Functional Foods, Aging, and Degenerative Disease

Functional foods, ageing and degenerative disease

Related titles from Woodhead's food science, technology and nutrition list: Functional foods, cardiovascular disease and diabetes (ISBN 1 85573 735 3) Cardiovascular disease and diabetes pose a serious and growing risk to the health of the population in the developed world. This important collection reviews dietary influences on these diseases and the ways individual functional foods can help prevent them. Dictionary of nutraceutical and functional foods (ISBN 0-8493-1572-7) This is the first reference of its kind for this rapidly developing field. It provides clearly written, concise, science-based information on approximately 150 nutraceutical and functional food products and compounds. Each entry lists the most current information on the product or compound and its role in the promotion of health or the prevention of disease, as well as peer-reviewed literature references. Chemical structures are provided for more than 100 compounds. Phytochemical functional foods (ISBN 1 85573 672 1) Phytochemicals are non-nutritive components that provide plants with colour, flavour and toxicity to pests. There is now a growing body of research that also suggests they may also help to reduce the risk of chronic diseases such as cancer, osteoporosis and heart disease. Edited by two leading authorities, this collection provides an authoritative review of the range of phytochemicals. The first part of the book considers individual groups of phytochemicals such as phenolic compounds and their health benefits. Other parts of the book discuss how functional benefits are tested, and ways of producing phytochemical functional products. Details of these books and a complete list of Woodhead's food science, technology and nutrition titles can be obtained by: · visiting our web site at www.woodhead-publishing.com · contacting Customer Services (email: [email protected]; fax: +44 (0) 1223 893694; tel.: +44 (0) 1223 891358 ext. 30; address: Woodhead Publishing Limited, Abington Hall, Abington, Cambridge CB1 6AH, UK) Selected food science and technology titles are also available in electronic form. Visit our web site (www.woodhead-publishing.com) to find out more. If you would like to receive information on forthcoming titles in this area, please send your address details to: Francis Dodds (address, tel. and fax as above; e-mail: [email protected]). Please confirm which subject areas you are interested in.

Functional foods, ageing and degenerative disease Edited by C. Remacle and B. Reusens

Published by Woodhead Publishing Limited Abington Hall, Abington Cambridge CB1 6AH England www.woodhead-publishing.com Published in North America by CRC Press LLC 2000 Corporate Blvd, NW Boca Raton FL 33431 USA First published 2004, Woodhead Publishing Limited and CRC Press LLC ß 2004, Woodhead Publishing Limited The authors have asserted their moral rights. This book contains information obtained from authentic and highly regarded sources. Reprinted material is quoted with permission, and sources are indicated. Reasonable efforts have been made to publish reliable data and information, but the authors and the publishers cannot assume responsibility for the validity of all materials. Neither the authors nor the publishers, nor anyone else associated with this publication, shall be liable for any loss, damage or liability directly or indirectly caused or alleged to be caused by this book. Neither this book nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, microfilming and recording, or by any information storage or retrieval system, without permission in writing from the publishers. The consent of Woodhead Publishing Limited and CRC Press LLC does not extend to copying for general distribution, for promotion, for creating new works, or for resale. Specific permission must be obtained in writing from Woodhead Publishing Limited or CRC Press LLC for such copying. Trademark notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation, without intent to infringe. British Library Cataloguing in Publication Data A catalogue record for this book is available from the British Library. Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress. Woodhead Publishing Limited ISBN 1 85573 725 6 (book); 1 85573 901 1 (e-book) CRC Press ISBN 0-8493-2538-2 CRC Press order number: WP2538 The publisher's policy is to use permanent paper from mills that operate a sustainable forestry policy, and which have been manufactured from pulp which is processed using acid-free and elementary chlorine-free practices. Furthermore, the publisher ensures that the text paper and cover board used have met acceptable environmental accreditation standards. Project managed by Macfarlane Production Services, Markyate, Hertfordshire (e-mail: [email protected]) Typeset by MHL Typesetting Limited, Coventry, Warwickshire Printed by TJ International Limited, Padstow, Cornwall, England

Contents

Contributor contact details . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

2

Regulatory context in the EU . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . P. Berry Ottaway, Berry Ottaway and Associates Ltd, UK 1.1 Introduction: the EU and food legislation . . . . . . . . . . . . . . . . . . . . 1.2 The regulation of novel foods and novel ingredients in the EU . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3 EU food law and regulation of food health claims . . . . . . . . . . . 1.4 National initiatives to regulate food health claims . . . . . . . . . . . 1.5 Approval and substantiation of health claims . . . . . . . . . . . . . . . . 1.6 Medicinal products and EU legislation . . . . . . . . . . . . . . . . . . . . . . . 1.7 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Diet and the prevention of degenerative disease . . . . . . . . . . . . . . . . L. Kalbe, B. Reusens and C. Remacle, Universite Catholique de Louvain, Belgium 2.1 Introduction: epidemiological studies and the influence of diet in early life . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 Foetal and neonatal nutritional requirements . . . . . . . . . . . . . . . . . 2.3 The effects of supplement intake . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4 The role of functional foods: nutrition during pregnancy and infancy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5 Safety concerns of functional foods . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6 Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.7 Sources of further information and advice . . . . . . . . . . . . . . . . . . . 2.8 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

xv 1 1 4 7 10 13 14 15 17

17 21 30 33 39 41 43 43

vi

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3

New functional foods for age-related diseases . . . . . . . . . . . . . . . . . . . D. Rivera, University of Murcia and C. OboÂn, University Miguel HernaÂndez, Spain 3.1 Introduction: the Mediterranean diet and healthy living . . . . . . 3.2 Mediterranean foods and their functional properties . . . . . . . . . 3.3 The functional properties of Mediterranean herbs, spices and wild greens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4 Diet and age-related diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5 Methods of identifying and analysing plant extracts . . . . . . . . . 3.6 Developing supplements for healthy ageing and other future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.7 Sources of further information and advice . . . . . . . . . . . . . . . . . . . 3.8 Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.9 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Part I 4

5

57 57 60 65 66 68 70 72 72 72

Bone and oral health . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

81

Diet and the control of osteoporosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . K. D. Cashman, University College Cork, Ireland 4.1 Introduction: definition and epidemiology of osteoporosis . . . 4.2 Bone growth and factors affecting bone mass . . . . . . . . . . . . . . . . 4.3 Dietary strategies for preventing osteoporosis: minerals . . . . . . 4.4 Dietary strategies for preventing osteoporosis: vitamins, proteins and lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5 Preventing osteoporosis: the impact of genetic variation and diet . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.6 Conclusions and future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.7 Sources of further information and advice . . . . . . . . . . . . . . . . . . . 4.8 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

83

Phytoestrogens and the control of osteoporosis . . . . . . . . . . . . . . . . . S. Lorenzetti and F. Branca, Instituto Nazionale di Ricerca per gli Alimenti e la Nutrizione (INRAN), Italy 5.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2 Osteoporosis: prevention and treatment . . . . . . . . . . . . . . . . . . . . . . 5.3 Mechanisms of action of phytoestrogens in bone metabolism 5.4 Phytoestrogen action on bone cells . . . . . . . . . . . . . . . . . . . . . . . . . . 5.5 Investigating phytoestrogen action on bone: animal and human studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.6 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.7 Sources of further information and advice . . . . . . . . . . . . . . . . . . . 5.8 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

83 85 87 95 100 105 106 106 115 115 116 120 122 124 126 127 129

Contents 6

7

8

9

Vitamin D fortification and bone health . . . . . . . . . . . . . . . . . . . . . . . . L. Ovesen, Institute of Food Safety and Nutrition, Denmark 6.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2 Vitamin D: sources, metabolism, function and deficiency . . . 6.3 Vitamin D fortification and osteoporosis . . . . . . . . . . . . . . . . . . . . . 6.4 Dietary intake of vitamin D . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.5 Strategies to improve vitamin D supply . . . . . . . . . . . . . . . . . . . . . . 6.6 Food fortification: reducing deficiency diseases . . . . . . . . . . . . . . 6.7 Issues in vitamin D fortification of food . . . . . . . . . . . . . . . . . . . . . 6.8 Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.9 Sources of further information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.10 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Calcium citrate (TCC) and bone health . . . . . . . . . . . . . . . . . . . . . . . . . S. Edelstein, The Weizmann Institute of Science, Israel 7.1 Introduction: bone formation and calcium fortification . . . . . . . 7.2 Calcium citrate (TCC) as a calcium supplement . . . . . . . . . . . . . 7.3 Measuring the effectiveness of TCC . . . . . . . . . . . . . . . . . . . . . . . . . 7.4 TCC fortification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.5 Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.6 Sources of further information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.7 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Diet, functional foods and oral health . . . . . . . . . . . . . . . . . . . . . . . . . . . M. Edgar, formerly The University of Liverpool, UK 8.1 Introduction: key dietary factors in oral health . . . . . . . . . . . . . . . 8.2 The effects of ageing on oral health . . . . . . . . . . . . . . . . . . . . . . . . . 8.3 Dietary strategies for oral health . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.4 Functional foods for promoting oral health . . . . . . . . . . . . . . . . . . 8.5 Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.6 Sources of further information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.7 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Sweeteners and dental health . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . K. K. Makinen, University of Turku, Finland 9.1 Introduction: the relationship between dental caries and dietary carbohydrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2 Xylitol and the prevention of dental caries . . . . . . . . . . . . . . . . . . . 9.3 The relationship between sucrose consumption and dental caries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.4 Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.5 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

vii 139 139 140 144 147 153 155 156 163 164 164 174 174 177 178 180 181 181 182 184 184 187 188 192 195 196 196 200 200 202 208 216 216

viii

Contents

Part II Obesity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

221

10

223

11

12

Nutrient-gene interactions in the control of obesity . . . . . . . . . . . . C. Verdich, Copenhagen University Hospital, Denmark, K. Clement, INSERM, France and T. I. A. Sùrensen, Copenhagen University Hospital, Denmark 10.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.2 Genetic influences on obesity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.3 Nutrient-sensitive genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.4 Nutrient-gene interaction and the development of obesity . . . . 10.5 Managing obesity: dietary and other strategies . . . . . . . . . . . . . . . 10.6 Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.7 Sources of further information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.8 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nutrition, fat synthesis and obesity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F. Foufelle and P. FerreÂ, INSERM, France 11.1 Introduction: fat synthesis and nutrition . . . . . . . . . . . . . . . . . . . . . . 11.2 Regulation of glycolytic/lipogenic enzymes . . . . . . . . . . . . . . . . . . 11.3 Molecular mechanisms involved in controlling glycolytic/lipogenic genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.4 Improving lipogenesis using functional foods . . . . . . . . . . . . . . . . 11.5 Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.6 Sources of further information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.7 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.8 Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

223 224 234 236 244 247 248 251 260 260 264 266 270 272 273 273 277

Satiety and the control of obesity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . W. A. M. Blom, A. Stafleu and C. de Graaf, TNO Nutrition and Food Research, The Netherlands 12.1 Introduction: satiety and obesity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.2 Factors influencing satiety and satiation . . . . . . . . . . . . . . . . . . . . . 12.3 The impact of different food components on satiety . . . . . . . . . 12.4 Developing biomarkers of satiety . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.5 Future trends: using biomarkers to assess weight-control foods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.6 Sources of further information and advice . . . . . . . . . . . . . . . . . . . 12.7 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

286 287 287

Part III Gut health and immune function . . . . . . . . . . . . . . . . . . . . . . . . . .

293

13

295

Functional foods for gut health: an overview . . . . . . . . . . . . . . . . . . . R. Tahvonen and S. Salminen, University of Turku, Finland 13.1 Introduction: the human gut . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13.2 The structure of the gut and its immune system . . . . . . . . . . . . .

