STATION FED DE RECH. EN PROD. ANIMALE CH 1726 POSIEUX SWITZERLAND Tel: ++26 40 77 275 Fax: ++26 40 77 275 [email protected] KING Janet USDA, ARS, WHNRC Ca 94129 DAVIS USA Tel: ++1 530 752 5268 Fax: ++1 530 752 5271 [email protected] KLEVAY Leslie M. USDA, ARS Human Nutrition Research Center 58202 9034 GRAND FORKS, ND USA Tel: ++1 701 795 8464 Fax: ++1 701 795 8220 [email protected] KLOS Anna MILITARY INSTITUTE OF HYGIENE & EPIDEMIOLOGY 4 Kozielska St. 01 163 WARSAW POLAND Fax: ++48 22 8381069 [email protected] KNOWLES Scott AG RESEARCH GRASSLANOS RESEARCH CENTRE Private Bag 11008 PALMERSTON NORTH NEW ZELANDE Tel: ++64 6 351 8066 Fax: ++64 6 351 8003 [email protected]

List of Participants

1137

KOCHEL Bonawentura WROCLAW UNIVERSITY OF MEDICINE Dept of Toxicology PL-50417 WROCLAW POLAND Tel: ++48 71 36437068 Fax: ++48 71 3437068 KOMOROWSKI James AMBI INC. 4 Manhattanville Rd 10577 PURCHASE, NEW YORK USA Tel: ++1 914 701 4500 Fax: ++1 914 1096 0860 KORKINA Ludmilla VITAMIN RESEARCH INSTITUTE Russian State Medical University 117513 MOSCOU RUSSIA Tel: ++7 95 434 7187 Fax: ++7 95 434 7187 [email protected] KOROPATNICK James UNIVERSITY OF WESTERN ONTARIO Dept of oncology N6A 4L6 LONDON, Ontario CANADA Tel: ++1 5196858654 Fax: ++1 5196858646 [email protected] KOUDRINE Alexei V. LAMMERSMITH HOSPITAL Imperial College School of Medicine W12 ONN LONDRES ENGLAND Tel: ++44 171 527 5238 Fax: ++44 171 181 383 2066 [email protected] KOVATSI Leda ARISTOTLE UNIVERSITY OF THESSALONIKI Laboratory of Forensic Medicine & Toxicology 54006 THESSALONIKI GRECE Tel: ++30 31 999 201 Fax: ++30 31 999 686 [email protected] KRACHLER Michael RESEARCH CENTER OF JUELICH, INSTITUTE For applied Physical Chemistry D-52425 JUELICH GERMANY Tel: ++49 24 61 61 31 79 Fax: ++49 24 61 61 24 93 [email protected] KRAUSSE Antje FACULTY OF FRIEDRICH-SCHILLER Hilgenfeldweg 8 D-07743 JENA GERMANY KREJPCIO Zbigniew AGRICULTURAL UNIVERSITY Department of Food Hygiene and human Nutrition PL 60 624 POZNAN POLAND Tel: ++48 61 8487336 Fax: ++48 61 8487332 [email protected] KRETSCH Mary USDA, Agricultural Research Service, WHNRC P.O. Box 29997 CA 94129 PRESIDIO OF SAN FRANCISCO USA Tel: ++1 415 556 6225 Fax: ++1 415 556 1432 [email protected] KROL Alain INSTITUT DE BIOLOGIE MOLECULAIRE ET CELLULAIRE UPR CNRS 9002 67084 STRASBOURG FRANCE Tel: ++3 88 41 70 50 Fax: ++3 88 60 22 18 [email protected] KRUSE-JARRES J.D KATHARINENHOSPITAL Institut fur Klinische & Laboratoriumsmedizin D-70174 STUTTGART GERMANY Fax: ++49 711 278 4809 [email protected] KURASAKI Masaaki HOKKAIDO UNIVERSITY Grad. Sch. Environ. earth Sci 060 0810 SAPPORA JAPAN Tel: ++81 11 706 2243 Fax: ++81 11 717 0629 [email protected] KVICALA Jan INSTITUTE OF ENDOCRINOLOGY Narodni 8 116 94 PRAHA 1 CZECH REPUBLIC Tel: ++42 224 905 242 Fax: ++42 224 905 325 [email protected] KWUN In Sook ANDONG NATIONAL UNIVERSITY Dept of Food and Nutrition 760 749 ANDONG KYUNGBUK SOUTH KOREA Tel: ++571 850 5917 Fax: ++571 841 1625 [email protected] L’ABBE Mary R. HEAD MICRO NUTRIENTS SECTION Nutrition Research Health Canada K1A OL2 OTTAWA, Ontario CANADA Tel: ++1 613 957 0924 Fax: ++1 613 941 6182 mary_l’[email protected] LACHILE Brahim UNIVERSITE DE BATNA Faculte de Medecine 05000 BATNA ALGERIE LAGARDA BLANCH Maria Jesus FACULTAD FARMACIA Area de Nutricion y Bromatologia 46100 BURJASSOT (VALENCIA) SPAIN Tel: ++34 963 864 956 Fax: ++34 963 864 954 [email protected]

1138

List of Participants

LAIRES Maria José FACULDADE MOTRICIDADE HUMANA Cruz Quebrada 1499 LISBOA PORTUGAL Tel: ++351 414 91 33 Fax: ++351 41 49 129 LALANNE Karine UNIVERSITE JOSEPH FOURIER Domaine de la Merci 38700 LA TRONCHE FRANCE Tel: ++4 76 63 71 16 LANGENECKERT Anja ROCHE CONSUMER HEALTH 5 chemin de la parfumerie CH 1214 VERNIER/GENEVA SWITZERLAND Tel: ++41 22 780 90 89 Fax: ++41 22 780 90 03 [email protected] LANOUE Louise UNIVERSITY OF CALIFORNIA One Shield Avenue 95616 8669 DAVIS USA Tel: ++1 530 752 2639 Fax: ++1 530 752 8966 LAPORTE François CHU A. MICHALLON Laboratoire de Biochimie C 38043 GRENOBLE FRANCE Tel: ++4 76 76 54 80 Fax: ++4 76 76 51 81 LARSEN Torben DANISH INSTITUTE OF AGRICULTURAL SCIENCES Dept of Animal Health & Weltare DK-8830 TJELE DANEMARK Tel: ++45 89 99 15 01 Fax: ++45 89 99 15 00 [email protected] LAVEDRINE Florence LBSO Service de chimie Analytique 38700 LA TRONCHE FRANCE Tel: 04 76 63 71 00 LE FRANCOIS Patrice SCERCAT 11 bis rue de Groussay 78120 RAMBOUILLET FRANCE Tel: ++1 34 57 69 40 Fax: ++1 34 57 61 54/55 LEBRETON Pascal IPA GROUPE CHASSOT Z.I du Cantubas 69170 TARARE FRANCE Tel: ++4 74 05 37 50 Fax: ++4 74 05 37 55 [email protected] LECCIA Marie Therese C H U Albert Michallon Service de Dermatologie 38043 GRENOBLE Cedex 9 FRANCE Tel: ++4 76 76 55 08 Fax: ++4 76 76 55 58 [email protected] LEMONNIER Daniel CHU Charles Nicolle Groupe de Recherches 76031 ROUEN FRANCE Tel: ++2 32 88 87 66 [email protected] LENNON Maureen US BORAX Inc 26877 Tourney Road 91355 1847 VALENCIA, CA USA Tel: ++1 661 287 5559 Fax: ++1 661 287 5566 maurennlennon@ compuserve.com LESCURE Alain CNRS 15 Rue René Descartes 67084 STRASBOURG FRANCE Tel: ++3 88 41 70 69 Fax: ++3 88 60 22 18 [email protected] LINDIAR Maria C. CALIFORNIA STATE UNIVERSITY Dept of Chemistry + Biochemistry 92834-6866 FULLERTON, CA USA Tel: ++1 7142782472 Fax: ++1 7142785316 [email protected] LINDMARK MANSSON Helena SWEDISH DAIRY ASSOCIATION Scheelevagen 18 S-22370 LUND SWEDEN Tel: ++46 46 19 1345 Fax: ++46 46 137040 [email protected] LLOPIS Juan UNIVERSIDAD DE GRANADA Instituto de Nutricion y technologia de alimentos 18071 GRANADA SPAIN Tel: ++34 958244174 Fax: ++34 958281164 [email protected] LONNERDAL Bo UNIVERSITY OF CALIFORNIA Dept of Nutrition CA 95616 DAVIS USA Tel: ++1 530 7528347 Fax: ++1 5307523564 [email protected] LOPES Paula Alexandra UNIVERSIDADE DE LISBOA Faculdade de Ciencias 1700 LISBOA PORTUGAL Tel: ++35 117573141 Fax: ++35 117500028 [email protected] LUO David UNIVERSITY OF CALIFORNIA Dept of Environmental Health Sciences 90095 1772 LOS ANGELES USA Tel: ++1 310 825 8429 Fax: ++1 310 825 8429 [email protected]

List of Participants

1139

MACKENZIE Sandy HARPER ADAMS AGRICULTURAL COLLEGE Harper Adams University College TF10 8NB SHROPSHIRE ENGLAND Tel: ++44 1952 820280 Fax: ++44 1952814783 [email protected] MACPHERSON Allan SCOTTISH AGRICULTURAL COLLEGE Auchcruive KA7 2XN AYR SCOTLAND Tel: ++44 1292525156 Fax: ++44 1292525177 [email protected] MALECKI Elise The Pennsylvania State University Dept of neuroscience & Anatomy 17033 HERSHEY, PA USA Tel: ++1 717 531 4716 Fax: ++1 717 531 5184 [email protected] MARA Michal HOSPITAL IN PRAGUE Medical Faculty of Charles university PRAGUE CZECH REPUBLIC MARTINEZ Isabel FACULTAD DE VETERINARIA Campus de Espinardo 30071 MURCIA SPAIN Tel: ++34 968364798 Fax: ++34 968364147 [email protected],es MARTINEZ GRACIA Carmen FACULTAD DE VETERINARIA Area de nutricion y bromatologia 30071 MURCIA SPAIN Tel: ++ 34 968 364798 Fax: ++34 968 364147 [email protected] MATHAI Jaya SBCH M 69 Commercial Center 110048 NEW DELHI INDIA Tel: ++91 11 6483561 Fax: ++91 11 6216247 MAZUR André INRA Theix 63122 ST GENES CHAMPANELLE FRANCE Tel: ++4 73 62 42 34 Fax: ++4 73 62 46 38 [email protected] MC ARDLE Harry J. ROWETT RESEARCH INSTITUTE Greenburn Road AB21 95B ABERDEEN SCOTLAND Tel: ++44 122 471 66 28 Fax: ++44 122 471 66 22 [email protected] MC CORD Joe M. UNIVERSITY OF COLORADO HEALTH SCIENCES Box C321 4200 E CO 80262 DENVER USA Tel: ++1 303 315 6257 Fax: ++1 303 315 8541 [email protected] MC NEILL J.H. UNIVERSITY OF BRITISH Faculty of Pharmaceutical Sciences V6T 1Z3 VANCOUVER CANADA Tel: ++1 604 822 93 73 Fax: ++1 604 822 80 01 [email protected] MERTZ Walter 12401 St. James Road MD 20850 ROCKVILLE USA Tel: ++1 301 424 8814 MEYER James UNIVERSITY OF PRETORIA Dept Arim + Wildlife Sci 0002 PRETORIA SOUTH AFRICA Tel: ++27 12 420 4018 Fax: ++27 12 420 3290 jwrcp@msmperl .up.ac.za MIELCARZ Gregorz K. Marcinkowski Univ. Med. Sciences Dept General Chem. 60780 POZNAN POLAND Tel: ++48 618658619 Fax: ++48 618520455 [email protected] MINAMI Takeshi NARA MEDICAL UNIVERSITY Laboratory of Cell Biology, Dept Anatomy 634 KASHIHARA, Nara JAPAN Tel: ++81 744223051 Fax: ++81 744248432 [email protected] MISSIAEN Erwin PHARMA NORD Excelsiorlaan, 21 B-1930 ZAVENTEM BELGIUM Tel: ++32 2 720 51 20 Fax: ++32 2 720 51 60 emissiaen@ pharmanord.com MOMCILOVIC Berislav INST MED RES OCCUP HEALTH Ksaverska cesta 2 10000 ZAGREB CROATIA Tel: ++ 3851 4673188 Fax: ++ 3851 4673303 [email protected] MORENO Victor Ste KELATRON CORPORATION 1675 WEST 2750 SOUTH 84401 OGDEN, Utah USA Tel: ++1 801 394 4558 Fax: ++1 801 394 4559

1140

List of Participants

MORO Renata Dipartimento scienze fisiche 80126 NAPOLI ITALY Tel: ++39 061 676348 Fax: ++39 081 676346 [email protected] MORVAY Steven AMBI INC 4 Manhattanville Rd NY 10577 PURCHASE USA Tel: ++1 914 701 4500 Fax: ++1 914 696 0860 [email protected] MOSTERT Volker MEDIZINISCHES INSTITUT FUR UMWELTHYGIENE Auf’m Hennekamp 50 40627 DUSSELDORF GERMANY Tel: ++49 211 3389 217 Fax: ++49 211 3190 910 [email protected] MOULIS Jean Marc CEA/GRENOBLE DBMS/MEP 38054 GRENOBLE Cedex 9 FRANCE Tel: ++4 76 88 56 23 Fax: ++4 76 88 58 72 [email protected] MYKHAYLYK Olga UKRAINIAN ACADEMY OF SCIENCES Institute for Applied Problems in physics & Biophysics 252001 KYIV UKRAINE Tel: ++380 44 2525771 Fax: ++380 44 462 48 88 [email protected] Mc LAUGHLIN James VETERINARY RESEARCH LAB. Abbotstown DUBLIN 15 IRELAND Tel: ++353 1 60723785 Fax: ++353 1 8220363 [email protected] MCDONALD Janet us FOOD AND DRUG ADMINISTRATION 1431 Harbor Bay Parkway 94502 7070 ALAMEDA, CA USA Tel: ++1 510 337 6845 Fax: ++1 510 337 6708 [email protected] McMASTER Dorothy THE QUEEN’S UNIVERSITY OF BELFAST Institute of Clinical Science BT12 6BJ BELFAST NORTH IRELAND Tel: ++44 1 232 240 503 Fax: ++44 1 232 329899 [email protected] NABET BELLEVILLE Francine HOPITAL BRABOIS Laboratoire de Biochimie B 54035 NANCY FRANCE Tel: ++3 83 85 19 70 Fax: ++3 83 85 27 85 NAKRY Soch UNIVERSITY OF CALIFORNIA, LOS ANGELES Dept of Envir. Sci. CA 90095 1712 LOS ANGELES USA Tel: ++1 310 825 8429 Fax: ++1 310 825 8429 [email protected] NASU Tameyuki TOKYO COLLEGE OF WELFARE I-14-26 Kyodo 156 0052 TOKYO JAPAN Tel: ++81 03 3429 1209 Fax: ++81 03 3429 1209 NAVARRO-BLASCO Inigo UNIVERSIDAD DE NAVARRA Fac. Ciencias—Dept Quimica 31080 PAMPLONA (Navarra) SPAIN Tel: ++34 48 425600 Fax: ++34 48 425649 [email protected] NAVES Andreia Rua Val de Palmas 302 03161-080 SAO PAULO BRAZIL Tel: ++55 11 69 66 60 89 Fax: ++55 11 50 84 37 58 [email protected] NEGRETTI DE BRATTER Virginia HAHN MEITNER INSTITUT BERLIN Gmbh Holsteinische Str. 49 D-10717 BERLIN GERMANY Tel: ++49 308062 2262 Fax: ++49 308062 2781 [email protected] NEVE Jean UNIVERSITE LIBRE DE BRUSSELS Institut de Pharmacie B-1050 BRUSSELS BELGIUM Tel: +32 2 650 51 77 Fax: +32 2 650 52 49 [email protected] NIELSEN Forrest H. USDA, ARS Grand Forks Human Nutrition Research Center ND 58202-9034 GRAND FORKS USA Tel: ++1 701 795 8456 Fax: ++1 701 795 8230 [email protected] NIKONOV Alexis TRACE ELEMENT—INSTITUT POUR L’UNESCO 1, place de 1’Ecole 69342 LYON Cedex 07 FRANCE Tel: 04 72 80 82 90 Fax: 04 78 58 86 71 NISHIJO Muneko KANAZAWA MEDICAL UNIVERSITY Dept Public Health 920-0293 ISHIKAWA JAPAN Tel: ++81 76 2862211 Fax: ++81 762863728 [email protected] O’CONNOR Jacqueline UNIVERSITY OF ULSTER NICHE—Biomedical Sciences BTS2 1SA COLERAINE NORTHERN IRELAND Tel: ++44 1265 324883 Fax: ++44 1265 324965 [email protected]

