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 (Halli