278 278 278 282 284

295 296

Contents 13.3 13.4 13.5 13.6 13.7 13.8 13.9 14

15

16

ix

Nutrients and gut function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nutrients and the gut immune system . . . . . . . . . . . . . . . . . . . . . . . . Nutrition and gut health . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The role of functional foods in promoting gut health . . . . . . . . Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sources of further information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

305 308 309 312 313 317 318

Analysing gut microflora . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . M. Blaut, German Institute of Human Nutrition Potsdam-Rehbruecke 14.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14.2 Molecular based methods for identifying gut micro-organisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14.3 Methods of characterising human gut microbiota . . . . . . . . . . . . 14.4 Using denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) for characterising microbiota . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14.5 Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14.6 Sources of further information and advice . . . . . . . . . . . . . . . . . . . 14.7 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

325

Dietary lipids and immune function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . P. C. Calder, University of Southampton, UK 15.1 Introduction: the immune system in health, disease and ageing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15.2 Dietary fatty acids: nomenclature, sources and intakes . . . . . . . 15.3 Fatty acid composition of immune cells and the immune function: eicosanoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15.4 Dietary fatty acids and immune function: mechanisms of action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15.5 Other mechanisms of action of dietary fatty acids not involving eicosanoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15.6 Dietary fatty acids and inflammatory diseases . . . . . . . . . . . . . . . 15.7 Targeting the immune function and inflammation: fatty acid-enriched functional foods . . . . . . . . . . . . . . . . . . . . . . . . . . 15.8 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15.9 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Improving gut health in the elderly . . . . . . . . . . . . . . . . . . . . . . . . . . . . . K. M. Tuohy, E. Likotrafiti, K. Manderson, G. R. Gibson and R. A. Rastall, University of Reading, UK 16.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16.2 Successional development of gastrointestinal microflora . . . . . 16.3 Modification of the gut microflora: probiotics, prebiotics and synbiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

325 326 334 340 341 344 344 349 349 354 361 364 372 375 378 382 382 394 394 395 399

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Contents 16.4 16.5 16.6 16.7 16.8

17

Factors affecting gut microflora in old age . . . . . . . . . . . . . . . . . . Immunosenescence and susceptibility to colon cancer in old age . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

402 405 408 409 410

Probiotics, prebiotics and gut health . . . . . . . . . . . . . . . . . . . . . . . . . . . . L. De Vuyst, L. Avonts and L. Makras, Vrije Universiteit Brussel, Brussels, Belgium 17.1 Introduction: defining probiotics and prebiotics . . . . . . . . . . . . . . 17.2 Types of probiotics and prebiotics and their influence on gut health . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17.3 Investigating the effectiveness of probiotics and prebiotics: the case of antimicrobial function . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17.4 Improving the effectiveness of probiotics and prebiotics in optimising gut health . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17.5 Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17.6 Sources of further information and advice . . . . . . . . . . . . . . . . . . . 17.7 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17.8 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

461 463 464 464 464

Part IV Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

483

18

19

Anti-angiogenic functional food, degenerative disease and cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . J. N. Losso and R. R. Bansode, Louisiana State University, USA 18.1 Introduction: mechanisms of degenerative disease . . . . . . . . . . . 18.2 Genetic/endogenous risk factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18.3 Environmental/exogenous risk factors . . . . . . . . . . . . . . . . . . . . . . . . 18.4 Angiogenesis, body function and degenerative disease . . . . . . . 18.5 Anti-angiogenic functional food compounds . . . . . . . . . . . . . . . . . 18.6 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18.7 Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18.8 Sources of further information and advice . . . . . . . . . . . . . . . . . . . 18.9 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Synbiotics and colon cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . M. F. Bennet, Y. E. Clune, F. Shanahan, G. O'Sullivan and J. K. Collins, University College Cork, Ireland 19.1 Introduction: probiotics, prebiotics and synbiotics . . . . . . . . . . . 19.2 Gut microflora . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19.3 Colon cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19.4 Risk factors and prevention of colon cancer . . . . . . . . . . . . . . . . .

416 416 420 427

485 485 486 493 495 501 511 511 513 513 524 524 528 533 541

Contents 19.5 19.6 19.7 19.8 19.9 19.10 20

21

22

xi

Screening of colorectal cancers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Diagnosis and treatment of colorectal cancers . . . . . . . . . . . . . . . Pre- pro- and synbiotic influences on colon carcinogenesis . . Predicting tumour formation: biomarkers . . . . . . . . . . . . . . . . . . . . Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

544 546 552 558 561 563

Identifying antimutagenic constituents of food . . . . . . . . . . . . . . . . . . S. KnasmuÈller, B. J. Majer and C. Buchmann, University of Vienna, Austria 20.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20.2 Methods for identifying antimutagenic constituents in foods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20.3 Limitations of methods for identifying antimutagenic compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20.4 Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20.5 Sources of further information and advice . . . . . . . . . . . . . . . . . . . 20.6 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

581

Glucosinolates and the prevention of cancer . . . . . . . . . . . . . . . . . . . . F. Kassie, University of Giessen, Germany and S KnasmuÈller, University of Vienna, Austria 21.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.2 The role of glucosinolates in the prevention of cancer . . . . . . . 21.3 Mechanisms of action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.4 Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.5 Sources of further information and advice . . . . . . . . . . . . . . . . . . . 21.6 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Dietary fiber and the prevention of cancer . . . . . . . . . . . . . . . . . . . . . J. Slavin, University of Minnesota, USA 22.1 Introduction: defining dietary fiber . . . . . . . . . . . . . . . . . . . . . . . . . . . 22.2 The relationship between dietary fiber intake and cancers of the gastrointestinal tract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22.3 Epidemiological evidence on the protective role of dietary fiber . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22.4 Dietary fiber and hormonally related cancers . . . . . . . . . . . . . . . . 22.5 Clinical studies of the protective role of dietary fiber . . . . . . . . 22.6 The relationship between dietary fiber intake and different cancers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22.7 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22.8 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

581 583 600 603 604 605 615 615 617 620 623 623 623 628 628 630 634 637 638 639 640 641

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Contents

23

24

Phytoestrogens and the prevention of cancer . . . . . . . . . . . . . . . . . . . Y. Ungar and E. Shimoni, Israel Institute of Technology 23.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23.2 Phytoestrogens in food: the effects of food processing and storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23.3 The role of phytoestrogens in the prevention of different cancers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23.4 Mechanisms of action of phytoestrogens . . . . . . . . . . . . . . . . . . . . . 23.5 Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23.6 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Food phenolics and cancer chemoprevention . . . . . . . . . . . . . . . . . . . F. Shahidi, Memorial University of Newfoundland, Canada 24.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24.2 Functional properties of plant phenolics and polyphenolics . . 24.3 The role of phenolic compounds in the prevention of cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24.4 Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24.5 Sources of further information and advice . . . . . . . . . . . . . . . . . . . 24.6 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

645 645 646 649 655 658 659 669 669 670 674 676 676 677

25

Vitamins and the prevention of cancer . . . . . . . . . . . . . . . . . . . . . . . . . . 681 C. A. Northrop-Clewes and D. I. Thurnham, University of Ulster, UK 25.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 681 25.2 The role of vitamins in the prevention of cancer . . . . . . . . . . . . . 683 25.3 Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 700 25.4 Sources of further information and advice . . . . . . . . . . . . . . . . . . . 701 25.5 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 701

26

Probiotics in inflammatory bowel disease . . . . . . . . . . . . . . . . . . . . . . . J. McCarthy, B. Sheil, L. O'Mahony, M. M. Anwar and F. Shanahan, National University of Ireland 26.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26.2 Managing inflammatory bowel disease: the role of probiotics 26.3 Analysing the effectiveness of probiotics in inflammatory bowel disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26.4 Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26.5 Source of further information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26.6 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26.7 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

708 708 709 713 721 722 722 722

Contents 27

xiii

Assessing the effectiveness of probiotics, prebiotics and synbiotics in preventing disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G. C. M. Rouzaud, The University of Reading, UK 27.1 Introduction: diet and gastrointestinal diseases . . . . . . . . . . . . . . . 27.2 Definitions of probiotics, prebiotics and synbiotics . . . . . . . . . . 27.3 Safety issues in the use of probiotics and prebiotics . . . . . . . . . 27.4 Methods for determining mode of action and effectiveness . . 27.5 Evidence for the effects of pro-, pre- and synbiotics on acute and chronic diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27.6 Sources of further information and advice . . . . . . . . . . . . . . . . . . . 27.7 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27.8 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

740 744 745 746

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

753

726 726 729 733 735

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Contributor contact details

Chapter 1

Chapter 3

Mr P. Berry Ottaway Berry Ottaway and Associates Ltd 1a Fields Yard Plough Lane Hereford HR4 0EL UK

Dr D. Rivera Department of Plant Biology University of Murcia E-30100 Espinardo Murcia Spain

Tel: +44 (0) 1432 270886 Fax: +44 (0) 1432 270808 E-mail: [email protected]

Chapter 2 Dr L. Kalbe, Dr B. Reusens and Professor C. Remacle Laboratoire de Biologie Cellulaire Universite Catholique de Louvain Louvain-la-Neuve Belgium Tel: +32 10 47 35 22 Fax: +32 10 47 35 15 E-mail: [email protected]

E-mail: [email protected] Dr C. Obon Department of Applied Botany University Miguel Hernandez E-03312 Orihuela Alicante Spain E-mail: [email protected]

Chapter 4 Professor K. D. Cashman Department of Food and Nutritional Sciences and Department of Medicine

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Contributors

University College Cork Ireland

Tel: + 972 5 0269814 Fax: +972 3 9655505 E-mail: [email protected]

Tel: +353 21 4901317 Fax: +353 21 4270244 E-mail: [email protected]

Chapter 8

Chapter 5 Dr S. Lorenzetti and Dr F. Branca Istituto Nazionale di Ricerca per gli Alimenti e la Nutrizione (INRAN) Via Ardeatina, 546 00178 Roma Italy Tel: +39 06 51494 - 571/521 Fax: +39 06 51494 - 550 E-mail: [email protected] [email protected]

Chapter 6

Professor M. Edgar School of Dentistry The University of Liverpool Daulby Street Liverpool L69 3GN UK Tel: +44(0) 151 706 5262 Fax: +44(0) 151 706 5937 E-mail: [email protected]

Chapter 9 Professor K. K. MaÈkinen SepaÈnkatu 10 FIN-23500 Uusikaupunki Finland

Dr L. Ovesen The Danish Heart Foundation 10 Hauser Plads 1127 Copenhagen K Denmark

Tel: 358 40 5561 063 Fax: 358 2 844 2571 E-mail: [email protected]

Tel: +45 3367 0010 Fax: +45 3393 1245 E-mail: [email protected]

Chapter 10

Chapter 7 Dr S. Edelstein Department of Biological Chemistry The Weizmann Institute of Science Rehovot Israel

Dr C. Verdich and Professor T. I. A Sorensen Danish Epidemiology Science Centre Institute of Preventive Medicine Copenhagen University Hospital DK 1399 Copenhagen K Denmark Tel: 45 3338 3760/3860 Fax: 45 333 4240 E-mail: [email protected]

Contributors [email protected] Dr K. Clement INSERM `Avenir' EA 3502 Paris VI University Nutrition Department Hotel-Dieu Place du parvis Notre-Dame 75004 Paris France Tel: +33 142 34 8670 Fax: +33 140 51 0057 E-mail: [email protected]

xvii

Chapter 13 Dr R. Tahvonen and Professor S. Salminen Department of Biochemistry and Food Chemistry Functional Foods Forum University of Turku 20014 Turku Finland Tel: +358 2 333 6840 E-mail: [email protected] [email protected]

Chapter 14 Chapter 11 Dr F. Foufelle and Professor P. Ferre Unit 465 INSERM, Centre de Recherches Biomedicales des Cordeliers Universite Paris 6 15 rue de l'Ecole de Medecine 75270, Paris cedex 06 Tel: +33 14234 69 22/23/24 Fax: +33 14051 85 86 E-mail: [email protected]

Chapter 12

Professor M. Blaut Department of Gastrointestinal Microbiology German Institute of Human Nutrition Potsdam-Rehbruecke Arthur-Scheunert-Allee 114-116 14558 Nuthetal Germany Tel: +49 33200 88470 Fax: +49 33200 88407 E-mail: [email protected]

Chapter 15

W. A. M. Blom, Dr A. Stafleu and Dr C. de Graaf TNO Nutrition and Food Research PO Box 360 3700 AJ Zeist The Netherlands

Professor P. C. Calder Institute of Human Nutrition School of Medicine University of Southampton Bassett Crescent East Southampton SO16 7PX UK

Tel: +31 30 694 43 41 Fax: +31 30 695 79 52 E-mail: [email protected]

Tel: +44 8059 4223 Fax: +44 8059 5489 E-mail: [email protected]

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Contributors

Chapter 16

Chapter 19

K. M. Tuohy, E. Likotrafiti, K. Manderson, G. R. Gibson and R. A. Rastall Food and Microbial Sciences Unit School of Food Sciences PO Box 226 The University of Reading Reading RG6 6AP