List of Participants

1141

OBERLEAS Donald TEXAS TECH UNIVERSITY & HOWARD UNIV. 3404 88th Street, Lubbock 79423-2706 LUBBOCK, TEXAS USA Tel: ++l 8067962614 Fax: ++1 8067962614 [email protected] ODUOR Fredrick D. O. UNIVERSITY OF NAIROBI Dept of Chemistry NAIROBI KENYA Tel: ++254 2 44 9004 Fax: ++254 2446138 OERNSRUD R. DIRECTORATE OF FISHERIES Institute of Nutrition N-5002 BERGEN NORWAY Tel: ++47 55 23 81 38 Fax: ++47 55 23 80 95 [email protected] OHLY Patricia DIABETES RESEARCH INSTITUT Auf’M Hennekamp 65 D-40225 DUSSELDORF GERMANY Tel: ++49 211 3382234 Fax: ++49 211 3382603 [email protected] OHTA Hisayoshi KITASATO UNIVERSITY Dept of occupational Health 228-8555 SAGAMIHARA City/Kanagawa JAPAN Tel: ++81 427 788 070 Fax: ++81 427 788 070 [email protected] OKABE Masashi HOKKAIDO UNIVERSITY Grad. Sch. Environ. Earth Sci. 060 0810 SAPPORO JAPAN Tel: ++81 11 706 2244 Fax: ++81 11 706 0629 [email protected] ONNING Gunilla LUND UNIVERSITY Chemical Center S-22100 LUND SWEDEN Tel: ++46 462224532 Fax: ++46 46 2224532 [email protected] OPRENDEK Marie France MEDECINS DU SPORT ENDOCRINO & NUTRIT. Institut Nationale des Sports 94300 VINCENNES FRANCE Tel: 01 43 98 07 23 Fax: 01 43 28 46 00 OTHMANE Ali FACULTE DE MEDECINE 5000 MONASTIR TUNISIE Tel: ++216 3462200 Fax: ++216 3460737 PAGLIUCA Maria Gabrielle Via pansini 5 80131 ITALY Tel: ++390817463205 Fax: ++390817463150 [email protected] PALLAUF Josef INSTITUT FUR TIERERNAHRUNG Der Justus-Liebig-Universitat D-35390 GIESSEN GERMANY Tel: ++49 641 993 92 30 Fax: ++49 641 993 92 39 [email protected] PAVAO Maria Leonor UNIVERSIDADE DOS ACORES R. Mae de Deus 9500 PONTA DELGADA/ACORES PORTUGAL Tel: ++351 966 531 55 Fax: ++351 966 536 40 [email protected] PENLAND James G. USDA ARS Grand Forks Human Nut. Res. Center POB 9034 ND 58202-9034 GRAND FORKS USA Tel: ++1 701 795 8471 Fax: ++1 701 795 8220 [email protected] PEPIN Denise FACULTE DE PHARMACIE 63000 CLERMONT FERRAND FRANCE Tel: ++4 73 60 28 84 50 Fax: ++4 76 60 28 84 55 [email protected] PERETZ Anne CHU BRUGMANN 4, place Van Gehuchten B-1020 BRUSSELS BELGIUM Tel: ++32 2 477 2383 Fax: ++32 2 477 2178 [email protected] PEREZ GALLARDO Lucia Luisa UNIVERSITY OF VALLADOLID Area de Bioquimica y Biologia Molecular E-42003 SORIA SPAIN Tel: ++34 975 224 350 Fax: ++34 975 229 385 [email protected] PFANNHAUSER Werner GRAZ UNIVERSITY OF TECHNOLOGY Institut Biol Food Chemistry A-8010 GRAZ AUTRICHE Tel: ++43 316 871 64 70 Fax: ++43 316 873 69 70 [email protected] PHILLIPS Clive UNIVERSITY OF CAMBRIDGE Department of Clinical Veterinary Medicine CB3 0ES CAMBRIDGE ENGLAND Tel: ++44 1223337678 Fax: ++44 1223330886 [email protected]

1142

List of Participants

PIASEK Martina INST FOR MEDICAL RES. AND OCCUP. HEALTH 2 Ksaverska St. HR 10 001 ZAGREB CROATIA Tel: ++385 14673188 Fax: ++385 1 4673303 [email protected] PIETTE Jacques UNIVERSITY OF LIEGE SART TILMAN Laboratory of Virology— Inst. of Pathology B-4000 LIEGE BELGIUM Tel: ++32 43 66 2442 Fax: ++32 43 66 2433 [email protected] PILECKI Adam MEDICAL UNIVERSITY Department of Biochemistry 85 092 BYDGOSZCZ POLAND Tel: ++48 52 341 33 99 Fax: ++48 52 341 59 33 PIZENT Alica INST. FOR MEDICAL RESEARCH & OCCUPATIONAL HEALTH Ksaverska c. 2, 10001 ZAGREB CROATIA Tel: ++385 14 67 31 88 Fax: ++385 14 67 33 03 [email protected] POLJAK BLAZI Marija RUDER BOSKOVIC INSTITUTE Bijenicka C. 54 10000 ZAGREB CROATIA Tel: ++385 14 56 10 17 Fax: ++385 14 68 00 94 POUPON Carole CHI EAUBONNE—MONTMORENCY Laboratoire de biochimie 95602 EAUBONNE FRANCE Tel: ++1 34 16 55 20 Fax: ++1 34 16 56 48 POUPON Joel HOPITAL F. WIDAL Laboratoire Biochimie Toxicologie 75475 PARIS Cedex 10 FRANCE Tel: ++1 40 05 42 17 Fax: ++1 40 05 48 78 [email protected] PROHASKA Joseph R. UNIVERSITY OF MINNESOTA Biochemistry & Molecular Biology MN 55812 DULUTH USA Tel: ++1/2187267502 Fax: ++1/2187266181 [email protected] RAAB Andrea HAHN MEITNER INSTITUT Department of Trace elements in Health & Nutrition D-14109 BERLIN GERMANY Tel: ++30 8062 3020 Fax: ++30 8062 2781 [email protected] RACHIDI Walid UNIVERSITE JOSEPH FOURIER Faculte de Pharmacie 38700 LA TRONCHE FRANCE Tel: ++4 76 63 71 16 RAINER Jonathan BORAX EUROPE 170 Priestley road GU2 SRQ GUILDFORD, SURREY ENGLAND Tel: ++44 1483 242 160 Fax: ++44 1483 242 083 [email protected] RAINEY Charlene NUTRITION RESEARCH GROUP 4199 Campus Drive, Suite 550 CA 92612 IRVINE USA Tel: ++1 949 497 60 66 Fax: ++1 949 497 91 66 [email protected] RAYMAN Margaret UNIVERSITY OF SURREY School of Biological Sciences GU2 5XH GUILDFORD ENGLAND Tel: ++44 148 356 28 82 Fax: ++44 148 330 03 74 [email protected] RAYSSIGUIER Yves INRA Laboratoire des Maladies Métaboliques 63122 ST GENES CHAMPANELLE FRANCE Tel: ++4 73 62 42 30 Fax: ++4 73 62 46 38 REA Irene Maeve QUEEN’S UNIVERSITY OF BELFAST Department of Geriatric Medicine BT97 BL BELFAST NORD IRELAND Tel: ++1232 272156 Fax: ++1232 663045 [email protected] REDDY Sheela Department of health SEI 6LH LONDON ENGLAND Tel: ++44 171 972 5236 Fax: ++44 171 972 553 [email protected] REID Steve UNIVERSITY OF SASKATCHEWAN Department of Chemistry S7N 5C9 SASKATOON, SK CANADA Tel: ++1 306 966 4673 Fax: ++1 306 966 4730 [email protected] REILLY Conor OXFORD BROOKES UNIVERSITY 10 Litchfield Close 0X7 4LB ENSTONE ENGLAND Tel: ++44 1608 677245 [email protected] REUL Benedicte PHARMA NORD Excelsiorlaan 21 B1930 ZAVENTEM BELGIUM Tel: ++32 2 720 51 20 Fax: ++32 2 720 51 60

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1143

RICHARD Marie Jeanne CHU A. MICHALLON Laboratoire de Biochimie C 38043 GRENOBLE Cedex 9 FRANCE Tel: ++4 76 76 51 47 Fax: ++4 76 76 56 64 [email protected] RICHARZ Andrea HAHN MEITNER INSTITUT BERLIN Glienicker Str 100 14109 BERLIN GERMANY Tel: ++49 30 8062 2294 Fax: ++49 30 8062 2781 [email protected] RIMER Ulf LIMHAMNS LAKARGRUPP Jarnvagsgatan 56 S-216 16 MALMO SWEDEN ROBBERECHT Harry UNIVERSITY OF ANTWERP Dept Pharmaceutical Sciences B-2610 WILRIJK BELGIUM Tel: ++32 38 202 731 Fax: ++32 38 202 734 [email protected] ROCK Edmond INRA—CENTRE DE RECHERCHE Laboratoire des maladies metaboliques 63122 ST GENES CHAMPANELLE FRANCE Tel: ++4 73 62 41 69 Fax: ++4 73 62 46 38 [email protected] RODRIGUEZ Adela EUROPEEN COMMISSION JOINT RESEARCH CENTRE Retieseweg B-2440 GEEL BELGIUM Tel: ++32 14 571207 Fax: ++32 14 584273 [email protected] ROSSINI Eliane UNIVERSITE JOSEPH FOURIER UFR de Pharmacie 38700 LA TRONCHE FRANCE Tel: ++4 76 63 71 16 ROTH Hans-Peter INSTITUT FUR ERNAHRUNGSPHYSIOLOGIE Technische Universitat Munchen D-85350 FREISING-WEIHENSTEPHAN GERMANY Tel: ++49/8161 713879 Fax: ++49/8161 713999 ROUSSEL Anne Marie UNIVERSITE JOSEPH FOURIER UFR de Pharmacie 38700 LA TRONCHE FRANCE Tel: ++4 76 63 71 31 Fax: ++4 76 63 71 80 [email protected] RUCKER Robert B. UNIVERSITY OF CALIFORNIA DAVIS One Shields Avenue 95616 DAVIS, CA USA Tel: ++1 916 752 20 89 Fax: ++1 916 752 89 66 [email protected] RUKSAN Bruna Estefania INSTITUTO DE PATOBIOLOGIA—CICV—INTA CC 77 Moron CP 1708 1439 BUENOS AIRES ARGENTINE Tel: ++54 1 605 1091 Fax: ++54 1 638 2220 SAARI Jack USDA ARS Grand Forks Human Nutrition Research Center 58202 GRAND FORKS, ND USA Tel: ++1 7017958499 Fax: ++1 7017958395 [email protected] SABATIER Magalie USDA ARS WHRNC P.O Box 29997 CA 94129 SAN FRANCISCO USA Tel: ++1 415 556 04 38 Fax: ++1 415 556 14 32 [email protected] SAITO Shigeru ST MARIANNA UNIVERSITY Depart of Preventive Medicine 2168511 KAWASAKI JAPAN Tel: ++81 449 77 8111 Fax: ++81 449 77 8356 ssaito@marianna-u .ac.jp SAITO Takeshi UNIVERSITY SCHOOL & MEDICINE Dept of Hygiene & preventive 060 8638 SAPPORO JAPAN Tel: ++81 11 706 5065 Fax: ++81 11 706 7819 [email protected] SALA PEINADO Maria Jose PIERRE FABRE MEDICAMENT La Chartreuse 81106 CASTRES Cedex FRANCE Tel: ++5 63 71 45 47 Fax: ++5 63 71 43 99 SAMPSON Barry CHARING CROSS HOSPITAL Clinical Chemistry W6 8RF LONDRES ENGLAND Tel: ++44 181 846 7080 Fax: ++44 181 846 7007 [email protected]