Y. E. Clune University College Cork Cork Ireland

Chapter 17 Professor L. De Vuyst VUB-IMDO Pleinlaan 2 B-1050 Brussels Belgium Tel: +32 02 629 32 45 Fax: +32 02 629 27 20 E-mail: [email protected]

Chapter 18 Dr J. N. Losso and R. R. Bansode Food Protein Biotechnology Laboratory Department of Food Science Louisiana State University Agricultural Center 111 Food Science Building Baton Rouge LA 70803 USA Tel: 225 578 3883 Fax: 225 578 5300 E-mail: [email protected]

E-mail: [email protected] Dr M. F. Bennett, Professor F. Shanahan, Professor G. O'Sullivan and Professor J. K. Collins University College Cork Cork Ireland

Chapter 20 Dr S. KnasmuÈller, Dr B. J. Majer and Dr C. Buchmann Institute of Cancer Research University of Vienna Austria E-mail: siegfried.knasmueller@ meduniwien.ac.at

Chapter 21 Dr F. Kassie Institute of Indoor and Environmental Toxicology University of Giessen Germany E-mail: [email protected] Dr S. KnasmuÈller Institute of Cancer Research University of Vienna Austria E-mail: siegfried.knasmueller@ meduniwien.ac.at

Contributors

xix

Chapter 22

Chapter 25

Professor J. Slavin Department of Food Science and Nutrition University of Minnesota 1334 Eckles Avenue St Paul MN 55108 USA

Dr C. A. Northrop-Clewes and Dr D. I. Thurnham Northern Ireland Centre for Food and Health University of Ulster Coleraine BT52 1SA UK

Tel: 612 624 7234 Fax: 612 625 5272 E-mail: [email protected]

Chapter 23 Mrs Y. Ungar and Dr E. Shimoni Department of Biotechnology and Food Engineering Israel Institute of Technology Haifa 32000 Israel Tel: +972-4-8292484 Fax: +972-4-8293399 E-mail: [email protected]. ac.il

Chapter 24 Dr F. Shahidi Department of Biochemistry Memorial University of Newfoundland St John's. NL A1B 3X9 Canada Tel: + (709) 737-8552 Fax: + (709) 737-4000 E-mail: [email protected]

Tel: +44(0) 2870 324473 Fax: +44(0) 2870 344965 E-mail: [email protected]

Chapter 26 J. McCarthy, B. Sheil, L. O'Mahony, M. M. Anwar and F. Shanahan Department of Medicine and Alimentary Pharmabiotic Centre National University of Ireland Cork Ireland Tel: +353-21 4901226 Fax: +353-21 4345300 E-mail: [email protected]

Chapter 27 Dr G. C. M. Rouzaud Food and Microbial Sciences Unit School of Food Sciences PO Box 226 The University of Reading Reading RG6 6AP Tel: +44(0)118 935 7215 Fax: +44(0)118 935 7222 E-mail: [email protected]

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1 Regulatory context in the EU P. Berry Ottaway, Berry Ottaway and Associates Ltd, Hereford, UK

1.1

Introduction: the EU and food legislation

The European Union (EU) has been evolving since March 1957 when the Treaty of Rome was signed by six states. At an early stage it was accepted that differences in the approaches to food legislation between the member countries of the European Economic Community (EEC), as it was first called, formed a significant barrier to free trade within the Community. At the beginning of the 1960s there was an ambitious programme to harmonise the food laws across the Community. The focus was on the development of compositional criteria (recipes) for a diverse range of product categories such as cocoa and chocolate products, fruit juices, jams, jellies and marmalade. Progress was slow and by the beginning of the 1980s much of the planned legislation had not been completed. In 1985 a new initiative was adopted. This was based on a concept of five horizontal, or framework, directives designed to incorporate identified requirements of public health and safety, consumer information and general food control measures. It was agreed that the legislation should be developed around these five framework directives, which were: · · · · ·

food labelling and presentation food additives materials and articles in contact with foods foods for particular nutritional uses official control of foodstuffs.

The idea was that these framework directives would lay down the general principles for control, and specific technical directives and regulations would be developed as adjuncts to the framework directives where necessary.1 However,

2

Functional foods, ageing and degenerative disease

even with the more simplified system introduced after 1985, much of the proposed legislation was not in place when the Single Market officially came into existence in January 1993. Seven years later, in January 2000, the European Commission published its White Paper on Food Safety.2 This included details of 84 pieces of food legislation that were still outstanding and a revised timetable for completion was included in the paper. All but two items were given completion dates between July 2000 and December 2002. However, by the end of 2002 many of the deadlines had not been met, including those for legislation on food claims and the micronutrient fortification of food. Until the middle of the 1990s, the principal aim of the European food law was to harmonise the existing laws of the member states to enable free and unrestricted trade between the countries. From about 1995 onwards there has been an increasing proportion of new legislation that was not previously on the statutes of any of the member states. A number of laws affecting functional foods fall into this category. Until European legislation is adopted and comes into force, the member states of the EU are allowed to retain their national laws. This means that although the harmonisation of European food law has been in progress for over forty years, there are some aspects of the legislation that are still regulated by disparate national legislation. 1.1.1 Member States of the EU Since the signing of the Treaty of Rome in 1957, membership of the EU has increased in stages to a total of 15 member states in the 1990s. The 15 states covered almost all Western Europe from Northern Scandinavia to the Mediterranean, with the exception of Norway and Switzerland who voted not to join. Within the 15 member states there are disparate cultures, dietary habits and different approaches to food legislation. It is this disparity that often makes it difficult to achieve unanimity on legislative proposals relating to food and is the main reason that some legislation has taken many years before agreement is reached. From May 2004 the EU will further enlarge to include another ten states, bringing the total membership to 25, with a combined population of over 450 million (Table 1.1). The new entrants will include a number of states formerly in the Soviet sphere of influence. As part of the condition of entry, each new state will have to work to the European food legislation already adopted and, to this end, some of the countries have for some years been adjusting their new food laws to harmonise with those of the EU. 1.1.2 Food safety and its assessment The EU has adopted a number of principles in relation to food safety. These are laid down in law and form the basis of specific food safety legislation. The cornerstone of these principles is that a high level of protection of human health

Regulatory context in the EU Table 1.1

3

Membership of the European Union

EU member states prior to May 2004

Additional EU member states from May 2004

Austria Belgium Denmark Finland France Germany Greece Ireland Italy Luxembourg Netherlands Portugal Spain Sweden United Kingdom

Czech Republic Cyprus Estonia Hungary Latvia Lithuania Malta Poland Slovakia Slovenia

and life should be assured in the pursuit of Community policies. To this end, all food safety policy should be based on a comprehensive integrated approach, not only on an EU basis between member states, but also internationally. In its White Paper on Food Safety published in early 2000, the European Commission introduced the concept of control from `farm to table' covering all sectors of the food chain. The Commission believes that a successful food policy demands that foods and their ingredients are fully traceable and that procedures are in place to facilitate traceability and to permit effective recalls. This requirement has been incorporated into the law that becomes fully effective on 1 January 2005. The other key aspect of the food safety policy is risk analysis, with food safety legislation being based on a risk analysis and not on general conjecture. Risk analysis is seen as being comprised of three components: risk assessment based on information analysis and scientific advice; risk management by regulation and control; and risk communication. This is considered to be capable of providing a systematic methodology for the determination of effective, targeted and proportionate measures to protect health. There is a need for the risk assessments to be undertaken in an independent, objective and transparent manner and on the basis of the best available scientific data and information. It is, however, recognised that in some cases, scientific risk assessment on its own cannot provide all the necessary information for a risk management decision and that other relevant factors may have to be taken into consideration. Such factors may include societal, economic, ethical and environmental aspects and the feasibility of controls. In cases where it is found that the scientific evidence is inconclusive, uncertain or insufficient, and where a preliminary objective scientific evaluation indicates that there may be possible unacceptable effects on human health and

4

Functional foods, ageing and degenerative disease

safety, or on the environment, the precautionary principle may be applied. The precautionary principle was introduced in 2002 as part of European general food law. Its introduction follows a judgment from the Court of Justice in 1998 that stated `where there is uncertainty as to the existence or extent of risks to human health, the (European) institutions may take protective measures without having to wait until the reality and seriousness of these risks have become fully apparent'. Risk management measures taken on the basis of the precautionary principle are regarded as provisional and are expected to be kept under review while the relevant scientific evidence is being obtained. The law requires that the review should be conducted within a reasonable period of time. This requirement will be governed by the nature of the possible risk to life and health and the type of scientific information required to clarify the areas of uncertainty that would allow a more comprehensive risk assessment to be conducted. Within the EU there has been a long history of risk assessment and risk management, particularly with respect to the evaluation of food additives. The Scientific Committee for Food of the European Commission (SCF) first issued its guidelines for the safety assessment of food additives in 1980. This document has since been superseded, but most of the original principles remain today.3

1.2 The regulation of novel foods and novel ingredients in the EU Towards the end of the 1980s the European Commission and a number of member state governments became concerned at the lack of control over the introduction of new ingredients, other than food additives, into the food chain. Ingredients made from genetically modified organisms were just beginning to emerge and this heightened the concerns. In an explanatory memorandum, which accompanied the first proposals for the regulation of new ingredients in early 1989, the Commission stated that the existing situation was acceptable when food technology and ingredients were based on a long tradition of safe use. However, developments in new raw materials and ingredients, and new food production processes, which led to fundamental changes in food components, were rapidly evolving from the research stage to the market-place. These novel food components, which were often present in food in much larger quantities than additives, were at the time not required to be subjected to any scientific assessment at European Community level. The Commission proposed that novel foods and ingredients should be examined for safety and subject to authorisation before being offered for sale. The draft proposal for a European Council regulation on novel foods and novel food ingredients was revised 12 times between 1989 and July 1992, when the formal proposal was presented by the Commission. This proposal became controversial and debate and discussion between the European Parliament and the Commission continued over the following 18 months, with a considerably

Regulatory context in the EU

5

amended proposal being circulated in December 1993. Agreement could still not be reached and eventually, after further debate, a Common Position was released in September 1995. This was not adopted and the proposal went to the Conciliation Committee with a final text being agreed in December 1996. A few months earlier, in September 1996, the European Commission had discovered that soya and maize supplies en route from the USA to Europe had been contaminated with varying amounts of genetically modified material. As neither the soya nor maize constructs had been approved for food use in the EU the Commission was faced with a dilemma as there was no formal requirement for authorisation of such food ingredients. This accentuated the need for regulation. The Regulation on Novel Foods and Novel Ingredients was finally adopted on 27 January 1997 as Regulation (EC) No 258/97 and it came fully into effect on 15 May 1997.4 The scope of the regulation is very broad and it applies to all foods and food ingredients that `have not hitherto been used for human consumption to a significant degree in the [European] Community'. The six categories of foods and ingredients that fell under the control of the regulation were given as: (a) foods and food ingredients containing or consisting of genetically modified organisms within the meaning of Directive 90/220/EEC; (b) foods and food ingredients produced from, but not containing, genetically modified organisms; (c) foods and food ingredients with a new or intentionally modified primary molecular structure; (d) foods and food ingredients consisting of or isolated from micro-organisms, fungi or algae; (e) foods and food ingredients consisting of or isolated from plants and food ingredients isolated from animals, except for foods and food ingredients obtained by traditional propagating or breeding practices and having a history of safe food use; (f) foods and food ingredients to which has been applied a production process not currently used, where that process gives rise to significant changes in the composition or structure of the foods or food ingredients which affect their nutritional value, metabolism or level of undesirable substances. When considered carefully, it can be seen that the list covers almost every source of a food or ingredient likely to come onto the market. Category (e) has been interpreted by the authorities to include all botanical sources (herbs, extracts, etc.) intended for use under food law and which did not have a significant history of human food use in the EU before 1997. The law requires that all foods, ingredients and in certain instances, processes that fall within the scope of the regulation be assessed for safety before being placed on the European market. The safety review has to be carried out by an expert committee in the EU country of intended first sale of the food or ingredient. The procedure laid down requires that the applicant submit a detailed dossier to the member state of intended first sale. A summary of the information has to be provided to the

6

Functional foods, ageing and degenerative disease

European Commission and the Commission is required to forward copies of this summary to the other member states. The member state receiving the application has to carry out an initial assessment and the report of the assessment also has to be sent to the Commission and forwarded to the other member states. The requirements for the format of the dossier and the safety data required to support an application are given in a Commission document published in 1997.5 This also provides a series of decision trees relating to the data required for the six categories of foods and ingredients. Different sets of data are needed to support those derived from genetic modification. The regulation makes provision for a simplified assessment process where the food or ingredient can be demonstrated to be substantially equivalent to one which is well established in the food chain. In practice, however, it has been found that the concept of substantial equivalence can be successfully applied only to foods and ingredients derived from genetic modification where, for example, the genetic changes have been introduced for agricultural purposes (e.g. pest resistance or herbicide resistance) and where the wholesomeness of the food can be demonstrated to be unchanged. Although the procedures for assessment are defined in the legislation, they have not worked well in practice. In the first five years after the regulation came into force, there had only been a total of 37 applications. Of these, two were withdrawn and one was submitted in error. Of the 34 that underwent initial assessment, only six were authorised and two were refused. The remainder (26) were either waiting for the completion of the initial assessment or had been referred to the Scientific Committee on Food (SCF) for an opinion. The time period allowed in the regulation for the initial assessment is three months from the receipt of the application. The report on the assessment should be sent by the reviewing committee to the European Commission within this period and the Commission is required to forward it to the other member states. The member states then have a further 60 days to make comments or to present a reasoned objection to the marketing of the food or ingredient concerned. If an allowance is made for the time needed to distribute the report, the total time taken should be in the order of 160 to 170 days. In reality, the quickest approval in the first six years took over a year and the others were between two and three years, with the average time being just over two years. For the majority of the applications filed in the first five years, it was found that the applicants had not supplied all the necessary data and the applications were held up until all the information was available. In addition, a number of assessments generated comments and objections from the other member states who were not reviewing the applications and in many cases this resulted in the dossiers being referred to the SCF for an opinion. The SCF is not bound by the time limits given in the regulation. A development not anticipated at the adoption of the regulation was that the member states not undertaking the review were, in most cases, not content with the summaries of the dossier and requested the complete dossier. This has resulted in multiple and sometimes contradictory reviews of the data.

Regulatory context in the EU

7

In July 2002, just over five years after the regulation came into force, the European Commission published a discussion paper on the implementation of the law.6 This covered most of the issues described above and provided some options for change. One change that had already been agreed in 2001 was the removal of the first two categories relating to genetic modifications from the scope of the regulation. A new regulation is being brought in to cover the authorisation of all new foods and ingredients derived from genetically modified organisms. It is envisaged that Regulation 258/97 would then cover the other novel foods and ingredients. Another likely change is the transfer of responsibility for assessment of the dossiers from the national committees to a centralised committee under the European Food Safety Authority.

1.3

EU food law and regulation of food health claims

European food law prohibits the attribution to any food the property of preventing, treating or curing a human disease or any reference to such properties.7 Under medicines law in the EU any substance or combination of substances for treating or preventing a disease in human beings or animals is defined as a medicinal product. This means that any references to a human disease or adverse condition, whether expressed or implied, that are made in the labelling, presentation or promotion of a food are illegal under food law and at the same time may make the product an unauthorised medicine, which is an offence under medicines law.8 This legislation has been very strictly interpreted and enforced. Whilst it has been accepted that statements about the biological functions of nutrients in a healthy body can normally be made, any reference to the effect of a food or one of its components on an unhealthy body, organ or tissue is invariably illegal. The law prohibits any allusion to a role of the food or a component of a food in the prevention of a disease, even if the claim has good scientific support such as for the role of folic acid in preventing neural tube defects. This example has already brought about an interesting situation in the United Kingdom where the Chief Medical Officer wrote to food supplement companies asking them to include a 400lg folic acid tablet in their range for women expecting to become pregnant, or in early stages of pregnancy. However, at the same time, the companies were informed that it would be illegal if the reasons for the product were stated on the label or in advertising. It is legal to state that `calcium is essential for healthy teeth and bones' but the statement `extra calcium may prevent osteoporosis' would break the law. In some countries of the EU there is either specific national legislation covering medicinal claims, or a more stringent interpretation of the law. For example, there is specific legislation in the United Kingdom, over 60 years old, which makes it illegal to make any reference to cancer in respect of a product or medical device.

8

Functional foods, ageing and degenerative disease

1.3.1 Food claims Claims made for foods can be classified as follows: · Nutrition claims: These, in general, relate to the nutrient content of the food and include statements for high or low contents such as `rich in protein' or `low fat'. Such claims relate to the presence or absence of a nutrient, normally with quantification. · Nutrient function claims: Claims in this category relate to the function of a nutrient, or group of nutrients, in a normal, healthy human. These can be as diverse as `glucose for extra energy' and `iron to help maintain healthy blood'. · Health claims: These are claims that state or imply that there is a relationship between the consumption of a food, or a component of a food, and health. Health claims make a reference to the health of the individual, who may benefit from the consumption of appropriate amounts of the food and can include claims for the reduction of disease risk. Health claims have to be carefully worded to ensure that the distinction is made between the `prevention' of a disease and the `significant reduction of a disease risk factor'. The former implies that the food may play a part in the prevention of a disease or condition, whilst the latter relates to the effect of the food on the management of aspects or factors known to play a part in the development of a disease. Legislation on food and health claims has been a controversial issue in the European Union for well over 20 years. The first proposal for a directive on food claims was circulated by the European Commission in 1980. Agreement on the content could not be reached by the then smaller group of member states and the proposal was dropped. Almost 12 years passed before the issue was resurrected with a revised proposal from the European Commission in 1992. This draft turned out to be even more contentious than the earlier one and no agreement could be reached, even though it was considerably amended during the discussions. This second draft was withdrawn by the Commission in 1995. In mid-2001, over five years later, a new discussion paper was produced that covered only nutrient and nutrient function claims. The important area of health claims was completely avoided in this document even though a number of member states (such as the Netherlands, Sweden, and United Kingdom) had already introduced internal procedures for the review and approval of such claims. A year later, in June 2002, the Commission published a draft proposal for a regulation on the control of nutrition, functional, and health claims made for food. This proposal, for the first time, included health claims that were defined as those that state or imply a relationship between a category of food, a specific food, or one of its constituents, and health. Following consultations with member states, consumer groups, and the food industry, the Commission withdrew the draft for further revision. A revised version was published by the Commission in March 2003, which was further revised in June before the Commission formally adopted the proposal in the middle of July 2003.9 Whilst

Regulatory context in the EU

9

there is provision for claims for the reduction of disease risk, the categories of functional claims and enhanced function claims in earlier drafts have been deleted, leaving only two: nutrition claims and health claims. Nutrition claims are those relating to the presence, absence, or increased or reduced levels of nutrients in a food. Such claims will be allowed only if the food meets all the conditions laid down in an annex to the proposal. For example, a claim for `no added sugar' may be used only if the product does not contain any added mono- or disaccharides or any other food used for its sweetening properties. Thus, the claim cannot be made for a food that does not contain any added sucrose if other carbohydrates with a sweetening effect (e.g. fructose, glucose, syrup, or honey) have been added. A claim for a natural source of a vitamin or mineral will be allowed only when the product contains at least 15% per 100g or 100ml of the recommended daily allowance (RDA) given in the directive on nutrition labelling 90/496/EEC.10 A claim that the food is high in vitamins or minerals will require at least 30% of the RDA per 100g or 100ml. A real danger in the proposal is contained in Article 11, which lists a number of prohibited claims. The first relates to general, non-specific benefits of a food or its nutrients for overall good health or well-being. In its preamble to the proposals the Commission has given examples of claims that will be banned. These include `helps your body resist stress', ` has a harmonising effect on your metabolism', `reinforces the body's resistance', and `has a positive effect on well-being'. The Commission also believes that there may be cases of scientifically truthful but highly specialised claims that should be prohibited and gives as an example the claim that `folate may help normalise plasma homocysteine levels'. It is also intended that the prohibition should include all claims making reference to psychological and behavioural functions. This is surprising in light of the EU consensus on functional foods published in 1999, which discusses and accepts such functions.11 A draconian prohibition is that preventing the reference to slimming and weight control in the labelling, presentation or advertising of foods. Also banned will be references to the rate or amount of weight loss that may result from the use of a product or any reduction in the sense of hunger or increase in sense of satiety resulting from its consumption. References to the reduction of the available energy from the diet will also not be allowed. The proposal also states that claims must not make a reference to the advice of doctors or other health professionals, or their professional associations, or charities, or suggest that health could be affected by not consuming the food. There is a general requirement that the use of both nutrition and health claims must not be false or misleading, give rise to doubt about the safety and/or the nutritional adequacy of other foods or state or imply that a balanced or varied diet cannot supply appropriate quantities of nutrients in general. The reference to changes in bodily functions in `improper or alarming terms' either in the text or through pictorial, graphical or symbolic representations will also be prohibited. Health claims will be permitted but will be subjected to a number of very stringent conditions. There is provision for health claims that describe the role of

10

Functional foods, ageing and degenerative disease

a nutrient or of another substance in the growth, development, and normal functions of the body and which are generally accepted by scientific data and are well understood by the consumer to be listed in a register maintained by the European Commission. Claims will then be allowed without prior approval, provided all the conditions laid down for the claim are met. To enable the register to be compiled, each member state of the EU will be required to provide the Commission with a list of claims acceptable in their territories. The national lists are required to be with the Commission within one year from the date the proposed regulation enters into force. For other health claims that are not on the register, there will be an official approval procedure. Although the detailed requirements are not given in the draft, all the indications are that a significant amount of scientific data will be required to substantiate the claim. An application for approval must also include proposals for the wording of the claim in all the languages of the EU. Achieving this may be a complex exercise as it has already been found that there are difficulties in accurately translating some relatively straightforward statements into the major European languages. There is also a specific requirement that claims for the reduction of a disease risk must include a statement indicating that diseases have multiple risk factors and that altering one of these factors may, or may not have a beneficial effect. The scientific aspects of the application and wording of the proposed claim will be evaluated by the European Food Safety Authority. There is provision in the draft for public comment on the Authority's decision, which must be made within 30 days of its publication. It is highly unlikely that the application route will be an easy option for the food industry, and the cost of obtaining the clinical data to substantiate the claim may make it non-viable for even the larger companies. The time scale before the Commission proposal comes into effect is not totally predictable, but it is unlikely that it will have passed through all the stages in the European Parliament and Council before the end of 2004 or the early part of 2005. It will come into effect on the first day of the sixth month following its publication in the Official Journal of the European Union. Foods placed on the market before the date of publication that do not comply may remain on the market until the last day of the eleventh month following the publication.

1.4

National initiatives to regulate food health claims

In the absence of EU legislation on claims, a number of member states have introduced their own systems for the approval of claims. 1.4.1 Sweden Sweden was the first of the EU countries to adopt a self-regulating procedure and the first initiative to regulate health claims was agreed by the industry and

Regulatory context in the EU

11

came into effect in August 1990. This was monitored for three years, until July 1993, and revised rules were introduced in August 1996, coming into effect from January 1997.12 According to the Swedish rules, a health claim must consist of two parts. The first must provide information on the diet and the health relationship of the food. The second part consists of information on the composition of the product for which the claim is made. For example, the permitted claims for iron and omega-3 fatty acids would be: · Part 1. Iron deficiency is common among women but can be prevented by good dietary habits. Part 2. Product X is an important source of the type of iron that is readily absorbed by the body. · Part 1. Omega-3 fatty acids have a positive effect on blood lipid and can therefore help protect against cardiovascular disease. Part 2. Fish product X is rich in omega-3 fatty acids. The Swedish system also provides a list of approved nutrient function claims such as `this product contains zinc, which is a component in many of the body's enzyme systems'. Such nutrient function claims can only be made for products that contain a significant amount of the nutrient claimed. A significant amount is generally considered to be 15% of the recommended daily intake per 100g or 100ml of the product, or of a package that contains one serving. Whilst the Swedish procedure is very prescriptive in the sense that it lays down the rules under which specified claims can be made, other countries have developed voluntary agreements between government, the food industry, and enforcement agencies, which allows for a prior approval for health claims. 1.4.2 United Kingdom The United Kingdom is operating a claims approval process under the auspices of the Joint Health Claims Initiative (JHCI).13 The JHCI developed from a review of the British market for functional foods and associated health claims carried out by the government's Food Advisory Committee in 1996. The JHCI was convened with the objective of establishing a UK Code of Practice for the use of health claims for foods and is a joint venture between the enforcement authorities, industry trade associations, and consumer organisations. The JHCI provides an independent scientific opinion on the validity of claims to help ensure that claims do not mislead the consumer or contravene food law. Whilst the JHCI code is not part of UK food legislation, compliance with the Code assists companies to establish a defence of due diligence in the event of a challenge or prosecution over the truthfulness of a claim. The claims approval process is based on a detailed and systematic review of the scientific evidence (section 1.5) by an independent panel of suitably qualified experts.