1144

List of Participants

SANDSTEAD Harold, H. UNIVERSITY OF TEXAS, Medical Branch Preventive Medicine & Community Health TX 77555-1109 GALVESTON USA Tel: ++1 409 772 46 61 Fax: ++1 409 772 62 87 [email protected] SANZ Inmaculada NATIONAL SCHOOL OF PUBLIC HEALTH C/Sinesio Delgado 8 28029 MADRID SPAIN Tel: ++34 91 387 78 58 Fax: ++34 91 387 78 72 [email protected] SCARINO Maria Laura ISTITUTO NAZIONALE DELLA NUTRIZIONE Via Ardeatina 546 I-00179 ROMA ITALY Tel: ++39 650 425 89 Fax: ++39 650 315 92 [email protected] SCHAEFER Klaus INSTITUT FUR TIERERNAHRUNG Brummerstr. 34 D-14195 BERLIN GERMANY Tel: ++30 838 3964 SCHALLER Dominique NOVARTIS CONSUMER HEALTH Fabrikstr. 10 3176 NEUENEGG SWITZERLAND Tel: ++41 31 377 2416 Fax: ++41 31 377 2421 [email protected] SCHLEGEL-ZAWADZKA Margorzata Dept Food Chemistry & Nutrition 30-688 KRAKOW POLAND Tel: ++48 126543949 [email protected] SCHRAMEL Peter GESELLSCHAFT FUR STRAHLEN & UM WZLTSCHUTZ Institut fur okologische Chemie D-91465 ERGERSHEIM-NEUHERBERG GERMANY [email protected] SENEGAL Pierre Etienne L’HOPITAL DE MONTREAL POUR ENFANTS 2300 Tupper, Bureau D-466 H3H IP3 MONTREAL CANADA Tel: ++1 514 344 1409 Fax: ++1 514 934 4477 [email protected] SENTENAC Xavier LABORATOIRE DE BIOLOGIE DU STRESS OXYDANT Faculte de Pharmacie 38700 LA TRONCHE FRANCE Tel: ++4 76 63 71 16 SERFASS Robert Earl UNIVERSITY OF TEXAS MEDICAL BRANCH Dept of Prev. Medic. & Com. Health TX 77555 1109 GALVESTON USA Tel: ++1 409 747 4575 Fax: ++1 409 772 6287 [email protected] SERRA BELTRAN Miguel-Angle JOINT RESEARCH CENTRE Environment Institute I-21020 ISPRA (Varese) ITALY Tel: ++39 0332 789353 Fax: ++39 0332 785336 SEVE Michel CHU A. MICHALLON Laboratoire de biochimie C 38043 GRENOBLE FRANCE Tel: ++4 76 76 52 78 Fax: ++4 76 76 56 64 [email protected] SHEEHAN T.M. REGIONAL TOXICOLOGY LABORATORY City Hospital NHS B18 7QH BIRMINGHAM ENGLAND Tel: ++44/1215075204 Fax: ++44/1215547386 SHENKIN Alan ROYAL LIVERPOOL UNIVERSITY HOSPITAL Department of Clinical Chemistry L69 3BX LIVERPOOL ENGLAND Tel: ++44 1517064232 Fax: ++44 151 7065813 [email protected] SHIMADA Akinori TOTTORI UNIVERSITY Dept of Veterinary pathology 680 0945 TOTTORI JAPAN Tel: ++81 875 31 5422 Fax: ++81 875 31 5422 [email protected] SHRIMPTON Derek COUNCIL FOR RESPONSIBLE NUTRITION 63 Hampton Court Way KT7 0LT SURREY ENGLAND Tel: ++44 181 398 9888 Fax: ++44 181 398 6906 [email protected] SIERRO Christian Cabinet medical CH 1964 CONTHEY SWITZERLAND Tel: ++41 27 346 77 77 Fax: ++41 27 346 77 77 SIES Helmut UNIVERSITY OF DUSSELDORF Institut Physiological Chemistry D40001 DUSSELDORF 1 GERMANY Tel: ++49 211 311 27 07 Fax: ++49 211 311 30 29 [email protected]

List of Participants

1145

SIEVERS Erika KLINIKUM DER CHRISTIAN ALBRECHTS Universitat zu Kiel 24105 KIEL GERMANY Tel: ++49 4 315970 Fax: ++49 4 31 597 1831 SILIO Fernando FUNDACION ENTORNO Y SALUD C/Modesto Lafuente 16 BajoB 28010 MADRID SPAIN Tel: ++34 914416845 Fax: ++34 914416845 [email protected] SIMA Andrea INSTITUTE OF B10 Food and Chemistry 8010 GRAZ AUTRICHE Tel: ++43 316 8736498 Fax: ++43 312 873 6970 [email protected] SKINNER Nicola WALTHAM Waltham on the wolds, Melton LE14 4RT LEICESTERSHIRE ENGLAND Tel: ++44 166 441 53 05 Fax: ++44 166 441 54 40 [email protected] SPEARS Jerry W. NORTH CAROLINA STATE UNIVERSITY Box 7621 27695-7621 RALEIGH, NC USA Tel: ++1 9195154008 Fax: ++1 9195154463 [email protected] STADTMAN Theresa NIH NHLBI Laboratory of Biochemistry MA 20892 BETHESDA USA Tel: ++1 301 496 3002 Fax: ++1 301 480 0357 STANGL Gabriele TECHICAL UNIVERSITY MUNICH Institute of Nutrition Science D-85350 FREISING GERMANY Tel: ++49 8161 713116 Fax: ++49 8161 713999 [email protected] STARSKA Krystyna NATIONAL INSTITUTE OF HYGIENE Chocimska 24 00 791 WARSAW POLAND Tel: ++48 22 49 40 51 Fax: ++48 22 49 74 84 [email protected] STASTNA Marie CHARLES UNIVERSITY Faculty of Sciences—Dept chemistry 120 00 PRAGUE 2 CZECH REPUBLIC Tel: ++42 2 643 3931 [email protected] STEWART Peters ROYAL PRINCE ALFRED HOSPITAL Missenden Road Camperdown 2050 CAMPERDOWN AUSTRALIA Tel: ++61 2 95157162 Fax: ++61 2 95157931 [email protected] STIBILJ Vekoslava JOSEF STEFAN INSTITUTE Dept of Environmental Sciences 1000 LJUBLJANA SLOVENIA Tel: ++386 61 1885354 [email protected] STOECKER Barbara J. OKLAHOMA STATE UNIVERSITY 425 Human Environmental Sciences OK 74078 STILLWATER, Oklahoma USA Tel: ++1 405 744 50 40 Fax: ++1 405 744 71 13 [email protected] STRAIN J.J UNIVERSITY OF ULSTER Cromdre Road BT52 ISA COLERAINE IRELAND Tel: ++44 12 653 24795 Fax: ++44 126 532 49 65 [email protected] STRONG Phillip L. U.S. Borax Inc. 26877 Tourney Road 91355-1847 VALENCIA, CALIFORNIA USA Tel: ++1 661 287 5634 Fax: ++1 661 287 5542 pstrong@ compuserve.com STRUYS-PONSAR Cecile UNIVERSITE CATHOLIQUE DE LOUVAIN Laboratoire Biologie Cellulaire B-1348 LOUVAIN LA NEUVE BELGIUM Tel: ++32 10 47 35 18 Fax:++32 10 47 35 15 SUCK Catherine LABCATAL 7, rue Roger Salengro 92120 MONTROUGE FRANCE Tel: ++1 46 54 27 92 Fax: ++1 46 54 22 21 SUNDE Roger A. UNIVERSITY OF MISSOURI 217 Gwynn Hall 65211 COLUMBIA, MO USA Tel: ++1 5738824526 Fax: ++1 5738820185 roger-sunde@ muccmail.missouri.edu SUSLIKOV Vikentij Leonidivich CHUVASH STATE UNIVERSITY Department of Ecology and Hygiene 428022 CHEBOKSARY RUSSIA Tel: ++7 835 2 23 20 59 SUTHERLAND Barbara UNIVERSITY OF CALIFORNIA Berkeley 94605 OAKLAND, CA USA Tel: ++1 510 633 6287 Fax: ++1 510 642 0535 [email protected]

1146

List of Participants

SUZUKI Kazuo T. CHIBA UNIVERSITY Faculty of Pharmaceutical Sciences 263 8522 CHIBA JAPAN Tel: ++81 432902891 Fax: ++81 432902891 [email protected] SWENSON Connie ZINPRO 10500 City West Parkway Suite 300 MN 55344 EDEN PRAIRIE USA Tel: ++1 612 944 2736 Fax: ++1 612 943 5828 [email protected] TAHIRI Maha INRA—CENTRE DE RECHERCHE Laboratoire des Maladies Metaboliques 63122 ST GENE CHAMPANELLE FRANCE Tel: ++4 73 62 42 81 Fax: ++4 73 62 46 38 TALASZ Heribert INST FOR MEDICAL CHEM. & BIOCHEMISTRY Fritz-Pregl Str 3 A-6020 INNSBRUCK AUTRICHE Tel: ++43 512/507 3522 Fax: ++43 512/507 2876 [email protected] THIELE Dennis J. UNIVERSITY OF MICHIGAN MEDICAL SCHOOL Biological Chemistry MI 48109 0606 ANN ARBOR USA Tel: ++1 734 763 5717 Fax: ++1 734 763 4581 [email protected] THOMAS Christoph MICROMASS GmbH Dieckerhofsweg 12 D-58239 SCHWERTE GERMANY Tel: ++49 2304 219442 Fax: ++49 2304 219443 [email protected] THOMPSON Katherine H. THE UNIVERSITY OF BRITISH COLUMBIA Chemistry Department V6T 1Z1 VANCOUVER, B;C CANADA Tel: ++1 604 822 1776 Fax: ++1 604 822 2847 [email protected] THOMSON Christine UNIVERSITY OF OTAGO Dept of Human Nutrition DUNEDIN NEW ZEALAND Tel: ++64 3 479 7943 Fax: ++64 3 479 7958 [email protected] TRUMBO Paula NATIONAL ACADEMY OF SCIENCES Food and Nutrition Board WASHINGTON, DC USA Tel: ++1 202 334 1314 Fax: ++1 202 334 2316 TURCK Dominique CHU DE LILLE Service de pédiatrie 59038 LILLE FRANCE TURNLUND Judith R. USDA, ARS Western Human Nutrition Research Center CA 94129 PRESIDIO OF SAN FRANCISCO USA Tel: ++1 4155565662 Fax: ++1 4155561437 [email protected] URIU ADAMS Janet UNIVERSITY OF CALIFORNIA One Shield Ave., CA 95616 8669 DAVIS USA Tel: ++1 530 752 4658 Fax: ++1 530 752 8966 [email protected] USUDA Kan OSAKA MEDICAL COLLEGE Department of Hygiene & Public Health 569 OSAKA JAPAN Fax: ++81 5726846519 VAN BERGEN Manfred J. FACULTY OF EARTH SCIENCES, UTRECHT UNIV. Budapestlaan 4 3584 CD UTRECHT THE NETHERLANDS Tel: ++31 30 2535036 Fax: ++31 30 2535030 [email protected] VAN DAEL Peter NESTLE RESEARCH CENTER P.O. Box 44 CH-1000 LAUSANNE 26 SWITZERLAND Tel: ++41 21 785 8944 Fax: ++41 21 785 8563 [email protected] VAN DEN HEUVEL E.G.H.M TNO Nutrition and Food Research Institute P.O. Box 360 PB-3700 AJ ZEIST THE NETHERLANDS Tel: ++31 30 6944940 Fax: ++31 30 6944928 [email protected] VAN DYCK Kristien UNIVERSITY OF ANTWERP (UIA) Dept of Pharm. Sc B-2610 ANTWERP (WILRIJK) BELGIUM Tel: ++32 38202732 Fax: ++32 38202734 [email protected] VAN RYSSEN J.B.J. UNIVERSITY OF PRETONIA Department Animal & Wildlife Sciences 0002 PRETONIA SOUTH AFRICA Tel: ++ 27 124203268 Fax: ++27 124203290 ryssen@nsnperl .up.ac.za

List of Participants

1147

VAQUERO Pilar INST. NUTRICION Y BROMATOLOGIA Fac. Farmacia E-28040 MADRID SPAIN Tel: ++34 091 373 9356 Fax: ++34 091 549 5079 [email protected] VARELA Pilar INSTITUTO DE NUTRICION (CSIC-UCM) Facultad de Farmacia E-28040 MADRID SPAIN Tel: ++34 15490038 Fax: ++34 15495079 [email protected] VENEGAS V. Guillermo UNIVERSIDAD DE CONCEPCION Urrutia Manzano 330 Casille 60-2 CONCEPCION CHILE Tel: ++56 41 22 90 08 [email protected] VERDURA Tomas INSTITUTO FINLAY Ave 27 Nro 19805 La Lisa 11600 LA HAVANA CUBA Tel: ++53 7 336075 VIEGAS CRESPO Ana Maria UNIVERSIDADE DE LISBOA Faculdade de Ciencias 1700 LISBON PORTUGAL Tel: ++38 71 7573141 Fax: ++38 717500028 [email protected] VIGEZZI Jean François H.I.A Val de Grâce 74, rue Boulevard de Port Royal 75230 PARIS Cedex 05 FRANCE Tel: ++1 40 51 46 16 Fax: ++1 40 51 50 57 VINCZER Peter BERES RESEARCH CENTER P.O. Box 270 H-1300 BUDAPEST HUNGARY Tel: ++361 250 72 51 Fax: ++361 250 72 51 [email protected] VIRGONA Nantiga NATIONAL INSTITUTE OF HEALTH & NUTRITION Division of Applied Food Research 162-8636 TOKYO JAPAN Tel: ++81 3 32035602 Fax: ++81 3 3205 6549 [email protected] VIRTANEN Vesa IRMM Retieseweg B-2440 GEEL BELGIUM Tel: ++32 14571256 Fax: ++32 14584273 [email protected] VIVOLI G. Franco UNIVERSITY OF MODENA Institut of Hygiene I-41100 MODENA ITALY Tel: ++39 59360084 WALKER Ron UNIVERSITY OF SURREY School of Biological Sciences GU2 5XH GUILFORD, SURREY ENGLAND Tel: ++44 1483 3000800 Fax: ++44 1483 576978 [email protected] WALKER Ann ROYAL FREE & UNIVERS COLLEGE MEDICAL Royal Free campus NW3 2PF LONDON ENGLAND Tel: ++44 171 794 0500 Fax: ++44 171 830 2631 [email protected] WARD David UNIVERSITY OF ADELAIDE Department of Chemistry 5005 ADELAIDE AUSTRALIA Tel: ++61 8 8303 5496 Fax: ++61 8 8303 4358 [email protected] WASOWICZ Wojeiech INSITUTE OF OCCUPATIONAL MEDICINE 8 Teresy St 90-850 LODZ POLAND Tel: ++48 42314631 Fax: ++48 42568331 [email protected] WASTNEY Meryl E. GEORGETOWN UNIVERSITY MEDICAL CENTER Neonatology D.C 20007 WASHINGTON USA Tel: ++1 2026875004 Fax: ++1 2027844747 [email protected] WEDEKIND Karren HILL’S PET NUTRITION, INC P.O. Box 1658 KS 66601 TOPEKA USA Tel: ++1 7852868095 Fax: ++1 7852868014 karen-wedekind@ hillspet.com WERMAN Moshe J. TECHNION ISRAEL INSTITUTE OF TECHNOLOGY Dept Food Eng. & Biotechnol 32000 HAIFA ISRAEL Tel: ++972 4 8293073 Fax: ++972 48320742 [email protected] WINDISCH Wilhelm INSTITUTE OF NUTRITION PHYSIOLOGY Technological University of Munich D-85350 FREISING GERMANY Tel: ++49 8161 713673 Fax: ++49 8161 713999 [email protected]