12

Functional foods, ageing and degenerative disease

1.4.3 The Netherlands A similar Code of Practice has been introduced into the Netherlands under the auspices of a number of interested organisations representing the food manufacturers, retailers, consumer associations and the Netherlands Nutrition Centre (Voedingscentrum).14 Although this Code has not been officially adopted by the Dutch government, representatives of the government have declared official support for it. Under this Code, health benefit claims have to be assessed by an independent panel of experts appointed by the Netherlands Nutrition Centre. There is an essential requirement that the supporting evidence for the claim must be based on relevant data from human subjects. 1.4.4 Belgium In Belgium the work on a Code of Conduct on Health Claims was undertaken by Fevia, the food industry federation.15 Unlike the British and Dutch codes, this does not call for an independent assessment of the claim but requires that the claim must be scientifically justifiable. All the data used to provide substantiation for the claim must be collected into a dossier, which must be retained by the person responsible for making the claim and made available on request to the food inspection service. The Code has a section that lays down in some detail the criteria that should be used for the scientific justification. 1.4.5 Spain In 1998 an agreement on health claims for foods was reached by the Spanish Ministry of Health (Ministerio de Sonidad y Consumo) and the Spanish Federation of Food and Drinks Manufacturers (FederacioÂn de Industrias de AlimentacioÂn y Bebidas FIAB).16 This agreement is voluntary and clarifies the situation relating to health claims within the legislation. There is a general requirement that all health claims must be truthful and be able to be clearly substantiated by scientific evidence. Where a claim is made for the beneficial properties of an ingredient rather than for a food, the nutrient or component that is claimed to have the beneficial properties must be present in sufficient quantities to produce the claimed effect. The same rule applies to claims for the absence of a specific nutrient or component (e.g. saturated fat). There is also a requirement that whenever a health claim is made in the promotion or labelling of a food, it should be accompanied by a statement on the importance of maintaining a healthy and balanced diet. As part of the Spanish agreement a joint committee of officials from the Ministry and representatives from the food industry has been set up to review proposed claims that can be submitted on a voluntary basis before marketing. 1.4.6 France In September 1997 the French National Dietary Council (Conseil national de l'alimentation) considered their position on claims linking diet and health.17

Regulatory context in the EU

13

Following a wide consultation, which included consumer groups, the food and drink industry, and food scientists, the council concluded that provided certain principles were maintained, health claims could be acceptable. The Council felt that health claims would require prior authorisation before marketing unless they appeared on an approved list. It also recommended that for new claims, and possibly for all claims, the validation of the scientific data that forms the basis of the claims should be undertaken by independent organisations such as the French Human Nutrition Research Centre. The scientific justification should be of the highest standard.

1.5

Approval and substantiation of health claims

Whilst there have been a number of approaches to the validation of health claims around the world, a common denominator is that they all require a high level of scientific substantiation for the claim. The type of claim being made will, to some extent, determine the type of evidence required. For example, claims relating to the role of a nutrient in the body can normally be supported by generally accepted and established scientific knowledge, whereas a claim for the reduction of a disease risk factor may require more substantial data from largescale human studies. The approval of a health claim requires that there be significant scientific agreement among appropriately qualified experts. This can be obtained only from a critical review of the totality of the evidence. This requires a systematic and totally objective compilation of all the available evidence. There are now accepted procedures for ensuring that all the evidence relating to a scientific question is identified, collected and evaluated for its relevance. These are outlined in a consensus paper prepared by the members of a European project team working on the process for the assessment of scientific support for claims on foods (PASSCLAIM).18 This paper emphasises that the compilation of the evidence, including published scientific literature, must be carried out in a balanced and unbiased way to ensure that all relevant data, both positive and negative, has been included. Whilst it can be expected that some studies may provide negative or contradictory results, the weight of the evidence must clearly substantiate the claim. The form and nature of the scientific evidence is critical for the support of a claim. The design of any study and its methodological soundness, execution and analysis is of paramount importance when assessing its value in support of the claim. In general, the scientific evidence in support of a health claim is likely to be obtained from three groups of studies: 1. 2.

Human intervention studies. These are experimental human studies, preferably based on well-designed, randomised controlled trials. Human observational studies (epidemiological studies). These may be either prospective or retrospective. In prospective studies, the subjects can be

14

3.

Functional foods, ageing and degenerative disease selected and observed prior to the outcome of the study whereas in retrospective studies the subjects are interviewed and their records retrieved after the outcome has occurred. Animal studies and in vitro studies. These studies may be able to provide evidence to support a health claim, particularly for evidence on doseresponse relationships, mechanisms of action and on the processes that can cause the disease. Animal studies can suffer from the problems of differences in the metabolisms of the test animal and humans, which can lead to problems with the extrapolation of the results to the physiological effects in humans.

In terms of a hierarchy of evidence, human studies are accorded greater weight than animal and in vitro studies, and human intervention (clinical) trials have greater weight than observational (epidemiological) studies. However, there are a number of factors that can influence the validity of the studies. The relevance of a study is improved if the subjects are representative of the target group intended to be covered by the claim and where they have consumed a reasonable amount of the food or the active component of the food at a reasonable frequency. A study should be large enough to demonstrate the expected beneficial effect and its duration should be long enough to demonstrate the beneficial effect is a long-term and not a short-term reaction. The outcome of any studies should be the same or similar to the claimed effect when measured according to standard procedures. If the claim relates to the reduction of a risk factor for a disease, a measurement of that risk factor should be incorporated into at least some of the studies used to substantiate the claim. It is important that during the evaluation of the studies, possible confounding factors are taken into account. For example, the age of the subjects could be a confounding factor when looking for an association between a food and a beneficial effect.

1.6

Medicinal products and EU legislation

Whilst the European definition of a food is very broad, there are a number of substances and products that have been determined to be medicinal and which cannot be sold under food law. Medicinal products need an official authorisation to market them in the EU. The definition of a medicine/medicinal product in EU legislation is in two parts. The first part relates to the way a product is presented and the second relates to the function of a substance or product. The EU Directive on Medicines 2001/83/EC8 in 1965 defines a medicine as `any substance or combination of substances presented for treating or preventing disease in human beings or animals'. This part of the definition is generally known as the `presentation' section as it refers to the manner in which the product is presented or advertised. A second part, which refers to the function of the actual substance or product states that `any substance or combination of substances which may be administered to human beings or animals with a view

Regulatory context in the EU

15

to making a diagnosis or restoring, correcting or modifying physiological function in human beings or animals is likewise considered a medicinal product'. Thus, the law provides two definitions of a medicine, one relating to the presentation of a product and the other relating to the function. With regard to the second part of the definition, it can be explained in simple terms as `if a substance or product can be demonstrated to have a significant physiological effect on the body which is not one obtained from recognised nutrients or combination of nutrients, there is a strong possibility that the substance or product could be classified as a medicine by function.' A `medicinal by function' substance or product cannot be sold under food law, including sale as a supplement, in the EU, although there have been some inconsistencies of interpretation of the status of borderline substances between the member states. Although there is not a total consistency between member states, the majority regard most herbs or herbal extracts that exhibit a pharmacological effect to be medicines. This is in contrast to the USA where many medicinal herbs can be sold in food supplements. Whilst some products could fall under both parts of the definition, the European Court of Justice has confirmed its view that a product can be deemed to be a medicine if it falls into only one of the two parts. Thus, an innocuous product can be determined to be a medicine on the basis of labelling or promotional statements and a product with no marketing statements can be a medicine on the basis of its composition. When evaluating a functional substance, great care must be taken to confirm its status as this will determine whether it can be sold as a functional food or whether it will be controlled as a medicine.

1.7 1. 2. 3. 4. 5. 6. 7.

References BERRY OTTAWAY P (1995) `Harmonisation of European Food Legislation.' FT Management Report Series, Pearson Professional Publications, London. EUROPEAN COMMISSION (2000) `White Paper on Food Safety'. COM(1999) 719 Final of 12 January 2000. SCIENTIFIC COMMITTEE ON FOOD OF THE EUROPEAN COMMISSION (2001) `Guidance on Submissions for Food Additive Evaluations by the Scientific Committee on Food' SCF/CS/ADD/GEN/26 Final of 12 July 2001. European Parliament and Council Regulation (EC) No. 258/97 concerning Novel Foods and Novel Food Ingredients. O.J. of E.C. L43/1 of 14 February 1997. European Commission Recommendation 97/618/EC on Scientific aspects and presentation of information to support applications under Regulations (EC) No. 258/97. O.J. of E.C. L253/1 of 16 September 1997. EUROPEAN COMMISSION (2002) `Discussion paper on implementation of Regulation (EC) No. 258/97' SANCO D4 July 2002. European Parliament and Council Directive 2000/13/EC relating to labelling, presentation and advertising of foodstuffs. O.J. of E.C. L109/29 of 6 May 2000.

16 8. 9. 10. 11. 12. 13. 14. 15. 16.

Functional foods, ageing and degenerative disease European Parliament and Council Directive 2001/83/EC on medicinal products. O.J. of E.C. L311/67 of 28 November 2001. EUROPEAN COMMISSION (2003) `Proposal for a Regulation of the European Parliament and Council on nutrition and health claims made on foods' COM(2003) Final, Brussels. European Council Directive 90/496/EEC on nutrition labelling for foodstuffs. O.J. of E.C. L276/40 of 6 October 1990. DIPLOCK A T, AGGETT P J, ASHWELL M, BARNET F, FERN E B, ROBERFROID M B (1999) `Scientific concepts of functional foods in Europe: Consensus Document'. British J. Nut. 81 : S1-S19. FEDERATION OF SWEDISH FOOD INDUSTRIES et al. Health Claims in the Labelling and Marketing of Food Products. Food Industry's Rules (Self Regulating Programme) Revised programme of 28 Aug 1996. JOINT HEALTH CLAIMS INITIATIVE (UNITED KINGDOM) (1998) `Code of Practice on Health Claims on Foods'. Final Text 9 November 1998. VOEDINGSCENTRUM (NETHERLANDS). Code of practice assessing the scientific evidence for health benefits stated in health claims on food and drink products, April 1998. FEDERATIE VOEDINGSINDUSTRIE/FEÂ DEÂ RATION DE L'INDUSTRIE ALIMENTAIRE (BELGIUM).

Health Claims Code of Conduct, draft, 21 Oct 1998.

JOINT `MINISTERIO DE SONIDAD Y CONSUMO' (MINISTRY OF HEALTH, SPAIN) AND Â N DE INDUSTRIAS DE ALIMENTACIO Â N Y BEBIDAS (SPANISH FEDERATION OF FEDERACIO FOOD AND DRINK MANUFACTURERS).

17. 18.

March 1998.

Agreement on health claims on foods of 20

AlleÂgations faisant un lien entre alimentation et santeÂ. Avis No. 21, 30 June 1998, Paris. RICHARDSON D P, AFFERTSHOLT T et al. (2003) `PASSCLAIM Synthesis and Review of Existing Processes'. Eur. J. Nutr. 42 (Suppl 1) 1/96-1/111. CONSEIL NATIONAL DE L'ALIMENTATION (FRANCE).

2 Diet and the prevention of degenerative disease L. Kalbe, B. Reusens and C. Remacle, Universite Catholique de Louvain, Belgium

2.1 Introduction: epidemiological studies and the influence of diet in early life 2.1.1 The influence of diet in early life on susceptibility to degenerative disease Imbalances in maternal nutrition can adversely affect normal foetal growth and development. Impaired foetal growth is prevalent in developing countries and has been associated with negative short- and long-term outcomes such as increased perinatal morbidity and mortality, infant mortality and childhood morbidity. Children who experience impaired foetal growth are more likely to show poor cognitive development and neurological impairment. Some chronic adult diseases are hypothesized to originate in utero: cardiovascular disease, high blood pressure, obstructive lung disease, diabetes, high cholesterol concentration, renal damage (Barker, 1994).

2.1.2 Epidemiological studies pointing the concept of early origin of adult disease Twenty-five years ago, an association between poor maternal and neonatal health and subsequent disease many years later had already been established. For Forsdahl (1977), the link was the result of poverty during adolescence. However, Barker and Osmond (1986) proposed that poor nutrition in foetal and early life may be responsible for this association. Three years later they published the results of an epidemiological study of a population born in

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Hertfordshire for which the birth weight data were available. They found that there were increased death rates from ischaemic heart disease in men with low birth weight and weight at one year of age (Barker et al., 1989). The concept of foetal origin of adult diseases was then proposed. Low birth weight is the proxy of poor foetal nutrition. When poor nutrition occurs during foetal development, organ-selective changes in nutrient distribution have to take place so that the growth of some organs will be spared (e.g. the brain) to the detriment of other organs (e.g. the viscera), serving the purpose of enhancing postnatal survival under conditions of intermittent and poor nutrition. It was thus realized that some of the persisting effects of early undernutrition become translated into pathology, and thereby determine chronic diseases in later life. The notion of `programming' was defined by Lucas (1991) as the process whereby a stimulus or input during a sensitive period of development has permanent effects on the structure, physiology and metabolism of the organ. Since then, numerous retrospective and prospective epidemiological studies were undertaken aiming to support this hypothesis of the foetal origin of adult degenerative diseases. The association between low birth weight and coronary heart disease has now been confirmed in Sweden (Leon, 1998), Finland (Forsen et al., 1999), United States (Rich-Edwards et al., 1997) and in South India (Stein et al., 1996). The same correlation was found between low birth weight or weight at one and the subsequent metabolic syndrome (glucose intolerance, hypertension and raised plasma triglyceride concentration) as well as type 2 diabetes (Hales et al., 1991, Barker et al., 1993). Bavdekar et al. (1999) studied a cohort of eight-year-old Indian children to define the relationship between birth weight and cardiovascular risks factors, including insulin resistance. Here again, the highest levels of insulin resistance and LDL-cholesterol were in children of low birth weight. Likewise, the epidemiological studies on the offspring of the Dutch Famine also support the hypothesis of chronic degenerative disease (raised blood pressure, coronary heart disease, glucose intolerance, hypercholesterolaemia) having a possible origin in utero. This famine took place from October 1944 to April 1945 and the daily caloric intake fell from 1800 calories to less than 600 calories. Rosenboom et al. (2000) provided evidence that people exposed to famine in early gestation had more atherogenic lipid profiles, somewhat higher fibrinogen concentrations and reduced plasma concentrations of factor VII. The long-term effect of intrauterine undernutrition, however, depends upon its timing during gestation and on the tissues and systems undergoing critical periods of development at that time. These studies also suggested that maternal malnutrition during gestation may permanently affect adult health without affecting the size of the baby at birth. The relation between poor nutrition in utero and obesity is less clear. Ravelli et al. (1999) had analysed the offspring of the Dutch Famine and demonstrated that maternal malnutrition during early gestation was associated with higher Body Mass Index and waist circumference in 50-year-old women, but not in men, although this association had been previously demonstrated in young men

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19

in the same cohort (Ravelli et al., 1976). These findings suggest that perturbations of central endocrine regulatory systems established in early gestation may contribute to the development of abdominal obesity in later life. Not only undernutrition during foetal life may have lasting consequences for the health of the offspring. Overfeeding of the foetus as it occurs in gestational diabetes may have a dramatic impact for the progeny as well. Intrauterine exposure to diabetes conveys risk factors for type 2 diabetes and obesity in the Pima Indian population where diabetes has the highest prevalence (Dabelea et al., 2000). Indeed the risk of diabetes was higher in siblings born from diabetic mothers than those born before the mother became diabetic. Breast cancers have been linked to high birth weight (Michels et al., 1996; Vatten et al., 2002). It has been suggested that high concentrations of estrogen in pregnancy may play a role (Sanderson et al., 1996). Strong evidence for the influence of intrauterine factors in the future risk of breast cancer was provided by Vatten et al. (2002). A common feature of such factors would be their ability to promote foetal growth and simultaneously intrauterine development of the mammary gland. From a biological point of view, the concept of foetal programming of adult disease is not surprising. Starting from the zygote, the growth and development of the organism involves cell proliferation, and cells undergo usually several steps of commitment which progressively restrict the variety of the differentiation potential. These commitments are accompanied by mitotic runs. The lineage then evolves towards a differentiated, functional state, either definitely or reversibly. During development, not only cell divisions and differentiation occur, but also programmed cell death. Programmed cell death suppresses unnecessary cells during development, like cells of interdigital webbing or the muÈllerian ducts. It eliminates T and B lymphocytes that are not adequately programmed, neuronal and glial cells in excess in the developing nervous system, or cells with damaged DNA. During that period of coordinated growth and progressive acquiring of function, it may be expected that alterations of the milieu would have permanent consequences. They would indeed modify pools of precursors, changing the future potentiality of the organ. In fact, corresponding to critical periods of cell growth or regulation, differential vulnerability of different organs to teratogens has been known for many years. The same would be true for nutrient supply to the embryo, foetus and child. Appropriate supply of bulk nutrients must not only provide raw material for the construction of the organism, but also specific nutrients may interfere with the precise regulation of development. Numerous examples of gene regulation by nutrients are indeed known. It is the case, for example, of fatty acids on the transcription of a number of genes characteristic of adipocyte differentiation and function (Antras-Ferry et al., 1995), of glucose on three enzymes of the glycolytic-lipogenic pathway in liver and adipose tissue (Foufelle et al., 1992), or of cholesterol on LDL receptor gene expression (Wang et al., 1994). However, despite considerable progress in cell biology studies using tissue culture systems and the identification of different requirements for

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the growth and differentiation of varied cell types, we are still a long way from identifying all the stimulatory and inhibitory molecules that regulate human growth and development in vivo. 2.1.3 Animal models supporting the concept of foetal origin of adult diseases To prevent disease, we need to progress beyond epidemiologic associations to greater understanding of the cellular and molecular processes that underlie them. We need to know what factors limit the delivery of nutrients and oxygen to the human foetus, how the foetus adapts to a limited supply, how these adaptations programme the structure and physiology of the body, and by what molecular mechanisms nutrients and hormones alter gene expression. Further research requires a strategy of interdependent clinical, animal and epidemiological studies (Osmond and Barker, 2000). The concept that maternal or foetal nutrition can programme adult disease is now well established in animal models. Thirty years ago, Winick (1970) demonstrated that poor nutrition during gestation irreversibly led to reduced cell numbers in tissues such as the pancreas. Since then, different models of malnutrition occurring during different periods of development were set up; caloric restriction, protein restriction, and a high fat diet were the most used. In each model, alterations in organ development were observed and later consequences for the health of the progeny were reported. Few examples will be developed below, but more information can be found elsewhere (Lucas, 1998; Bertram and Hanson, 2001; Reusens and Remacle, 2001). Dobbling and Sands (1971), and Smart (1986) showed that malnutrition during a vulnerable period of brain development may have a permanent effect on brain size, brain cell number, behaviour and learning. More recently, Bennis-Taleb et al. (1999) reported permanently reduced brain vascularization after giving a low-protein diet to pregnant rats. Maternal low-protein models of foetal programming have been used to investigate the mechanisms linking maternal nutrition with impaired foetal growth and later cardiovascular disease, hypertension and diabetes. Depending on the source of carbohydrate, fat content, fatty acid composition and methionine, hypertension was observed at adulthood (Langley-Evans, 2000, Hales et al., 1996). If the low-protein diet was restricted to the pre-implantation period (Kwong et al., 2000) hypertension was also observed. The number of mature glomeruli in the kidney has been shown to be reduced at birth in such experimental conditions (Merlet-Benichou et al., 1994). Proteins play a key role in the development of the islets of Langherhans in utero. Foetuses and neonates from dams fed 8% protein instead of 20% exhibited a poor development of the endocrine pancreas including its vascularization; cell mass being reduced due to lower proliferation rate, more apoptosis and less IGF-I and -II, survival factors prevent apoptosis (Snoeck et al., 1990, Petrik et al., 1999). Islet insulin secretion was reduced at least by 50% in response to different

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secretagogues (Cherif et al., 1998). In addition, low-protein foetal islets were more vulnerable to the cytotoxic effect of IL-1 , a cytokine involved in the destruction of cells in type 1 diabetes (Merezak, et al., 2001). Despite feeding the animals with a normal diet after weaning, the adult offspring showed a lower plasma insulin level and insulin secretion after glucose challenge (Dahri et al., 1995). In that animal model of malnutrition, the lower level of taurine, an important amino acid during development, was proposed to be partially responsible for the various alterations because the simple supplementation of the maternal low-protein diet with taurine was sufficient to restore most of the altered parameters (Cherif et al., 1998, Merezak et al., 2001, Boujendar et al., 2002, 2003). A maternal low-protein diet also induced changes in zonation and enzyme activity in the liver of the pups that was not restored at adulthood even when the animals were fed a normal diet (Desai et al., 1995). An over-expression of the insulin receptors was observed in the liver, adipocytes and hepatocytes in such offspring (Ozanne and Hales, 2002). Caloric restriction during pregnancy in rats led to similar results. The -cell mass was also reduced, insulin secretion was blunted at three months and glucose intolerance appeared later (Garofano et al., 1997, 1999). Rats whose mothers had restricted food during the first two weeks of pregnancy became obese, but depending on the strain and the diet used, it was either the male or the females which were affected (Jones and Friedman, 1982, Anguita et al., 1993). Vickers et al. (2001) showed that severe maternal undernutrition throughout pregnancy in rats results in obesity, hypertension, hyperinsulinemia and hyperleptinemia in the offspring when they reach adulthood. It is thus obvious that these and other animal models are needed to reveal the specific mechanism by which foeto-maternal nutrition leads to degenerative disease in order to pave the way for simple nutritional preventive intervention.

2.2

Foetal and neonatal nutritional requirements

Maternal education is acknowledged to be one of the most important criteria for determining good public health. Irrespective of any other factor such as cultural, racial or religious background, it is the parents' and in particular the mothers' educational level that determines the type of nutritional criteria applied in her child's education, her capability to adapt to new imperatives, and ultimately the nutritional problems the child will suffer from. In addition to short-term effects on the growth, body composition and functions of the small child, appropriate nutrition of the mother during gestation and lactation, as well as of the young children is now considered of highest importance to reduce the increasing burden of adult degenerative disease. Malnutrition concerns more than mere shortage of food. Inadequate food intake, be it from lack or excess of food, from dietary imbalance, or from selective deficiency of micronutrients, may lead to programming of degenerative diseases. Since the unborn child and the infant have only limited stores of nutrients, as well as an immature metabolism and body functions that would enable its

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developing body to compensate for inadequate food supply, any lack, excess or imbalance of its prenatal and early postnatal diet will, therefore, have a determining impact. During pregnancy and lactation, nutritional requirements of the mother increase to support foetal and infant growth and development, as well as maternal metabolism and tissue development specific to reproduction (Picciano, 2003). Prenatal nutrition of the mother is also of importance for providing adequate availability of a number of nutrients. The mother's organism constitutes indeed a reservoir from which the foetus and infant will extract what is needed for his or her growth and development. Total nutrient requirements are not necessarily the simple sum of those accumulated in maternal tissues, products of pregnancy and lactation and those attributable to maintenance of the foetus even though this sum is sometimes used to derive estimates of recommended nutrient intakes. Pregnancy and lactation are anabolic states that are orchestrated via hormones to produce a redirection of nutrients to highly specialized maternal tissues characteristic of reproduction (placenta, mammary gland, etc) and their transfer to the developing foetus or infant (Picciano, 2003). From a continuous supply of substrates through the placenta during pregnancy, to a short period of food withdrawal after birth, followed by intermittent feeding with milk and finally weaning with its transition to solid foods, the infant has to repeatedly adapt to profound changes of nutrition (Girard et al., 1992). This requires changes in the metabolism of most organs at a period of its life that is characterized by rapid growth and maturation of its entire organism. Different nutrients are needed for each task. 2.2.1 Prenatal life: placenta Placental development and function determine nutrient transfer to the foetus during this period of life. Programmed cell death of uterine cells allows blastocyst implantation and placentation (Welsh, 1993). Two pathways have evolved for nutrient transfer from the mother to the foetus; histiotrophic and hemotrophic nutrition. In human pregnancy, histiotrophic nutrition lasts for most of the first trimester of pregnancy, i.e. until the 10th±12th week (Burton et al., 2002). During this period, when maternal circulation to the placenta is not fully established, oxygen tension within the placenta is low (Rodesch et al., 1992) and the metabolism of the foetal and placental tissues is largely anaerobic (Jauniaux et al., 2001). During this period of organogenesis, uterine glands are the major source of nutrients for the foetus. These glands secrete glycogen, glycoproteins such as mucin and glycodelin that are broken down, and the constituent sugars and amino acids are then transported to the foetus via the secondary yolk sac (Burton et al., 2002). This provides the foeto-placental unit with a rich source of building blocks to meet its biosynthetic requirements and obviates the need for energy-dependent specific transport systems (Burton et al., 2002). By week ten of pregnancy, the secondary yolk sac shows signs of cellular degeneration (Jones, 1997) and foetal nutrition becomes increasingly hemotrophic as circulation is established on the maternal side of the placenta. In this