1148

List of Participants

WING Anncatherine KUWAIT UNIVERSITY Faculty of dentistry 131 10 SAFAT KOWAIT Tel: ++965 531 2300 Fax: ++965 532 6049 [email protected] WING Kenneth KUWAIT UNIVERSITY Faculty of Dentistry 13110 SAFAT KUWAIT Tel: ++965 531 2300 Fax: ++965 532 6049 [email protected] WOLF Christian HAHN MEITNER INSTITUT BERLIN Department Trace Elements in Health and Nutrtion D-14109 BERLIN GERMANY Tel: ++49 30 8062 2262 Fax: ++49 30 8062 2781 [email protected] WONG Heng Kuan UFR DE PHARMACIE Laboratoire de Biologie du Stress Oxydant 38700 LA TRONCHE FRANCE Tel: ++4 76 63 71 16 WRIGHT Cody NORTH STATE UNIVERSITY Department of Animal Science 27695-7621 RALEIGH USA Tel: ++1 9195154013 Fax: ++1 9195154463 [email protected] WUNDERLICH Shahla M. MONTCLAIR STATE UNIVERSITY 306 Charm Court 07747 2218 ABERDEEN, NJ USA Tel: ++1 7325836346 Fax: ++1 7322901860 [email protected] XIA Yiming INSTITUT OF NUTRITION & FOOD HYGIENE CHINAse Academy of preventive Medicine 100050 BEIJING CHINA Tel: ++86 1063040634 Fax: ++86 1063011875 [email protected] YATES Allison NATIONAL ACADEMY OF MEDICINE Food and Nutrition Board WASHINGTON, DC USA Tel: ++1 202 334 1732 Fax: ++1 202 334 2316 [email protected] YEUDALL Fiona UNIVERSITY OF OTAGO Dept of Human Nutrtion DUNEDIN NEW ZEALAND Tel: ++6434797833 Fax: ++6434797958 [email protected] YSART Gillian MINISTRY OF AGRICULTURE, FISHERIES & FOOD Room 238 SW1P 3JR LONDON ENGLAND Tel: ++171 238 6337 Fax: ++171 238 5331 [email protected] ZACHARA Bronislaw A. MEDICAL UNIVERSITY Department of Biochemistry 85 092 BYDGOSZCZ POLAND Tel: ++48 52 341 33 99 Fax: ++48 52 341 59 33 [email protected] ZACHWIEJA Zofia JAGIELLONIAN UNIVERSITY MEDICAL COLLEGE Dept of Food Chemistry & Nutrition 30-084 KRAKOW POLAND Tel: ++48 12 637 56 72 Fax: ++48 12 633 07 91 ZAGRODZKI Pawek COLLEGIUM MEDICUM JAGIELLONIAN UNIVERSITY Dept of Food Chemistry & Nutrition 30 688 CRACOW POLAND Tel: ++4812 659 00 20 Fax: ++4812 657 02 62 [email protected] ZAWISLAK Richard FACULTE DE MEDECINE Lab de 1’Institut de chimie biologique 67085 STRASBOURG FRANCE Tel: ++3 88 37 12 55 Fax: ++3 88 35 56 02 ZIMMERMANN M. Eidgenossische Technische Hochschule ZURICH P.O Box 474 CH-8803 RUSCHILKON SWITZERLAND Tel: ++41 1 724 01 83 ZOUARI Nouri CHU HABIB BOURGUIBA service des Explorations fonctionnelles 3333029 SFAX TUNISIA

AUTHOR INDEX

Abel, J., 515, 899 Abou-Shakra, F., 1119 Abrams, S., 267 Aburto, E., 679, 695 Accominotti, M., 547 Adamowicz, A., 643 Adams, F. C., 1107 Adou, P., 587 Afanas’ev, I. B., 115, 119 Agte, V. V., 261, 305 Aguilar, A. E., 1039, 1040 Akesson, B., 250, 865, 892, 893 Alary, J., 232 Al-Awadi, F. M., 855 Alimonti, A., 332, 667 Allen, L. H., 267 Alperovitch, A., 442 Alvarez, J. I., 247 Amancio, O. M. S., 638, 639 Amano, R., 663 Ambe, F., 1099 Ambe, S., 1099 Amblard, P., 421 Ammerman, C. B., 283 Amouyel, P., 463 Anderson, R. A., 393, 449, 503, 504 Andrews, W. L., 829 Andriollo, M., 133 Angelow, L., 209 Anke, M., 209, 221, 229, 685, 777 Anke.S., 221, 777 Anzulovich, 181, 183 Aral, B., 917 Aranda, P., 340 Araneda, O., 399 Araos, M., 399 Araujo, C. L., 329 Argaud, D., 233 Armstrong, T. A., 729, 759, 1067 Arnaud, J., 138, 240, 377, 442, 449, 450, 467, 484, 534, 536, 641 Arnaud, M. J., 1115

Arpe.T., 987 Arteel, G. E., 71 Arthur, J. R., 843 Arveiler, D., 463 Atherton, C., 279, 877 Audette, R., 563 Avsar, B., 623 Azevedo, C. H., 315 Aziz, K., 829 Babosa, A. C., 690 Bacso, J., 569 Baj, A., 591 Baker, A., 597, 937, 943, 959 Bakowska, E., 1120, 1121 Balansard, B., 442 Barbera, R., 306 Barclay, D., 138, 195 Barlis, J., 611 Barnouin, J., 240 Barron, T. F., 601 Barthomeuf, C., 311 Barton, H., 242, 693 Basu, G. K., 645 Baucells, M., 183 Baxter, M., 279, 705, 877 Bayle, B., 571 Bazex, J., 437 Beani, J. C., 77, 129, 143, 421, 437 Beattie, J. H., 937, 961, 031 Becker, K., 1049, 1078 Beckett, G. J., 843 Behn, C., 399 Behne, D., 29 Belin, N., 897 Bell, A., 271 Bellanger, J., 287 Belleville, F., 1061, 1088 Bellisola, G., 873 Belonge, B., 308 Benderdour, M., 1061 Benn, I., 519 Bennett, A., 1049, 1077, 1078, 1087

Bensimon, G., 629 Bergdahl, I. A., 161 Bergmann, P., 1009 Bergomi, M., 581 Berntssen, M. H. G., 141 Berr, C., 442 Bertouze, M., 527 Bertrand, J. ch., 709 Bertrandt, J., 231, 244 Bettger, W., 323 Betts, H., 995 Bicho, M., 333 Bingham, M. J., 383 Birch, F., 741 Biswas, B. K., 645 Bittencourt, V. B., 405 Blanusa, M., 809 Bocca, B., 667 Bodgen, J. D., 407, 597 Bogucka-Sciezynskag, A., 309 Bonforte, G., 591 Bonham, M. P., 475, 937, 943 Bonnefont-Rousselot, D., 629 Booker, P., 1119 Bordin, G., 169, 1103 Borella, P., 581 Borla darve, G., 421 Bose, S. K., 67 Bosscher, D., 275 Bouabsal, A., 641 Bourdonnais, A., 819 Bourely, B., 629 Bouvard, S., 140, 497, 528, 532, 538 Braetter, P., 145, 180, 390 Bregante, G., 591 Bremner, I., 937, 961 Breska III, A. P., 1013 Briancon, S., 450, 484, 488 Briend, A., 245 Brines, J., 381 Briviba, K.,71 Brizard, C., 629 Brokken, K. A., 293

1149

1150 Bruce-Johnson, W., 383 Brulinska-Ostrowska, E., 252 Bureau, I., 142, 443, 449 Burgess, E., 563 Burk, R. F., 837 Butcher, L., 827 Butler, J., 175 Cadet, J., 77 Cajigal, J., 399 Calderoni, A. M., 691 Calomme, M., 634, 1111 Camargo, L. P., 511 Carbon, P., 849, 903 Caroli, S., 332, 667 Carrasco, A., 399 Carron, C., 687 Casey, N. H., 753 Cashman, K., 937, 943, 959 Casterline, J., 1053 Caulfiel, L., 347 Ceballos-Picot, I., 917 Cefalu, W., 503 Celedon, G., 399 Cesarini, J.P., 887 Cestnik, D., 178 Chakraborti, D., 645 Chanda, C. R., 645 Chappuis, P., 917 Charlton, C. J., 779, 787 Chassagne, M., 240 Chaud, D. M. A., 638, 639 Chen, J., 892 Chenevier, C., 709 Cheng, B., 855 Cherchenko, A. P., 813 Chernyaev, A. L., 568 Chicourel, E. L., 391 Chimienti, F., 1003, 1035 Chiplonkar, S. A., 261, 305 Chowdhury, T. R., 645 Chowdhury, U. K., 645 Christopherson, D. M., 339 Cigliano, S., 55 Ciriolo, M. R., 937 Clark, L. C., 575 Clegg, M., 186, 929 Clemente, G., 381 Cockell, A. K., 215, 313 Cöl, M., 1080 Colli. C., 315 Colls, A., 343 Colombatti, M., 873 Combs, G. F., 781 Cornelis, K. R., 153 Cos, P., 634, 1111 Coudray, C., 287, 311, 703, 707, 957 Coulter, J. S., 943

Author Index Cousins, R. J., 1 Coutinho, V. F., 405 Couzy, F., 138 Coyle, P., 43 Cozzolino, S. M. F., 65, 329, 331, 391, 405, 511, 625, 626 Crews, H., 279, 705, 877 Cribb, A., 679, 695 Cui, C. T., 186, 929 Cuppari, L., 626

Dreher, 899 Drobner, S., 209 Dubois, S., 215, 312 Ducimetiere, P., 463 Ducros, V., 232, 443, 527, 897 Dudchenko, N. A., 336, 813 Dudek, D., 607 Duffield, A. J., 869 Duprez, A., 1061 Dzondo-Gadet, M., 1088

D’Angelo, R., 591 D’Hases, P., 1111 Dabrio, M., 1116 Dahlen, G. M., 523 Dainty, J., 279, 877 Dale, S., 47 Daniels, L. A., 175, 359, 388 Dantas, E. L., 329 Danzeisen, R., 947 Darmon, N., 245 Daveloose, D., 144 David, A., 897 Davila, E. H., 399 Davis, C. W., 1025 De Broe, M., 1111 de Cremer, K., 153 de l’Argy, C., 705 de Lorgeril, M., 547 de Miguel Romera, R., 338 de Moura, J. M., 329 de Portela, M. L., 375 de Wayne, A., 819 Debraekeleer, J., 781 Declercq, L., 437 Deelstra, H., 275, 345 Deeva, I., 115 Delattre, J., 629 Demanneville, S., 887 DeMartino, A., 937 Deng, Z., 707 Deutch, B., 651 Devenyi, A. G., 601 Diaz, M., 267 Didier, C., 77, 143 Dieter, H. H., 655 Dincer, Z., 723 Doco, T., 703 Dohle, C., 515 Dohs, S., 1082 Donley, S., 184 Donovan, U. M., 323 Dopller, V., 629 Dorea, J. G., 690 Dorn, W., 209 Dote, T., 335 Dousset, B., 1061, 1088 Downing, G., 1085 Doyle, T. D., 343

Ebbrell, S. L., 787 Eckhert, C. D., 1049, 1077, 1078, 1082, 1087 Eder, K., 555, 565, 566 Effa, P., 271 Eide, D. J., 35 Elhan, A. H., 1080 Emonet-Piccardi, N., 77, 129 Emteborg, H., 1107 Endo, F., 59 Engberg, R. M., 763 Engle, T. E., 729, 759, 1067 Enomoto, S., 1099 Erçevik, E., 1080 Eretova, V., 385 Esashi, T., 330 Eslinger, P., 601 Estevez, L., 827 Estrada, M. I., 399 Evans, A., 463 Evans, R. W., 611 Evenson, J. K., 21 Fagerhol, M. K., 1031 Fairweather-Tait, S., 227, 255, 279, 877, 937, 959 Fakler, T. M., 530 Falnoga, I., 178, 895 Farre, R., 306, 381 Faughan, M. S., 943 Faure, H., 377, 443, 484, 641 Faure, P., 138, 140, 497, 527, 528, 532, 536 Favaro, D. I. T., 625 Favier, A., 77, 129, 133, 137, 138, 140, 143, 144, 240, 377, 421, 443, 448, 449, 450, 467, 484, 488, 497, 527, 528, 532, 534, 536, 538, 641, 687, 897, 907, 969, 1003, 1035 Favier, M., 377 Feillet-Coudray, C., 142, 957 Feldmann, J., 165 Ferle Vidovic, A., 135 Fernandez, J. A., 763 Fernandez, M. R., 1036 Ferrell, J. M., 809 Ferruzza, S., 63, 937

Author Index Fields, M., 543 Fieux, B., 488 Fisberg, M., 331, 391 Fischer, P. W. F., 308 Flaherty-Craig, C. V., 601 Fleischman, S., 375 Fleites, P., 240 Flynn, A., 937, 943, 959 Folta, M., 242, 693 Fong, A. K. H., 999 Fontecave, M., 83 Forastiere, F., 667 Forbes, I., 995 Fort, D. J., 1057, 1079 Fox, T., 227, 279, 877 Fracasso, G., 873 Fragoso, G., 1040 Francois, B., 634 Franvel, C., 917 Frederickson, C. J., 981 Friel, J. K., 829 Fuentealba, I. C., 679, 695 Fujita, H., 429 Gabriell, I . , 63 Galan, P., 450, 467, 484, 486, 488 Galhardo, A. P., 315 Gallaher, C. M., 293 Gallaher, D. D., 293 Galliot-Guilley, M., 709, 1122 Gambling, L., 383 Gao, B., 67 Garbe-Schonberg, D., 987 Garcia Arribas, O., 1 1 1 7 Garcia, M., 375 Garcia Obregon, O., 267 Garcia-Garcia, P., 446 Garrel, C., 137, 687 Garrow, T. A., 1013 Gautheret, D., 849, 903 Gawecki, J., 697 Gedik, Y., 469 Genç, Y., 1080 Gibson, R. A., 359, 388 Gibson, R. S., 323 Gill, I. M., 749, 782 Gilman, V., 1077 Gimenez, M. S., 181, 183, 691, 1036 Ginty, F., 937 Giray, B., 469, 471 Girigosavi, S., 305 Gitlin, J.D., 9 Glei, M., 209 Gleichmann, H., 515 Gleizes, V., 1122 Gokmen, I. G., 623, 627 Golden, B. E., 1031 Gomez, N. N., 1036