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next phase of pregnancy, the main maternal environmental factors that regulate foeto-placental delivery of substrates to the placental site are not only the maternal substrate level, but also the rate of placental-bed blood flow. Thus, maternal factors that change either substrate level or flow alter the foetoplacental growth rate (Clapp, 2002). The placenta has a high metabolic rate throughout pregnancy and actively synthesizes glycogen, lactate, fatty acids, cholesterol and proteins (Sibley and Boyd, 1992). The trophoblast functions of nutrient transport and protein synthesis generate a higher concentration of amino acids in the placental tissue than in either maternal or foetal plasma (Pearse and Sornson, 1969; Valazquez et al., 1976). Placental transport provides the foetus with the nutrients needed for growth and development and disposes of the metabolic wastes. Transplacental transport of free amino acids from the maternal blood to the exocoelomic cavity, for instance, goes against a concentration gradient. All amino acids are not transported with the same efficacy, however. Taurine, glycine, glutamic acid and alanine are the amino acids with the highest placental concentration, whereas alpha-amino butyric acid, tyrosine and histidine are partially retained by early placenta (Jauniaux et al., 1998). Placental function must keep pace with foetal growth, that is, unless placental size and transport capacity increases proportionally with foetal growth, the metabolic demands cannot be met. Insufficient transport of nutrients across the placenta will result in foetal malnutrition and hamper foetal growth. Foetal malnutrition may not only result from maternal malnutrition but also from deficient utero-placental vascularization if the mother is hypertensive (Shah, 2001) and/or smokes. In smokers, the placenta's ability to implant and the uterine vascularization are defective. Changes in both utero-placental and foeto-placental blood flow have been described that may be related to the vasoconstrictive effects of nicotine (Shiverick and Salafia, 1999). In addition, vasomotor tone in smokers is altered due to abnormal arterial uptake of atherogenic plasma proteins (Salafia and Shiverick, 1999). Placenta from smokers are in fact under-perfused, thus supplying the foetus with only limited amounts of nutrients. Preeclampsia is a pregnancy-specific condition that increases maternal and infant mortality and morbidity. It is diagnosed by new-onset increased blood pressure and proteinuria during gestation. The placenta appears to be the pregnancy component that leads to poor perfusion to the foetus in this disease. The epidemiology of preeclampsia, being more common in poor women, suggests that diet may play a role in preeclampsia (Roberts et al., 2003). Women with preeclampsia have reversible increase levels of triacylglycerols and LDLcholesterol and reduced HDL-cholesterol (Hubel et al., 1998). 2.2.2 Prenatal life: maternal nutrition Energy Energy needs during pregnancy are currently estimated by the sum of total energy expenditure of a non-pregnant woman plus the median change in total

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energy expenditure of 8 kcal/gestational week plus the energy deposition during pregnancy of 180 kcal/day. Because total energy expenditure does not change greatly and weight gain is minimal in the first trimester, additional energy intake is recommended only in the second and third trimester. Approximately an additional 340 and 450 kcal per day are recommended during the second and third trimesters, respectively (Institute of Medicine, 2002). Maternal weight and weight gain are remarkably resistant to supplementation. While dietary supplements during pregnancy have a large effect on birth weight in famine conditions, there is only a modest effect in non-famine situations and, in this case, this is not mediated by maternal energy deposition. On the contrary, declining peripheral fat stores in late pregnancy are associated with accelerated foetal growth, improved nutrition leading to lower fat stores. Rather, the component of maternal weight gain during pregnancy associated with foetal growth is water and presumably plasma volume (Rush, 2001). Ramadan fasting does not seem to influence the weight gain of the mother during pregnancy the infant birth weight (Salled, 1989). Anorexia in pregnant women, however, reduced significantly the birth weight of the infant. Complications including hypothermia, hypoglycaemia infection may ensue (James , 2001). Maternal obesity is also a risk factor for both the mother and her child. Pregnancy outcomes such as gestational diabetes, preeclampsia, caesarian section, infections, etc., are significantly more common in obese women (Sebire et al., 2001). Likewise, babies of obese, non-diabetic women were reported to be more likely to suffer from cardiac anomalies (Mikhail et al., 2002). Carbohydrates The best-studied substrate in human pregnancy is glucose and there is a direct relationship between maternal blood glucose, foetal glycaemia and size at birth (Catalano and Kirwan, 2001). Glucose is indeed the major energy source for the foetus, comprising around 90% of the energy supply. Therefore, maternal carbohydrate metabolism during pregnancy and the source of carbohydrate may be relevant to the optimal supply for the foetus. Altering the type of carbohydrate consumed (high- vs low-glycaemic sources) changes post-prandial glucose and insulin responses in pregnant and non-pregnant women, and a consistent change in carbohydrate type eaten during pregnancy influences both the rate of foeto-placental growth and maternal weight gain. A high-glycaemic carbohydrate diet leads to foetal overgrowth and excessive maternal weight gain, while low-glycaemic carbohydrates cause average to low birth weights and normal maternal weight gain. Since changing the type of carbohydrate ingested changes metabolic efficiency and substrate utilization (glucose vs lipid oxidation) this will favour either insulin resistance or sensitivity and may eventually increase or reduce the risk for later obesity or insulin resistance (Clapp, 2002). A high carbohydrate intake in early pregnancy suppressed placental growth, especially if combined with low dairy protein consumption in late pregnancy (Godfrey et al., 1996). The intake of small quantities of animal protein and

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plenty of carbohydrate or vice-versa in late pregnancy, has also been associated with reduced placental size and higher blood pressure in the adult offspring (Campbell et al., 1996). Gestational diabetes mellitus results in an excessive glucose concentration in maternal plasma and, to a certain extent, may be assimilated to a highcarbohydrate diet. It can lead to neonatal macrosomia associated with foetal death, pre-maturity, birth trauma, neonatal stress syndrome, hypoglycaemia, etc. (Cordero and Landon, 1993; Hod et al., 1991; Khwaja et al., 1986). Carbohydrate is transported into the foetus as glucose that is taken up from the maternal plasma by the GLUT1 transporter (Hay, 1994). In diabetic pregnancies, including those under glycaemic control, basal membrane GLUT1 expression and activity are responsible for an increased trans-placental glucose influx and may contribute to foetal macrosomia and other consequences of diabetic pregnancy (Gaither et al., 1999). Lipids The net increase in maternal body weight (free of conceptus) corresponds to the accumulation of fat deposits during the first two-thirds of pregnancy. In the third trimester of pregnancy, the mother's body switches to an accelerated breakdown of these fat deposits. Enhanced maternal insulin levels and changes in insulin sensitivity taking place throughout pregnancy may be responsible for the early anabolism and late catabolism present in maternal adipose tissue during pregnancy. Long-chain polyunsaturated fatty acids circulate in maternal plasma mostly associated to lipoprotein triglycerides, and in a minor proportion in the form of free fatty acids. Despite the lack of direct placental transfer of triglycerides, diffusion of their fatty acids to the foetus is ensured by means of lipoprotein receptors, lipoprotein lipase activity and intracellular lipase activities in the placenta. Maternal plasma free fatty acids, themselves an important source of long-chain polyunsaturated fatty acids to the foetus, are translocated at the placenta by a plasma membrane fatty acid-binding protein (Herrera, 2002). Pregnant women have high requirements for lipid-soluble vitamins and polyunsaturated fatty acids. During pregnancy, concentrations of blood lipids and their constituent fatty acids rise sharply (Al et al., 1995). All of the n-6 and n-3 fatty acid structure needed by the foetus must be supplied by the mother and cross the placenta either in the shape of the essential fatty acids linoleic acid (18:2, n-6) or -linolenic acid (18:3, n-3) or their long-chain polyunsaturated fatty acid derivatives such as arachidonic acid (20:4, n-6) or docohexaenoic acid (22:6, n-3). An adequate supply of essential fatty acids and of long-chain polyunsaturated fatty acids is essential for normal foetal development. Arachidonic acid is the main precursor of eicosanoids, prostaglandins and leukotrienes and is also essential for neonatal growth, whereas docosahexaenoic acid plays a role in brain development and visual function (Herrera, 2002). A high supply of long-chain n-3 fatty acids may be beneficial to the developing foetus for several reasons: their importance for neural tissue development (Koletzko, 1992), a lowering of pregnancy-induced hypertension that may

26

Functional foods, ageing and degenerative disease

induce some obstetric complications (Secher and Olsen, 1990), or an improvement of the average birth weight without adverse effects on foetal growth or the course of delivery (Olsen et al., 1986, 1992). However, fish oil or n-3 fatty acids may also feature adverse effects, such as higher blood loss on delivery due to suppression of platelet aggregation, and higher perinatal mortality (Olsen et al., 1986). The maternal diet, and hence the maternal concentration of individual fatty acids, can influence the delivery of polyunsaturated fatty acids and long-chain polyunsaturated fatty acids to the foetus, because placental selectivity for arachidonic acid increases with maternal arachidonic acid concentration. Placental selectivity for -linolenic acid and docosahexanoic acid, on the other hand, appears to be relatively unresponsive to changes in the fatty acid mixture in the maternal circulation (Haggarty et al., 1999). Excessive consumption of certain long-chain polyunsaturated fatty acids inhibits delta-5- and delta-6desaturases and leads to declines in arachidonic acid or docosahexanoic acid levels (Herrera, 2002). Excess dietary polyunsaturated fatty acids also enhance lipid peroxidation and reduce antioxidant activity (Herrera, 2002). Hence, additional studies are needed before recommendations to increase long-chain polyunsaturated fatty acid intake during pregnancy may be made. On the other hand, it is not clear to which degree the foetus is capable of desaturating and elongating fatty acids, but term and pre-term infants can synthesize long-chain polyunsaturated fatty acids from parental fatty acids (Herrera, 2002). Protein and amino acids Although little is known about the metabolic processes in early foetal organs, important needs for amino acids exist to maintain the intense formation and remodelling of new tissues during embryogenesis and organogenesis. Additional protein is needed during pregnancy to cover the estimated 21 g/day deposited in foetal, placental and maternal tissues during the second and third trimesters (Picciano, 2003). The recommended increment of protein intake over nonpregnancy values is higher than that of energy during pregnancy. In the first trimester of pregnancy, protein synthesis is similar to that of non-pregnant women and increases respectively by 15% and 25% in the second and the third trimester (Duggleby and Jackson, 2002). In less developed countries, however, as well as in specific populations even in Europe and North America, intrauterine growth retardation is clearly associated both with energy and protein deficiency (Kramer, 2002; World Health Organisation, 1995). Folic acid and homocysteine Since mammals cannot synthesize folic acid it must be provided by the diet or by intestinal microorganisms. It is essential for the biosynthesis of some amino acids, neurotransmitters, purines and pyrimidines, and hence DNA and RNA. Compromised maternal folate intake or status is associated with several negative pregnancy outcomes including low birth weight, abnormal placenta, spontaneous abortions, or neural tube defects. (Bung et al., 1995; Pietrzik et al., 1992; George et