1151 Gonthier, B., 144 Gonzalez, G., 399 Gonzalez, M., 399 Gorinstein, S., 242 Gouzoux, L., 707 Grace, N. D., 717, 768 Gralak, M. A., 309 Green, A., 769 Green, T., 323 Grizard, D., 311 Gromadzinska, J., 127, 369 Gueux, E., 142 Guillard, O., 425 Guiraud, P., 133, 904 Gunstheimer, G., 685 Gurbay, A., 144 Gurtler, H., 221, 777 Ha, P., 175 Hagmar, L., 250 Haguenau, M., 917 Halimi, S., 138, 140, 497, 527, 528, 532, 538 Hallfrisch, J., 519 Hallock, M., 827 Halpern, M. J., 333 Hambidge, M., 347, 977 Han, S. H., 407, 597 Hansen, J.C., 651 Hansen, M., 820 Hanson, S., 184 Hariveau, E., 377 Harrison, N., 705 Hartmann, E., 229 Harvey, J., 937 Harvey, L., 959 Hashiguchi, N., 335 Hata, A., 59 Haywood, S., 723 Hazegh-Azam, M., 827 Heath, A-L, 323 Hediger, M. A., 795 Heins, U., 379, 821 Heisterkamp, M., 1107 Hendrickson, K., 995 Henriques, G. S., 65, 329 Henry, P. R., 283 Hercberg, S., 437, 450, 453, 467, 484, 488, 799 Herman, D., 1053 Hess, K., 1061, 1088 Heumann, S., 239 Higashiguchi, K., 671 Hilario, M. O. E., 638, 639 Hill, A. D., 519 Hill, K. E., 837, 869 Hincal, F., 144, 469, 471 Hininger, I., 377 Hirunuma, R., 1099

Hodjberg-Bugel, S., 937 Holiday, B., 519 Holmes, J., 407 Holzinger, S., 209, 221 Horwath, B. J., 1085 Hosokawa, T., 59, 105, 429, 933 Howse, A. M., 737 Hunt, C. D., 1071 Hurrell, R., 587 Ibragimova, G. A., 119 Illing-Gunther, H., 221 Illingworth, D. V., 733, 741, 749, 782 Indumadhavi, M., 305 Jacimovi, R., 178 Jackson, D. W., 733, 741, 749, 782 Jackson, J., 481 Jahritz, M., 209 Jang, H. S., 559 Janghorbani, M., 175 Jaritz, M., 221, 229 Jaudon, M. C., 629 Jeejeebhoy, K. N., 503 Jiranek, J., 417 Jiranek, V., 473 Johansson, M., 169 Johnson, A. B., 530 Jones, L., 955 Jorgensen, B. A., 820 Jourdan, E., 77, 129 Jovanovic, I. B., 551 Judson, G. J., 737 Jumba, I. O., 786 Jurasovic, J., 341, 675 Juska, C., 827 Kaats, G. R., 503 Kabir, S., 645 Kakade, V., 305 Kamiyama, M., 330 Karlowski, K., 252 Kaup, S. M., 481 Keen, C. L., 186, 929, 1083, 1085 Kegley, E. B., 530 Kehoe, C., 475, 943 Keklik, A., 1080 Kemp, F. W., 597, 407 Kendall, N. R., 733, 741, 749, 769, 782, 784 Kerkeni, A., 534 Keyes, W. R., 951, 1115 Khatir, F., 377 Khatun, A., 906 Kido, T., 671 Kimber, M., 995 King, J.C., 189, 999, King, L. J., 611

1152 Kirchgessner, M., 173, 299, 555, 565, 566, 883, 991, 1021 Klevay, L. M., 339 Klos, A., 231, 244 Klotz, L.-O., 71 Knowles, S. O., 717, 768 Knudtson, M., 563 Koebnick, C., 379, 821 Kohrle, J., 899 Kono, K., 335 Korkina, L., 115, 119 Koropatnick, J., 47 Kostial, K., 809 Koudrine, A. V., 568, 640 Kovar, I. Z., 1031 Kovatsi, L., 177 Krachler, M., 365, 667 Kralj, M., 123 Krebs, N., 347, 977 Krejpcio, Z., 1 1 1 , 636, 697 Kretsch, M. J., 999 Krol, A., 849, 903 Krosniak, M., 607 Kroupova, V., 477 Kulasek, W., 309 Kurasaki, M., 59, 105, 429, 933 Kvasnicka, J., 385 Kvicala, J., 417, 473, 477 Kwa, P. F., 829 Kwon, C. S., 559 Kwun, I. S., 559 Kyriakopoulos, A., 29 L’Abbe, M. R., 215, 313 Lachili, B., 536, 641 Lacomblez, L., 629 Lagarda, M. J., 306 Laires, M. J., 333 Lalane, K., 140, 497 Lamberts, L., 1 1 1 1 Langford, N., 279 Langini, S. H., 375 Lanoue, L., 1083 Lansdown, A., 1038 Laporte, F., 443, 449, 897 Largarda, M. J., 381 Larsen, T., 763 Laskey, J. W., 809 Lassal, H., 917 Lassance, Ae.C., 391 Lastra, M. D., 1039, 1040 Latunde-Dada, G. O., 209 Laurenti, F., 332 Lavedrine, F., 232 Le Francois, P., 233 Lea, R. G., 383 Lebedev, E. A., 813 Leccia, M. T., 77, 421 Lecka, J., 574

Author Index Leconte, M., 528 Lee, J., 15, 717, 768 Lefevre, B., 701 Leitzmann, C., 379, 821 Len, J., 511 Leone, A., 55 Leontowicz, H., 309 Leontowicz, M., 309 Lerose, R., 55 Lescure, A., 849, 903 Levrat-Verny, M. A., 287 Lewis, C.G., 543 Lewis, J., 279, 877 Li, D., 429 Li, W., 407 Lincoln, S., 995 Linder, M. C., 184, 827, 955 Lindgarde, F., 865 Lindmark Mansson, H., 893 Lindmark Mansson, J., 892 Linke, K., 633 Litherland, A., 717 Littell, R. C., 283 Liu, N., 955 Llobet, S., 333 Llopis, J., 340 Lloyd, J., 597 Lo, L., 955 Lodh, D., 645 Lofthouse, S., 705 Lonnerdal, B., 353 Lopes, P. A., 479 Lopez, G., 227, 235 Lopez, H. W., 287 Lopez, L., 375 Lopez-Sobaler, A., 446 Lorentzen, M., 981 Losch, E., 229 Loughrey, C. M., 631 Loui, A., 390 Louria, D. B., 597 Lowe, L. B., 775 Lubyanova, I. P., 336 Luo, D., 1049, 1077 Maage, A., 141 MacDonald, A., 829 Machelon, V., 701 MacKenzie, A. M., 749, 782, 784 MacPherson, A., 248, 569 Maes, D., 437 Mafra, D., 625, 626 Mahadevan, I., 995 Majhoub, S., 534 Majsak-Newman, G., 937, 959 Malecki, E. A., 601 Malpuech-Brugere, C., 139 Malvy, D., 437, 450, 484, 488 Mandal, B. K., 645

Manitius, J., 643 Mara, M., 385 Marangunich, L., 775 Marova, E., 385 Marques Vidal, P., 333, 463 Marreiro, D. N., 331 Martin, A., 247 Martinez, C., 227, 235, 314 Martinez, I., 237 Martinez, L. D., 181, 183 Martinez-Costa, C., 381 Mathieu, J., 1003, 1035 Matic, G., 551 Matsuda, I., 59 Matsumoto, M., 511 Maxwell, H., 741 Mayap-Nzietchueng, R., 1061, 1088 Mazej, D., 895 Mazur, A., 97, 139, 142, 475, 571, 707, 937, 943, 957 McArdle, H. J., 383, 947 McCord, J. M., 67 McElhome, A., 293 McEneny, J., 131 McGregor, V. A., 737 McJelvey-Martin, V. J., 943 McKeown, A., 943 McLaughlin, K. E., 323 McMaster, D., 131, 433, 463, 631 McMullen, S., 769 McNeill, J. H., 491, 539 Meininger, V., 629 Meningaud, J. P., 709 Mercer, C., 131, 433, 631, 829 Meyer, E., 655 Meyer, J. A., 753 Micetic-Turk, D., 365 Middlemas, C., 741 Mielcarz, G., 573, 633 Mihailovic, M. B., 551 Miles, R. D., 283 Millat, F., 571 Miller, P. F., 705 Milne, D. B., 1025 Minami, T., 663 Mitchell, A. E., 186, 929 Miura, K., 671 Mizukoshi, K., 671 Momcilovic, B., 699, 1017 Monteiro, C. P., 333 Moreno, O., 375 Morikawa, Y., 671 Mosher, T. J., 601 Most, E., 901 Mostert, V., 899 Moundras, C., 571 Muijs, T., 441 Mukherjee, S. C., 645

Author Index Muller, A., 901 Muller, M., 209 Muller, R., 209 Muller, T., 723 Muller, W., 723 Murphy, A., 433 Murphy, S., 267 Murray, F. J., 1057, 1079, 1083, 1085 Mykhaylyk, O. M., 336, 813 Nabet, P., 1088 Nagano, K., 59 Najjar, F., 534 Nakagawa, H., 671 Nakagawa, N., 429 Nakamura, N., 429 Nartowicz, E., 574, 643 Navarro, I., 247 Navarro, M. P., 825 Ndigui, P., 271 Neve, J., 127, 317, 479, 859, 1009 Nguyen, A., 827 Nguyen, F., 827 Nicole, A., 917 Nielsen, F. H., 1043 Nishi, M., 671 Nishijo, M., 671 Nishiura, H., 335 Nogawa, K., 671 Nowacki, W., 139 Nowak, G., 607 Nyquist, L., 1053 O’Connor, D. L., 323 O’Connor, J. M., 475, 937, 943 O’Hare, M., 131 Oberleas, D., 823 Obladen, M., 390 Oduor, F. D. O., 786 Oernsrud, R., 891 Ogawa, K., 429 Ohly, P., 515 Ojeda, M. S., 1036 Okabe, M., 59, 105, 429, 933 Okazaki, Y., 663 Oktekin, R., 627 Oktem, A., 469 Olejnik, D., 697 Olesik, J., 175 Olivares, A. B., 314 Oliveira, G. L., 690 Oliveros, L. B., 181, 183 Onning, G., 161 Orlova, T. A., 336 Ortega, R. M., 446 Ortega Soler, C. R., 375 Orvig, C., 539

1153 Osman, M., 137 Ostrachovich, A., 119 Ostrachovich, E., 115 Othmane, A., 534 Pagliuca, M. G., 55 Palkar, S., 305 Pallauf, J., 901 Papadopoulos, T., 1009 Parat, M. O., 77 Parnell, W., 323 Pastelin, R., 1039, 1040 Pasti, F., 873 Patterson, K., 519 Pavao, M. L., 479, 859 Pavlovic, M., 341 Pedrosa, L. F. C., 329, 511 Pellerin, P., 703 Pelletier, B., 486 Penland, J. G., 981, 999, 1025 Pepin, D., 707 Peretz, A., 1009 Perez Calvo, M., 1117 Perez Gallardo, L., 338 Perez, S., 1039 Perez-Cristia, R., 240 Perez-Granados, A. M., 825 Periago, M. J., 235, 237 Persson-Moschos, L., 250, 865 Pesty, A., 701 Pesut, O., 551 Petrucci, F., 332, 667 Pfannhauser, W., 239 Pfeifer, H., 29 Philcox, J. C., 43 Phillips, C. J. C., 772 Piasek, M., 809 Pietrzak, Zb., 369 Piette, J., 89 Pilecki, A., 482, 643 Pineda, O., 819 Pinto, A. F., 391 Piotrowicz, J., 242 Pizent, A., 341, 675 Planells, E., 340 Poljak-Blazi, M., 123, 135 Popovic-Hadzija, M., 123 Porr, P. J., 617 Poupon, J., 701, 709, 1122 Pramuk, K., 481 Preziosi, P., 450, 467, 484, 486, 488 Price, F., 519 Prohaska, J. R., 909 Prosser, N., 323 Psaroulis, D., 177 Pyasetska, N. M., 336 Quamruzzaman, Q., 645

Raab, A., 145, 390 Radzanowski, G. M., 481 Rainey, C., 1053 Ralston, S. L., 530 Ramaekers, V., 634 Ramon, O., 497 Rapic, V., 135 Raub, R., 530 Ravel, A., 232 Rayman, M. P., 575, 611 Rayssiguier, Y., 97, 139, 142, 287, 311, 443, 475, 571, 703, 707, 937, 957, 943 Rea, I. M., 433 Redman, C. W. G., 611 Reid, C., 131 Reid, M. D., 937 Remesy, C., 287 Requejo, A. M., 446 Rezaee, L., 827 Ribas, B., 540 Ribas Ozonas, B., 1117 Ribuot, C., 536 Richard, M. J., 77, 129, 133, 138, 143, 421, 449, 450, 484, 536, 538, 641, 904, 1035 Richard, S., 1003 Richarz, A. N., 145 Richmond, P., 1031 Rim, H., 184 Riondel, J., 448 Robb, P., 705 Robberecht, H., 275, 345 Rock, E., 139, 142, 475, 937, 957, Rock, Y., 943 Rodriguez, A. R., 169, 1103, 1116 Rodway, R. G., 769 Rofe, A. M., 43 Rogers, R. L., 1057 Rohrig, B., 209 Rolfs, A., 795 Ros, G., 227, 235, 314 Rosado, J. L., 267 Rosick, U., 180 Ross, R., 519 Ross, W. H., 215, 313 Rossaro, L., 601 Rossini, E., 138, 538 Rossipal, E., 365 Roth, H. P., 991, 1021 Rother, C., 209 Rothlein, D., 29 Rotilio, G., 937 Rounce, J. R., 717, 768 Roura, M., 181 Roussel, A. M., 377, 409, 442, 443, 449, 450, 467, 484, 488, 497, 534, 641, 897 Rovesti, S., 581

1154 Rowe, A., 1038 Rowe, R., 1087 Roxborough, H. E., 131, 631 Roy, S., 645 Rucker, B., 929 Rucker, R. B., 186, 929 Ruksan, B. E., 775 Rydzynski, K., 127, 369 Saari, J. T., 523 Sabatier, M., 1115 Saha, K. C., 645 Saillant, G., 1122 Saito, S., 51, 105, 933 Saito, T., 59, 105, 429, 933 Sakita, Y., 663 Salachas, F., 629 Salen, P., 547 Samanta, G., 645 Sambuy, Y., 63, 937 Sampson, B., 1031, 1038 Sanchez-Mayoral, A., 338 Sanchez-Morito, N., 340 Sandoval, M., 283 Sandstead, H. H., 981 Sandstrom, B., 37 Sandstrom, S., 820 Santaella y Gaspar Ros, M., 237 Santos, A., 540 Santos, M. C., 479, 859 Sardinha, L., 333 Sargent, C., 705 Sasaki, H., 429 Satoh, M., 444 Sayli, B. S., 1080 Scarino, M. L., 63, 937 Schaafsma, G., 441 Schaffer, K., 711, 827 Schafer, U., 221 Schaller, U., 239 Schaub, J., 987 Schieke, S. M., 71 Schimmelpfennig, W., 655 Schlegel-Zawadzka, M., 607 Schleyerbach, U., 987 Schmidmayer, S., 991 Schramel, P., 1091 Schroeder, G. E., 767 Schutz, A., 250 Schwartzman, F., 5 1 1 Schwarz, F. J., 299 Sciutto, E., 1040 Sebastia, V., 306 Seifert, M., 209, 229 Sepulveda, D., 407 Sequeira, F., 855 Serfass, R. E., 789 Serougne, C., 571 Seto, T., 671

Author Index Seve, M., 536, 1003, 1035 Sheehan, T. M. T., 906 Shenkin, A., 201 Shimada, A., 444 Shimahara, M., 335 Shimizu, H., 105 Shiobara, Y., 157 Shrimpton, D. H., 228 Shuler, T. R., 339 Shulman, S., 293 Shurson, G., 293 Shurunov, B. S., 813 Siderova, V., 1009 Sies, H., 71 Sievers, E., 987 Silio, F., 540 Silvestre, M. D., 381 Sima, A., 239 Simmer, K., 388 Simmer, S., 359 Simmons, B., 829 Skalny, A. V., 568, 640 Skare, D., 135 Skeaff, C. M., 323 Skinner, N. D., 779, 787 Skurnick, J. H., 597 Sobajic, S., 551 Soch, N., 1077, 1087 Socha, M. T., 530 Sokari, S., 611 Sorenson, A., 519 Soria, R., 399 Soulie, C., 917 Souza, J. R., 690 Spears, J . W . , 729, 759, 1067 Spielvogel, H., 399 Spinola-Castro, A., 511 Srikumar, S., 855 Stadtman, T. C., 831 Stangl, G. I., 299 Starska, K., 252 Stavenow, L., 865 Stibilj, V., 178, 895 Stites, T., 929 Stover, E. L., 1057, 1079 Strain, J. J., 475, 923, 937, 943 Strojan, S. T., 772 Strong, P., 1085 Strong, P. L., 1057, 1079, 1083 Struys-Ponsar, C., 425 Sunde, R. A., 21 Surai, P., 248 Surian, M., 591 Sutherland, B., 999 Suzuki, K. T., 157 Suzuki-Kurasaki, M., 933 Swenson, C. K., 530 Szram, K., 369 Szymaczek, M., 607

Tabata, M., 655, 671 Tahiri, M., 311, 703 Takahashi, K., 429 Takeishi, H., 335 Tarwadi, K. V., 261, 305 Tchaparian, E. H., 186, 929 Telfer, S. B., 733, 741, 749, 782, 784 Telisman, S., 341, 675 Tellez, W., 399 Testart, J., 701 Tezic, T., 469 Thakkar, J., 855 Thiele, D. J., 15 Thompson, K. H., 539 Thomson, C. D., 343, 869 Thoulon, J., 377 Tian, X., 1107 Tiber, D., 407 Toffoletto, F., 591 Tohyama, C., 444 Tomczak, J., 369 Torboli, A., 873 Torresani, T., 587 Trafikowska, A., 643 Trafikowska, U., 643 Traktenberg, S., 242 Tran, T., 955 Tressol, J. C., 311, 703 Trupschuch, A., 685 Tsougas, M., 177 Tsoukali, H., 177 Turley, E., 943 Turnlund, J. R., 951, 1115 Tusek Znidaric, M., 895 Ukleja-Adamowicz, M., 574 Umlaufova, A., 385 Urakami, K., 59 Uria-Adams, J. Y., 1085 Uriu-Hare, J. Y., 186, 929 Usuda, K., 335 Uza, G., 617 Vaillant, L. Valdes, M., 873 Valeix, P., 486 Valera, P., 446 van Caillie-Bertrand, M., 275, 634 van Cauwenbergh, R., 345 van den Bosch de Aguilar, P., 425 van den Heuvel, E. G. H. M., 441 van Doesum, Y., 311 van Dokkum, W., 441 van Dyck, K., 275, 345 van Hoorebeke, C., 1 1 1 1 van Ryssen, J. B. J., 767 vanden Berghe, D., 1111

Author Index vanden Berghe, V., 634 Vaquero, M. P., 825 Vargas, E., 399 Vasiljevic, Z., 551 Veillon, C., 519 Verdura, T., 240 Vesterberg, O., 688 Viegas-Crespo, A. M., 479, 859 Villena, M., 399 Villet, A., 232 Vingerhoets, R., 1 1 1 1 Virgona, N., 330 Virtanen, V., 1103 Vivoli, G., 581 Vlad, M., 617 Vormann, J., 209 Vu, K., 827 Walczak, R., 849 Waldhauser, K., 565 Wandiga, S. O., 786 Wang, C., 407 Ward, D., 995 Wasowicz, W., 127, 369 Watanabe, T., 335 Wattam, S. L., 779

1155 Wedekind, K. J., 781 Weisstaub, A., 375 Wendelaar Bonga, S. E., 141 West, D. M., 717 Whanger, P., 175 Wiernsperger, N., 140 Wild, S., 555 Wilplinger, M., 239 Windisch, W., 173, 883 Woimant, F., 917 Wojciak, R., 111 Wojciak, R. W., 636, 697 Wojciechowska-Mazurek, M., 252 Wolf, C., 180 Wolkanin, P., 369 Wong, H. K., 448 Woodruffe, S., 343 Wright, C. L., 729, 759, 1067 Wunderlich, S. M., 387 Wyse, G., 563

Xia, Y., 175 Yamamoto, Y., 429 Yamanoshita, O., 933 Yano, T., 330

Yoshida, K., 51 Young, I. S., 131, 631 Ysart, G.E., 705 Zachara, B. A., 482, 574, 643 Zachwieja, Z., 242, 693 Zadrozna, M., 373 Zagrodzki, P., 373 Zalewski, P., 995 Zalups, R. K., 47 Zamany, M., 827 Zamorska, L., 373 Zamrazil, V., 417, 473 Zarebska, M., 486, 488 Zeder, C., 587 Zemser, M., 242 Zhavoronkov, A. A., 568 Zhonnie, S., 519 Zieba, A., 607 Zietek, K., 633 Zimmermann, M., 587 Zirbo, M., 617 Zivny, J., 385 Zochling, S., 239 Zouari, N., 534 Zozulya, Y. A., 813

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SUBJECT INDEX

Acid phenol compounds aluminum, 707 distribution, 707 ACTH, selenium, 855 Adolescent haptoglobin, 333 lipid, 333 magnesium, 333 obese, 331 zinc status, 331 Adriamycin asbestos, 119 cancer, 133 oncogene, 119 oxidative stress, 119 selenium, 133 Aging antioxidant intake, 446 copper, 421 deficit, 409 enzyme, 448 glutamate, 429 glutathione peroxidase, 421 metals, 339, 421 review, 409 selenium, 421, 450 skin, 421 status, 339, 446 superoxide dismutase, 421 supplementation, 409, 450 Werner Syndrome, 421 zinc, 429 ALS oxidative stress, 629 blood markers, 629 Altitude, oxidative stress, review, 399 Aluminum brain, 425 distribution, 707 glutamate, 425 MnSOD expression, 687 NFκB, 137 toxicity, 425

Alzheimer aluminum, 687 MnSOD expression, 687 Amino acid chelate anemia, 819 bioavailability, 819 iron, 819 Analysis AAS, 169 capillary electrophoresis, 1117 electro analytical method, 1116 electrothermal atomic Absorption spectrometry, 1122 food, 169, 252 heavy metals, 1120 HPLC-ICP-MS, 1119 ICP MS, 563, 1107, 1121 metallothionein, 1103 methods, 1099 new methods, 1091 quality control, 252 review, 145 speciation, 145, 1107 Anemia amino acid chelate, 819 iron, 385, 819 pregnancy, 375, 385 Antidepressant therapy, relation with status, 607 Antioxidant myocardial infarction, 551 photoaging, 437 skin, 437 supplementation, 484 aging, 409, 446 Aortic wall, metals, 568 Apoptosis cadmiun, 141 cellular regulation review, 969 complex, 123 HeLa cells, 1003, 1035 iron, 123 zinc, 969, 1003, 1035

1157

1158 Arsenic environment, 645 ICP/MS, 165 speciation, 165 water, 645 Asbestos adriamycin, 119 oncogene, 119 oxidative stress, 119 Ascorbate bioavailability, 267 iron, 267 ASSE interaction, 178 liver, 178 Atherosclerosis, 97 ATPases copper transport, 9, 59 human disease, review, 9 vanadium, 186 zinc, review, 1

Baby foods, bioavailability, 314 Balance infant, 390, 987 metals, 390 prematurity, 390 ultratrace, 209 zinc, 987 Betaine homocysteine S methyltransferase liver, 1013 zinc, 1013 Bioavailability baby foods, 314 beans, 306 calcium, 441 carbohydrate, 311 diet, 255, 267, 287, 305 flour, 287 iron, 306, 311, 314, 315, 379, 819, metals, 763 pregnancy, 379 selenium, 173, 279, 883, 891 silicon, 1111 vegetarian diet, 261 zinc, 43, 261, 275, 283, 287, 293, 311, 329 Biomarker ALS, 629 human, 175 newborn, 359 Biomedical application, ICP-MS, 1121 Body composition, chromium, review, 503 Bolus, copper, cobalt, selenium, sheep, 749 Bone boron, 1067 zinc supplementation, 1009 Boron bone, 1067

Subject Index Boron (cont.) deficiency, 1079, 1082, 1083, 1085 environment, 1080 essentiality, 1057 fertility, 1080 frog, 1057 gene expression, 1078 inflammation, 1071 intake, 1053 molecular biology, 1049, 1077 nude mouse, 1061 nutriture, 1043 pig, 1067 protein transcription, 1088 rat, 1085 turkish, 1080 wound healing, 1061 xenopus, 1079 yeast growth, 1087 zebrafish, 1082 Brain aluminum, 425 cadmium, 105 mercury, 105 metallothionein, 444 zinc nutriture, review, 981 permeability, 663 Brazil nut glutathione peroxidase, 405 selenium, 405 Breast milk composition, 481 infant, 482 selenium, 482

Cadmium apoptosis, 141 brain, 105 copper, 141, 145, 671 environment, 671 intake, 809 Japan, 671 metallothionein, 51 mortality, 671 oxidative stress, 141 phospholipid, 691 salmon, 141 speciation, 145 urinary copper, 671 zinc, 145 Calcium bioavailability, 275, 441, 763 environment, 569 heart disease, 569 interaction, 763 pig, 763 postmenopausal women, 441 zinc, 275

Subject Index Calmodulin regulated enzyme zinc deficiency, 991 rat, 991 Calprotectin infant, 1031 zinc, 1031 Cancer adriamycin, 133 complex, 135 gallium nitrate, 640 hemochromatosis, 67 iron, 135 lymphocytes, 640 middle-aged, 865 selenium, 133, 575, 623, 865 selenoprotein P, 865 zinc, 640 Capillary electrophoresis, metallothionein isoforms, 1103, 1117 Carbamylation gluthatione peroxidase, 131 superoxide dismutase, 131 Carbohydrate bioavailability, 311 iron, 311 zinc, 311 Cardiovascular disease copper, 543, 573 iron overload, review, 543 Cat and dog, ceruloplasmin, 779 Cattle chromium, 530 cobalt, 299 copper, 759 deficiency, 299 glucose metabolism, 530 growth, 759 lactating cattle, 786 water, 759 Cellular regulation apoptosis, 969 zinc, review, 969 Cellular zinc, zinquin, 995 Cerebrovascular disease, oxidative stress, 559 Ceruloplasmin, cat and dog, 779 Ceruloplasmin oxidase, pregnancy, 383 Chromium body composition, 503 elderly, 409 environment, 688 exercise, review, 393 glucose intolerance, 503 glucose metabolism, 530 manganese, 201 metabolism, 393 molybdenum, 201 parenteral nutrition, 503 review, 393, 503 sensitization indicators, 688

1159 Chromium (cont.) status, 591 Chronic heart failure, selenium, 547 Chronic liver disease iron, 601 manganese, 601 pallidi, 601 Chronic renal failure nutriture, 626 selenium, 625 zinc, 625, 626 Ciprofloxacin desferroxamine, 144 iron, 144 oxidative stress, 144 Cirrhosis copper, 180, 723 metallothionein, 180 sheep, 723 Cobalt analysis, 1122 cattle, 299 deficiency, 299 diabetes, 540 electrothermal atomic absorption spectrometry, 1122 environment, 688 GTF, 540 homocysteine, 299 oxidative stress, 299 sensitization indicators, 688 Cognitive function low zinc diet, 999 selenium, 442 zinc, 429, 999 Colorectal polyps copper, 633 zinc, 633 Complex apoptosis, 123 cancer, 135 copper, 119 flavonoid, 119 gene, 123 iron, 119, 123, 135 Copper absorption, 717 aging, 409, 421 ATPases, 9, 59 bioavailability, 287 cadmium, 141, 671 cardiovascular disease, 543 cattle, 759 ceruloplasmin oxidase, 383 cirrhosis, 180 complex, 119 copper oxidase, 947 deficiency, 142, 523, 543, 571, 573, 733, 924, 1079 diamine oxidase, 957

1160

Subject Index

Copper (cont.) elderly, 409 excess, 679 FOODCUE study, 475, 937, 943 flour, 287 ginseng, 338 grazing red deer, 733 intake, 639, 655, 679, 729, 959 interaction, 63, 142, 184, 186 iron, 63, 119, 543 lactaction, 381 LDL, 97 lipoperoxidation, 617, 695 lysyl oxidase, 186 lysyl oxidase activation, 924 metabolism, 15, 63, 933 metallothionein, 51, 105, 180 neurochemical alteration, 909 NO synthase, 105 oxidative stress, 141, 142, 617, 695 polyphenol, 142 pregnancy, 353, 383 rat embryo, 59 reviews, 409, 543, 909, 917 rodent, 933 ruminant, 768 salmon, 141 selenium, 201, 341 sheep, 723 silver, 184 sodium vanadate, 924 speciation, 145 stable isotope, 717, 768 status, 338, 627, 633, 638, 951 steers, 729 superoxide dismutase, 105, 421 supplementation, 409, 475 toxicosis, 723 transport, 9, 15, 59, 184 transport protein, 955 vanadium, 186 water, 759 Werner Syndrome, 421 Wilson Disease, 917 xenopus, 1079 zinc, 105, 145, 201, 338 Cow excretion, 767 protein source, 767 selenium, 477, 767 C-peptide, zinc deficiency, 1021 CTR, 15

Diabetes (cont.) GTF, 540 insulin, 138 insulin sensitivity, 497, 538 mineral water, 519 mouse, 515 navajo, 519 oxidative stress, 534 reviews, 491, 497 selenium, 527 stable isotope, 527 Tunisian, 534 vanadium, review, 491 vanadium compounds, 539 zinc, 140, 497, 511, 515, 527, 528, 532, 538 Diet ascorbate, 267 Austrian, 239 bioavailability, 255, 267, 287, 305 Brazil nut, 405 Cameroon, 271 copper, 287 Cuba, 240 flour, 287 glutathione peroxidase, 405 iron, 267, 271, 287 Mexico, 267 Poland, 244 primrose, 309 reviews, 221, 255 selenium, 405, 877 strontium, 229 vanadium, 221 vegetarian, 305 walnut, 232 Dietary exposure, platinum, 705 Dietary fats lipoprotein, 555 rat, 555 zinc, 555 Diplegic infant, hair, 636 Disulfiram, magnesium, 175 DNA oxidative stress, 129 selenium, 143 thioredoxin reductase, 143, 77 UV, 129, 77 zinc, 129 Dog ferritin, 787 requirement, 781 selenium, 781

Desferroxamine ciprofloxacin, 144 iron, 144 oxidative stress, 144 Diabetes endothelial cell, 140

ECC, copper intakes, 655 Egg, enrichment, 248 Elderly chromium, 409 cognitive function, 442 copper, 409

Subject Index Elderly (cont.) deficiencies, 409 glutathione peroxidase, 433 Ireland, 433 iron, 409 Poland, 244 selenium, 409, 417, 433,442 supplementation, 409, 417 zinc, 409 Electroanalytical method, metallothionein, 1116 Electrothermal atomic absorption spectrometry, cobalt, 1122 Embryo, copper, ATPases, 59 Enrichment food, 195, 215, 227, 235, 248, 313 iron, 227, 235 policy, 313 safety, 215 selenium, 248 weaning, 235 Enteral diets, bioavailability, 315 Environment arsenic, 645 Bangladesh, 645 boron, 1080 cadmium, 671 calcium, 569 chromium, 688 cobalt, 688 copper excess, 679 fertility, 1080 heavy metals, 651 infant, 667 Japan, 671 lead, 675 metals, 651, 688 new born, 651 nickel, 688 PGMs, 667 rat, 679 Turkish, 1080 Enzyme aging, 409, 448 redox centers, 83 EPO hemodialysis, 643 oxidative stress, 643 ESR iron, 336 newborn, 336 Exercise chromium, 393 food restriction, 407 iron, 393 lead, 407 metabolism, review, 393 zinc, 393 Exposure lead, 697, 703

1161 Exposure (cont.) metals supplementation, 697 RG-II, 703 Fat intake, iron metabolism, 825 Ferric ion, phytate, 823 Ferritin dog, 787 immunity, 827 inflammation, 827 iron, 787, 827 Ferrous ion, phytate, 823 Fertility boron, 1080 environment, 1080 lead, 701 mouse, 701 toxicity, 701 Turkish, 1080 Fetus glutathione peroxidase, 369 lactation, 365 metals, 365 poland, 369 pregnancy, 365, 369 superoxide dismutase, 369 Fish ICP/MS, 161 selenium, 161, 250 selenoprotein, 250 spain, 237 speciation, 161 thyroid, 250 Flavonoid, iron, 119 Flour, copper bioavailability, 287 Fluoride sheep, 753 water, 753 Food AAS, 169 analysis, 169, 252 Austrian, 239 balance, 209 copper requirement, 937 copper supplementation, 943 dietary exposure, 705 food enrichment, 227, 248, 313 enrichment, review, 195, 215 fish, 237 glutathione peroxidase, 892 goiter, 471 iron, 227, 235 metals, 252 persimmons, 242 platinum, 705 policy, 313 safety, 215 selenium, 169, 248, 279, 471 Spain, 237

1162 Food (cont.) speciation, 169 Turkey, 471 ultratrace, 209 weaning, 235 zinc, 245 Food restriction exercise, 407 lead, 407 Foodcue study copper, 475 food, 937, 943 supplementation, 475 Formula bioavailability, calcium, zinc, 275 infant, 247 Fortification, iron fortification, review, 799 Frog, boron, 1057 Fructose diet diabetes, 538 insulin sensitivity, 538 oxidative stress, 536 rat, 536 zinc, 538 Gallium nitrate cancer, 640 lymphocytes, 640 zinc, 640 Gene apoptosis, 123 boron, 1078 complex, 123 glutathione peroxidase, 21 HFE, 67 iron, 123 metallothionein, 55 regulation selenoprotein, 21 ZnT, 35 ZRT, 35 Ginseng, status, 338 Glial brain tumour iron, 813 non heme exchange, 813 Glucose intolerance, chromium, review, 503 Glucose metabolism, cattle, chromium, 530 Glutathione peroxidase aging, 421 carbamylation, 131 diet, 405, 892 gene, 21 HIV, 904 Ireland, 433 Poland, 369 pregnancy, 369 purification, 893 regulation, 21 selenium, 21, 359. 405, 421, 433, 892, 904 selenoprotein, 21

Subject Index Glutathione peroxidase (cont.) selenoprotein P, 71 stability, 892 TAT protein, 904 whey, 892 Goiter Côte d’Ivoire, 587 iodine, 471, 587 iron deficiency, 587 selenium, 469, 471 Turkey, 469, 471 water, 471 Gonad, zinc deficiency, 330 Grazing red deer copper deficiency, 733 supplementation, 733 Grazing sheep copper absorption, 717 molybdenum intake, 717 stable isotope, 717 Growth cattle, 759 copper, 759 infant, 829 iron, 829 supplementation, 829 water, 759 zinc, review, 977 GTF cobalt, 540 diabetes, 540

Hair aging, 339 Amazonia, 690 diplegic infant, 636 mercury, 690 metals, 339, 568, 636 speciation, 690 Haptoglobin adolescents, 333 lipid, 333 magnesium, 333 Heavy metals analysis, 1120 environment, 651 ICP-MS, 1120 newborn, 651 smoking, 651 transcription factor, 55 Heifer “enteque seco”, 775 deficiency, 775 selenium, 775 supplementation, 775 HeLa cells apoptosis, 1003, 1035 zinc chelation, 1003, 1035

Subject Index Hemochromatosis cancer, 67 HFE, 67 Hemodialysis chromium, 591 EPO, 643 oxidative stress, 643 selenium supplementation, 631, 643 therapy, 643 Hens distribution, 895 selenium, 178, 711, 895 tissular, trans fatty acids, 711 Hepatotoxicity, copper, 679 HFE cancer, review, 67 gene, 67 hemochromatosis, 67 HIV glutathione peroxidase, 904 oxidative stress, 904 selenium, 904 TAT protein, 904 Homocysteine cobalt, 299 oxidative stress, 299 Hypertension, metals, review, 581 ICP/MS analysis, 563 arsenic, 165 biomedical application, 1121 coronary artery disease, 563 fish, 161 heavy metals, 1120 lithium, 335 magnesium, 1115 metals, 1107 rat, 157 selenium, 157, 161 speciation, 157, 161, 165, 1107 stable isotope, 1115 urine, 335 Idiorhythmic dose rate variability, zinc, 1017 Igfl, zinc deficiency, 1021 IL-1 gene expression immunity, 1039 macrophage, 1039 zinc, 1039 Immunity deficiency, 139, 597, ferritin, 827 human, 47 IL-1 gene expression, 1039 inflammation, 827 iron, 827 macrophage, 1039 magnesium, 139 parasite infection, 1040

1163 Immunity (cont.) zinc, 47, 1039, 1040 Immunoassay glutathione peroxidase, 893 purification, 893 Incisional wounds, metals binding protein, 1038 Infant balance, 390, 987 Brazil, 391 breast milk, 482 calprotectin, 1031 diabetes, 511 diet, 247 environment, 667 formula, 247 growth, 829 iron, 391, 789 iron nutrition, 829 lead, 693 metabolism, review, 789 PGMs, 667 prematurity, 388, 390 selenium, 388, 482 status, 391, 693 supplementation, 388, 829 zinc, 511, 987, 1031 Inflammation acute phase response, 906 boron, review, 1071 ferritin, 827 immunity, 827 iron, 827 selenium, 906 Insulin sensitivity diabetes, 497, 538 fructose diet, 538 zinc, review, 497, 538 Intakes Austrian, 239 boron, 1053 cadmium, 809 Cameroon, 271 chronic renal failure, 625 copper, 639, 655, 679, 729, 595, 951 Cuba, 240 German, 655 hepatotoxicity, 679 infant, 247 iron, 271, 809 JRA, 639 military, 233 molybdenum, 951 oxidative stress, 859 RDA, review, 189 safety, 228 selenium. 625, 859, 877 steers, 729 steroidogenesis, 809 zinc, 625

1164 Interaction ASSE, 178 copper, 63, 142, 184, 186 lysyl oxidase, 186 magnesium, 340 metals, 685 oxidative stress, 77 silver, 184 supplementation, 685 thioredoxin reductase, 77 transport, 184 zinc, 77, 308 Iodine Côte d'Ivoire, 587 goiter, 471, 587 iron deficiency, 587 New Zealand, 343 nutriture, 486 thyroid, 343 Turkey, 471 water, 471 Iron amino acid chelate, 819 anemia, 385, 819 apoptosis, 123 ascorbate, 267 baby foods, 314 beans, 306 bioavailability, 261, 267, 287, 306, 311, 314, 315, 379, 819 Brazil, 391 Cameroon, 271 carbohydrate, 311 cardiovascular disease, 543 ciprofloxacin, 144 complex, 119, 123, 135 copper, 63, 543 deficiency, 587 desferroxamine, 144 dog, 787 elderly, 409 enrichment, 227, 235 enteral diets, 315 enzyme redox centers, 83 ESR, 336 exercise, 393 ferric ion, 823 ferritin, 787, 827 ferrous ion, 823 flavonoid complex, 119 fortification, 799 gene, 123 growth, 829 immunity, 827 infant, 391, 789, 829 intakes, 267, 271, 287, 809 interaction, 63, 308 lactation, 353 LDL, 97

Subject Index Iron (cont.) metabolism, 393, 789, 825 Mexico, 267 newborn, 336 NFkB, 89 nonheme exchange, 813 overload, 543 oxidative stress, 144 pallidi, 601 pregnancy, 353, 379, 385, 387 RDA, 387 reviews, 409, 789, 795 status, 336, 391, 601, 627, 820 transport, 67, 795 vegetarian diet, 261 weaning, 235 women, 323, 820 zinc, 261, 306, 308, 314, 323 Isotope, selenium, 175

JRA copper, 638, 639 intake, 639 status, 638 Lactation copper, 353, 381 fetus, 365 infant, 482 iron, 353 manganese, 353 metals, 365 pregnancy, 353, 365 zinc, 353 Lamb, zinc, cobalt, selenium bolus, 784 LDL postmenopausal women, 443 zinc deficiency, 566 Lead environment, 675 exercise, 407 exposure, 697, 703 food restriction, 407 oxidative stress, 111 rumen bacteria, 772 selenium status, 675 status, 693 thiamine, 1 1 1 toxicity, 701 Lipid copper, 695, 729 magnesium, 333 oxidative stress, 617, 695 steers, 729 adolescents, 333 zinc, 565 Lipoproteins copper, 97, 571 deficiency, 571

Subject Index Lipoproteins (cont.) dietary fats, 555 iron, 97 oxidative stress, 97, 443 selenium, 97, 897 selenomethionin, 897 zinc, 97, 555 Lithium, ICP, 335 Liver cells selenium, 899 selenoprotein P, 899 899 Lymphocytes cancer, 640 gallium nitrate, 640 zinc, 640 Lysyl oxidase activation, 924 copper, 186 vanadium, 186 Macrophage IL-1 gene expression, 1039 zinc, 1039 Magnesium deficiency, 139 disulfiram, 175 haptoglobin, 333 ICP-MS, 1115 immunity, 139 interaction, 340 lipid, 333 metabolism, 175 metals, 340 metals interaction, 685 stable isotope, 1115 supplementation, 685 Manganese chromium, 201 lactation, 353 metals interaction, 685 MnSOD expression, 687 molybdenum, 201 pallidi, 601 pregnancy, 353 status, 601 supplementation, 685 Menkes, copper transport, review, 9 Mercury Amazonia, 690 brain, 105 hair, 690 speciation, 690 Metabolism arsenic, 165 chromium, 393 copper, 15, 63, 933 disulfiram, 175 infant, 789

1165 Metabolism (cont.) iron, 393, 789, 825 magnesium, 175 selenium, 29, 873 zinc, 1, 35, 43, 393 Metallothionein analysis, 1103, 1116 brain, 444 cadmium, 51 capillary zone electrophoresis, 1103 cirrhosis, 180 copper, 51, 105, 180 diabetes, 515 electroanalytical method, 1116 expression, 444 gene, 55 gold, 51 isoform, 1 1 1 7 mouse, 515 rat, 105 silver, 51 transport, 1 zinc, 1, 43, 47, 51, 65, 515, 961, 1116 zinc finger protein, I ZnT, I Metals binding protein 1038 Middle-aged cancer, 865 selenoprotein P, 865 MnSOD expression aluminum, 687 Alzheimer, 687 Molecular aspect Wilson Disease, review, 917 boron, review, 1049 Molybdenum chromium, 201 grazing sheep, 717 induced deficiency, 741 intakes, 951 manganese, 201 stable isotope, 717 toxicity, 699 Monensin supplementation heifer, 775 selenium, 775 Mouse embryo, boron deficiency, 1083 Multitracer technique, analysis, 1099 Myocardial infarction, status, 551, 574

Neurochemical alteration, deficiency copper, review 909 Newborn environment, 651 glutathione peroxidase, 359 heavy metals. 651 iron, 336 selenium, 359

1166 Newborn (cont.) smoking, 651 status, 332, 336, 359 New methods, analysis, review, 1091 NfKB alunminium, 137 iron, 89 selenium, 89 SP1, 137 transcription factor, 89 zinc, 89 Nickel environment, 688 metals interaction, 685 sensitisation indicators, 688 supplementation, 685 No synthase, copper, 105 Nonheme exchange glial brain tumour, 813 iron, 813 NOX, oxidative stress, 127 Nude mouse, boron, 1061 Obese, adolescent status, 331 Oncogene adriamycin, 119 asbestos, 119 oxidative stress, 119 Oxidative stress actinic exposure, 887 adriamycin, 119 Algeria, 641 altitude, 399 antioxidant, 115, 142, 859 asbestos, 119 blood marker, 629 cancer, 67 ciprofloxacin, 144 cobalt, 299 copper, 141, 142, 617 copper load, 695 desferroxamine, 144 diabetes, 534 DNA, 129 EPO, 643 gene, 67 hemochromatosis, 67 hemodialysis, 643 HFE, 67 HIV, 904 iron, 67 LDL, 97, 443 lead, 111

NO X , 127 peroxynitrite, 71 preeclampsia, 641, 611 review, 67, 77 selenium, 77, 127, 643, 859, 887, 904 thioredoxin reductase, 77

Subject Index Oxidative stress (cont.) UV, 77 zinc, 77, 617, 536 Pallidi chronic liver disease, 601 status, 601 Parasite infection immunity, 1040 zinc, 1040 Parenteral nutrition chromium, review, 503 human, review, 201 Peroxynitrite, oxidative stress, 71 Persimmons, 242 Pets, status, 777 PGMS, infant, 667 Phenylketonuria status, 634 supplementation, 634 Phospholipid cadmium, 691 lung, 1036 pituitary gland, 691 rat, 691, 1036 zinc, 1036 Photoaging, antioxidant, 437 Photoreceptor dystrophy boron, 1082 zebrafish, 1082 Phytate, iron, 820, 823 Pig bioavailability, 763 bone, 1067 boron, 1067 calcium, carbonate, 763 metals, 763 Placenta selenoprotein, 373 zinc, 347 Placental cells, copper oxidase, 947 Platinum, food, 705 Policy, food, enrichment, 313 Pollution lead, 675 selenium, 675 Polyphenol, interaction, 142 Postmenopausal women bioavailability, 441 calcium, 441 high intakes, 1025 HRT, 449 oxidative stress, 443 status, 449 verbal memory, 1025 zinc, 1025 Pregnancy Algerian women, 641 anemia, 375

Subject Index Pregnancy (cont.) Argentinan women, 375 bioavailability, 379 ceruloplasmin oxidase, 383 copper, 353, 383 French women, 377 glutathione peroxidase, 369 iron, 353, 379, 385, 387, 611 lactation, 353, 365 manganese, 353 metals, 365, 377 oxidative stress, 641 Poland women, 369 preeclampsia, 611, 641 RDA, 387 zinc, review, 347 zinc, 347, 353, 387, 821 Prematurity balance, 390 selenium supplementation 388 Prime study, selenium, 463 Primrose, diet, 309

Quality control analysis, 252 food, 252

Rabbit selenium deficiency, 901 vanadium, 153 Rat fructose diet, 536 GH, 1021 glial brain tumour, 813 gonad, 330 hepatotoxicity, 679 IGF1, 1021 metals binding protein, 1038 metal supplementation, 697 phospholipid, 691, 1036 retinol, 181, 183 steroidogenesis, 809 thiamine, 111 RDA review, 189 pregnancy, 387 Redox centers, enzyme, 83 Regulation gene, 21, 55 glutathione peroxidase, 21 matallothionein, 55 selenoprotein, 21 Retinol deficiency, 181, 183 liver, 181, 183 rat, 181, 183 Rumen bacteria, lead, 772

1167 Ruminant copper absorption, 768 stable isotope, 768 Safety, supplementation, 228 Salmon bioavailability, 891 cadmium, 141 copper, 141 oxidative stress, 141 selenomethionine, 891 SECIS, review, 849 Selenium AAS, 169 absorption, 173 actinic exposure, 887 acute phase response, 906 aging, 421, 450 antioxidant, 127, 887 bioavailability, 173, 279, 883, 891 biochemistry. 843 biomarker, 175, 317, 359 body distribution, 29, 895 Brazil nut, 405 breast milk, 482 cancer, 133, 575 chronic heart failure, 547 cognitive function, 442 copper, 201, 341 cow, 767

Czech Republic, 477 deficiency, 775, 901 diabetes, 527 dietary intake, 405, 625, 859, 877 DNA, 143 dog, 781

egg, 248 elderly, 409, 417, 433, 442 "enteque seco", 775 excretion, 767, 873 fish, 161, 250 food, 169, 248, 279, 471 function, 831 gene, 21 glutathione peroxidase, 21, 359, 405, 421, 433, 892, 904 heifer, 775 hemodialysis, 643 hens, 178, 711, 895 H I V , 904 ICP/MS, 157, 161 infant, 388, 482 interventional studies, 453, 575 Ireland, 433 isotope, 175 lactation, 482 LDL, 97

liver cells, 899 metabolism, 29

1168 Selenium (cont.) middle-aged, 865 newborn, 359 NFkB, 89 NO x , 127 oxidative stress, 77, 127, 643, 887, 904 phenylketonuria, 634 prematurity, 388 regulation, 21 requirement, 781 salmon, 891 SECIS, 849 selenomethionine, 891, 897 selenoprotein, 21, 29, 71, 250, 373, 849 selenoprotein P, 71, 837, 865, 869, 899, 903 sheep, 753 speciation, 157, 161, 169, 175, 178 stable isotope, 279, 527 status, 317, 463, 467, 469, 473, 477, 574, 623, 631, 634, 675, 877, 906 status, review, 317 stress, 341 supplementation, 388, 409, 417, 643, 869 TAT protein, 904 thiol protection, review, 77 thioredoxin reductase, 77, 143 thyroid, 250 tissue, 711 Turkey, 469, 471, 623 UV.77, 143 water, 753 Werner Syndrome, 421 zinc, 201 zinc interaction, 77 Selenoprotein gene, 21 glutathione peroxidase, 21 human, 373 protein, 21 rat, 29 regulation, 21 review, 849 thyroid, 250 Selenoprotein P cancer, 865 function, 837 glutathione peroxidase, 71 liver cells, 899 middle-aged, 865 review, 837 structure, 903 supplementation, 869 899 thioredoxin reductase, 71 Sensitization indicators chromium, 688 cobalt, 688

environment, 688 nickel, 688

Subject Index Serum, analysis, 145 Sheep cirrhosis, 723 copper, 723 copper, cobalt, selenium bolus, 749, 782 fluoride, 753 selenium, 753 status, 749, 782 toxicity, 753 toxicosis, 723 Sheep/alpaca deficiency, 737 status, 737 Silicium Belgium, status, 345 bioavailability, 1 1 1 1 supplementation, 1 1 1 1 Skin aging, 421 antioxidant, 437 metals, 421 photoaging, 437 Smoking environment, 651 heavy metals, 651 newborn, 651 Sodium vanadate copper deficiency, 924 lysyl oxidase activation, 924 Spl aluminum, 137 NFkB, 137 Speciation AAS, 169 Amazonia, 690 arsenic, 165 cadmium, 145 copper, 145 hair, 690 ICP/MS, 157, 161, 165, 1107 mercury, 690 selenium, 157, 161, 169, 175, 178 serum, 145 vanadium, 153 zinc, 145 Stable isotope copper absorption, 717, 768 food, 279 magnesium, 1115 molybdenum intake, 717 ruminant, 768 selenium, 279, 527 zinc, 527 Status acute phase response, 906 adolescent, 331 aging, 339 analysis, 563

Subject Index Status (cont.) antidepressant therapy, 607 antioxidant, 446, 479, 551 boron, 1085 Brazil, 391 cancer, 623 cat and dog, 779 ceruloplasmin, 779 chromium, 591 chronic heart failure, 547 chronic liver disease, 601 colorectal polyps, 633 copper, 627, 633, 638, 923, 959 copper, cobalt, se. Bolus, 749 coronary artery disease, 563 cows, 477 Czech Republic, 477 diplegic infant, 636 ginseng, 338 HD, 591 hemodialyse, 631 HRT, 449 infant, 693 intake, 877, 959 iodine, 343, 469 iron, 336, 391, 601, 627, 820 JRA, 638 lactating cattle, 786 lamb, 784 lead, 693 manganese, 601 metals, 332, 339, 568, 597 metals, 636, 786 myocardial infarction, 551, 574 New Zealand, 343 pallidi, 601 pets, 777 phenylketonuria, 634 phytate interaction, 820 Portugal, 479 prime study, 463 selenium, 316, 463, 469, 477, 547, 574, 623, 631, 634, 877, 906 sheep, 749, 782 sheep/alpaca, 737 silicium, 345 thalassemia, 627 Turkey, 469, 623 zinc, 331, 338, 347, 607, 627, 633, zinc, cobalt, selenium bolus, 782, 784 Steers copper, 729 intake, 729 lipid, 729 Steroidogenesis cadmium, 809 intake, 809 iron, 809 rat, 809

1169 Stomatology tissular release, 709 titanium, 709 Stress copper, 341 selenium, 341 zinc, 341 Strontium, diet, 229 SU. VI. MAX interventional studies, review 453 iodine, 486 iron, 488 selenium, 467 antioxidant supplementation, 484 Superoxide dismutase aging, 421, 409 carbamylation, 131 copper, 105, 421 Poland, 369 pregnancy, 369 Supplement, silicon, 1 1 1 1 Supplementation aging, 450 bone, 1009 chromium, 409 copper, 409, 475, 733, 741 efficacy, 741 elderly, 417 elderly, review, 409 "enteque seco", 775 foodcue study, 475 grazing red deer, 733 growth, 829 heifer, 775 hemodialysis, 643 infant, 388, 829 iron, 409, 829 magnesium, 685 manganese, 685 metals, 685 molybdenum induced deficiency 741 nickel, 685 oxidative stress, 643 phenylketonuria, 634 placenta, 347 prematurity, 388 safety, 228 selenium, 388, 417, 450, 634, 643, 775, 869 selenoprotein P, 869 SU. VI. MAX, 484 toxicity, 699 zinc, 347, 409, 685, 1009

Tat protein glutathione peroxidase, 904 HIV, 904 oxidative stress, 904 selenium, 904

1170 Teratogenesis boron, 1079 copper, 1079 xenopus, 1079 selenoprotein P, 899 Thalassemia, status, 627 Therapy antioxidant, 643 EPO, 643

hemodialysis, 643 Thiamine, lead, 111 Thiol, interaction zinc/selenium, review, 77 Thioredoxin reductase glutathione peroxidase, 71 oxidative stress, 77 selenium, 77 selenoprotein P, 71 UV, 77, 143 Thyroid iodine, 343 New Zealand, 343 selenoprotein, 250 Tissue hens, 711 selenium, 711 trans fatty acids, 711 Tissular release stomatology, 709 titanium, 709 Titanium tissular release, 709 stromatology, 709 Toxicity aluminum, 425 brain, 425 fertility, 701 fluoride, 753 lead, 701 molybdenum, 699 selenium, 753 sheep, 753 supplementation, 699 water, 753 Toxicosis cirrhosis, 723 copper, 723 sheep, 723 Trans fatty acids hens, 711 selenium, 711 tissue, 711 Transcription factor, 137 heavy metals, 55 89 Transcuprein copper, 955 transport protein, 955 Transport ATPases, review, 9, 166

Subject Index Transport (cont.) copper, 9, 15, 59 interaction, 184 iron, 67 iron, review, 795 Menkes, 9 vanadium, 153 Wilson, 9 yeast, 35 zinc, 35 ZnT, 1 Transport protein copper, 955 transcuprein, 955 Transporter, zinc, review, 1 Ultratrace, balance, 209 Urine environment, 667 1CP, 335 infant, 667 lithium, 335 POMs, 667 UV DNA, 129, 143 oxidative stress, 77, 129 selenium, 77, 143 thiols, 77 thioredoxin reductase, 77, 143 zinc, 77, 129 Vanadium ATPases, 186 diabetes, review, 491 diet, 221 human, review, 221 liver, 186 lysyl oxidase, 186 rat, 186 speciation, 153 Vanadium compounds diabetes, 539 distribution, 539 Vegetarian diet bioavailability, 305 iron, 261 zinc, 261 Verbal memory postmenopausal women, 1025 zinc, 1025 Vitamin, French pregnant women, 377 Vitamin E deficiency, 901 rabbit, 901 selenium, 901 Walnut, composition, 232 Water arsenic, 645

Subject Index Water (cont.) Bangladesh, 645 cattle, 759 copper, 759 diabetes, 519 environment, 645 fluoride, 753 growth, 759 iodine, 471 Navajo, 519 selenium, 753 sheep, 753 toxicity, 753 Turkey, 471 Weaning, iron enrichment, 235 Werner Syndrome, 421 Whey glutathione peroxidase, 892 selenium, 892 Wilson ATPases transport, review, 9 molecular aspect, review, 917 Wound healing boron, 1061 nude mouse, 1061 Xenopus boron, 1079 copper, 1079 teratogenesis, 1079 Yeast copper, 15 transport zinc, 35 Yeast growth, boron, 1087 Zebrafish boron deficiency, 1082 photoreceptor dystrophy, 1082 Zinc absorption, 43 adolescent, 331 aging, 429 analysis, 1116 apoptosis, 969, 1003, 1035 apoptosis, review, 969 ATPases, review, 1 baby foods, 314 balance, 987 beans, 306 betaine homocysteine S methyltransferase, 1013 bioavailability, 261, 275, 283, 287, 293, 306, 311, 314, 329 brain nutriture, review, 981 Brazil, 626 cadmium, 145 calcium, 275

1171 Zinc (cont.) cancer, 640 carbohydrate, 311 cellular function, review, 961 chelation, 1003, 1035 cognitive function, 999 copper, 105, 145, 201, 338 deficiency, 323, 536, 565, 566, 991, 1021 diabetes, 140, 497, 511, 515, 527, 528, 532, 538 DNA, 129 elderly, review, 409 electroanalytical method, 1116 endothelial cell, 140 exercise, 393 finger protein, review, 1 formula, 275 ginseng, 338 glutamate, 429 gonad, 330 growth, 977 HeLa cells, 1003, 1035 high intake, 1025 human pregnancy, review, 347 immunity, 47, 1039, 1040 infant, 511, 987, 1031 insulin sensitivity, 497, 538 insulin sensitivity, review, 497 intake, 235, 625, 821 iron, 261, 306, 308, 314, 323 lactation, 353 LDL, 97, 555 lymphocytes, 640 macrophage, 1039 metabolism, 43, 393 metabolism, review, 1 metallothionein, 43, 47, 51, 65, 105, 515, 961, 1116 89 NO synthase, 105 obese, 331 oxidative stress, 77, 617 phospholipid, 1036 placenta, 347 postmenopausal women, 1025 pregnancy, 347, 353, 387, 821 RDA, 387 selenium, 77, 201 speciation, 145 stable isotope, 527 status, 331, 338, 347, 607, 627, 633, 821 stress, 341 superoxide dismutase, 105, 131, 409, 421 supplementation, 347, 409, 685, 1009 thiol protection, review, 77 thioredoxin reductase, 77 transport regulation, review 35 UV, 77 vascular protection, 528, 532, vegetarian, 261 women, 323

1172 Zinc (cont.) zinquin, 995 Zinc, cobalt, selenium bolus lamb, 784 sheep, 782 status, 782, 784

Subject Index ZnT transport, review, 1 ZRT, review, 35 Zrt gene, review, 35 ZnT, review, 35