Diet and the prevention of degenerative disease

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al., 2002; Bailey et al., 2003). Folate supplementation prevents the occurrence of neural tube defects (Bailey et al., 2003) and might reduce the incidence of low birth weight (de Onis et al., 1998). The recommended supplement is a daily dose of 400± 600 g/day (Institute of Medicine, 1998). Such intervention policy may also influence the programming of cardiovascular disease, since folate modulates the metabolism of homocysteine, which is supposed to increase the risk for atherosclerosis (Bailey et al., 2003). Homocysteine is an amino acid that is involved in several metabolic processes, including the methylation and sulfuration pathways. Blood concentrations of homocysteine are determined by dietary factors such as folic acid and vitamin B12, by altered physiology and by modifications of enzymatic activity due to genetic polymorphism. In normal pregnancy, like those of most of the amino acids, homocysteine concentrations fall (Hague, 2003). Choline A low availability of dietary choline during pregnancy alters foetal brain biochemistry and hippocampal development. This induces behavioural changes that persist throughout the lifetime of the offspring. Humans with choline deficiency but with an otherwise balanced diet develop liver damage due to programmed cell death. Because de novo synthesis of choline is not sufficient to compensate this lack of choline (Zeisel, 2000). In rats, dietary deficiency produced hepatocarcinoma. Female rats were less sensitive to this choline deficiency than males, perhaps because estrogen enhances their capacity to synthesize choline de novo from S-adenosylmethionine. However, pregnant rats were more vulnerable to the lack of choline than males, because maternal stores are depleted due to large-scale transfer of choline to the foetus across the placenta (Zeisel et al., 1995). At birth, plasma choline concentrations are considerably higher than in adult human and other mammalian species (Zeisel, 2000). 2.2.3 Postnatal life Growth rate, as well as physiological and developmental changes are considerable in a normal infant during its first months of life. The growth rate and the allocation of ingested nutrients for growth, development and maintenance change continuously rather than in discrete stages, but these changes are particularly rapid during these early months. Breast-milk provides all the nutrients needed to support adequate growth of the term infant during the first 4±6 months of life. It not only provides the recognized nutrients, but also a number of semi-essential nutrients such as enzymes, hormones, oligosaccharides and growth factors that also intervene in infant growth such as intestinal maturation (Koldovsky and Thornburg, 1987; Koldovsky and Strbak 1995). Carbohydrates This breast milk fraction contains, in addition to monosaccharides, oligosaccharides, nucleotide sugars, glycolipids, glycosphingolipids and

28

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glycoproteins. The high-molecular mass glycoprotein fraction in human milk and lactating tissue that contains up to 80% carbohydrates is thought to keep the ducts and lumen of the mammary glands open (Patton et al., 1995; Welply et al., 1994). Human milk contains more than 130 different oligosaccharides and is unique among mammalian species for its content of higher oligosaccharides (i.e. larger than lactose, the most abundant milk sugar), the oligosaccharide fraction being not only the third largest in human milk, but also larger than the protein fraction (Egge et al., 1983). Since oligosaccharides escape hydrolysis in the small intestine, they may have two functions. First, due to their intact absorption, they may serve as substrates for the maturation of organs such as the brain, where rapid synthesis of glycoproteins and glycolipids occurs (Kunz et al., 2000). Second, since the milk oligosacchrides are synthesized by the same glycosyltransferases as the oligosaccharide moieties of cell surface glycoproteins and glycolipids (Koletzko et al., 1998) they may act as analogues to host cell surface receptors for pathogens and therefore protect against infection (Anderson et al., 1986; Cravioto et al., 1991). Fatty acids The status of maternal very-short-chain polyunsaturated fatty acids during pregnancy is critical for the very-short-chain polyunsaturated fatty acid status in the newborn (Al et al., 1990). Newborn infants depend on a dietary supply of these fatty acids (Farquharson et al., 1992; Makrides et al., 1994). In contrast to most formulas, breast milk contains docohexaenoic acid and arachidonic acid. The concentration of polyunsaturated fatty acids varies, depending on the mother's diet. It is unclear how intakes of n-3 and n-6 long-chain polyunsaturated fatty acids affect the growth of specific tissues, but the fatty acid composition of certain organs and their plasma membranes, as well as their susceptibility to oxidants, growth and function depend on the composition of the diet (Suarez et al., 1996a, b). There is a positive correlation between body weight and plasma triacylglycerol content of arachidonic acid and total n-6 longchain polyunsaturated fatty acids (Koletzko and Braun, 1991). The arachidonic acid content of milk formula modulates the weight gain of the pre-term infants during the first year of life (Carlson et al., 1993). The nature and amount of the dietary lipids also determines the immune response to allergens, influences autoimmune disease or trauma by affecting both the cell-mediated and the humoral immunity due to the alteration of arachidonic acid metabolism, production of inflammatory cytokines and impairment of the reticulo-endothelial system (Endres, 1996; Hellerstein et al., 1996; Watanabe et al., 1994). With regard to excessive fat intake and maternal obesity, evidence from experimental models shows that they reduce fertility, increase the likelihood of difficult deliveries, reduce litter sizes and pup growth. They cause difficulties in initiating lactation and lower milk production and may lead to an increased risk of pup death (Rasmussen et al., 2001).

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Proteins Human milk contains a wide variety of proteins that contribute to its unique quality. Some of these proteins are digested and provide the amino acids needed for rapid growth. Others participate in the digestion and utilization of micro- and macronutrients. Some proteins are resistant to the proteolysis and may contribute to the defence of the infants, whereas others are involved in the development of the intestinal mucosa (LoÈnnerdal, 2003). Both total protein content and the concentrations of individual proteins in human milk change throughout the first year of lactation. Recent reassessments of estimated requirements have led to a lowering in the total protein recommendation. However, an emphasis is put on the provision of alpha-amino nitrogen because most of the non-nitrogen is not used for the maintenance or tissue deposition. It was shown that 13±15 g/l of total protein milk were able to promote adequate growth. This quantity corresponds to that found in breast milk (Dupont, 2003). Protein-calorie malnutrition, as well as micronutrient deficiencies are associated with impaired immunity, even when the deficiency state is relatively mild. Low-birth-weight infants, in particular, have a prolonged impairment of cell-mediated immunity that can be partly restored with zinc supplementation. Immunity is not only affected by under-nutrition, however. Over-nutrition and obesity also alter an individual's immunity (Chandra, 2002). Altered immunity renders malnourished populations, especially mothers and young children, more prone to infectious and metabolic disorders. Non-protein nitrogen Non-protein nitrogen represents around 20±30% of human milk nitrogen and comprises nucleotides, aminosugar oligosaccharides, free amino acids like taurine, arginine and glutamine, polyamines and amino alcohols of phospholipids (e.g. choline). The intake of nucleic acids depends on the number and quality of cells of the ingested food, since fish, meat and seeds are rich in nucleic acids, but fruits and vegetables are poor (Gil and Uauy, 1995). Preformed nucleotides may be important for the growth of tissues with a rapid turnover (Gil and Uauy, 1995) like bone marrow, leucocytes and intestinal mucosa that preferentially use the nucleotide salvage pathway to fulfil their nucleotide requirements (Cohen et al., 1984). For instance, the dynamic balance of cellular turnover in the developing human small intestine is controlled by AMP (Tanaka et al., 1996). Dietary nucleotides modulate gene transcription in the intestine (LeLeiko et al., 1995) as well as lipoprotein and fatty acid metabolism in early life; they also strengthen the immune response and promote the growth of intestinal bifidobacteria, thereby restricting enterobacterial growth in the gut of the newborns (Morillas et al., 1994; SaÂnchez-Pozo et al., 1994; Gil and Uauy, 1995). It should be noted, however, that the nucleotide profile of breast milk differs considerably from cows' milk or milk formula (Gil and Uauy, 1995). Free amino acids make up only 10% of all non-protein milk-nitrogen, but taurine and glutamine are, nevertheless, quite abundant. Taurine deficiency is

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detrimental to the pre-term infant due to its limited capacity to synthesize taurine (Gaull et al., 1977). Glutamine is not only the preferred fuel for rapidly proliferating cells like enterocytes and lymphocytes, but also a regulator of the acid-base balance through the production of NH3, a N-carrier between tissues. Glutamic acid is also a precursor of nucleotide aminosugars and proteins and is even required for maintaining the structure and function of the small intestine (Lacey and Wilmore, 1990; Newsholme and CarrieÂ, 1994). Ornithine, a degradation product of arginine, improves the nitrogen balance in acute and chronic malnutrition (Koletzko et al., 1998). In addition, arginine and ornithine are, respectively, precursors of nitric oxide and polyamines, the latter acting on the permeability and adaptive responses of the gut (Koletzko et al., 1998). It is noteworthy that polyamines themselves are a component of breast-milk, but not of milk formula (Dandrifosse et al., 2000). With respect to early malnutrition, it was shown that pre-weaning overfeeding affected nutrient utilization and body composition (Lewis et al., 1986) which increased the risk of obesity later on. The early diet has irreversible effects on body and gut size. A study in mice revealed that the intestinal size of the offspring depended on the supply of carbohydrates and protein their mothers had received during pregnancy and lactation. These differences not only persisted in adulthood, even when these offspring were switched onto a standard diet, but affected nutrient uptake as well (Karasov et al., 1985). Inadequate calorie and nutrient intakes from poorly planned vegetarian diets cause growth retardation, rickets, vitamin B12 deficiency, etc., especially if the diets do not include dairy products and eggs (Jacobs and Dwyer, 1988). More information on the nutritional requirements during lactation may be found in Udipi et al. (2000).

2.3

The effects of supplement intake

A large body of epidemiological evidence supports the association between maternal nutritional deficiency and maternal morbidity, length of pregnancy or foetal growth. It is important to differentiate the practical implications of these epidemiological associations from the effectiveness of pragmatic intervention during the reproductive period (Villar and Carroli, 1996). A tremendous number of nutritional interventions were and are still undertaken, but their evaluation during pregnancy is quite difficult because several factors may influence interpretation. Amongst these, a selection bias may exist in the targeted population due to covariables such as nutrition deficiency and infection. The effect of a nutritional deficiency or nutritional intervention may depend on the timing of occurrence during gestation (Villar et al., 2003) which will influence the nutrient transfer to the foetus. The effect of nutritional intervention is also linked to the length of the supplementation and the amount achieved. Dietary supplementation must be carefully weighed for its potential benefits and risks, because what favourably affects the risk for one disease may be

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detrimental with respect to another (Kelley and Erickson, 2003). Determination of the positive outcome of supplementation studies based only on increased birth weight of the offspring may not be as judicious as might seem and the consequences must be taken into account. Anaemia is very frequent in pregnant women, adolescent girls, low-birthweight infants and in populations in which malaria and parasite-induced blood losses are endemic. Iron depletion can be overcome by iron fortification, but iron supplements frequently fail to restore haemoglobin concentrations to normal during intervention periods with high iron requirements such as pregnancy. Anaemia can be overcome by adding vitamin A, riboflavin or folic acid to the iron supplement that is administered to pregnant or lactating women, as well as children (Allen, 2002). Iron supplementation during pregnancy not only increases the maternal iron stores during pregnancy and reduces the risk of adverse pregnancy outcomes (Allen, 2002), but also increases iron stores of the infant and of the mother postpartum (Preziosi et al., 1997). With respect to choline, there exists a time window in which young rats respond favourably to choline treatment through improvement of their spatial memory, as was mentioned for the prenatal period (Zeisel, 2000). Folic acid supplementation is widely carried out during pregnancy in order to reduce the risk of neural tube defects. These defects increase, however, with increasing prepregnancy weight, independent of the mother's folate intake (Werler et al., 1996). In other words, folate loses its protective effect in overweight and obese mothers (Prentice and Goldberg, 1996). Although taurine supplementation was never investigated during pregnancy and early life in humans, some arguments indicate a potential benefit. Taurine, a free amino acid that is not incorporated into proteins, is very important for development. Taurine is one of the most abundant free amino acids in human milk (Rassin et al., 1978). Since humans, and more specifically infants, depend on exogen sources of taurine (Gaull et al., 1977), Gaull et al. (1982) suggested that synthetic formulas be supplied with taurine. Taurine supplementation reduces hypercholesterolemia in adults consuming a high-cholesterol diet by stimulating cholesterol degradation and excretion of bile acid (Yokogoshi and Oda, 2002). Clinically, taurine has been used in the treatment of a wide variety of conditions, including cardiovascular diseases, epilepsy and other seizure disorders, macular degeneration, Alzheimer's disease, hepatic disorders and cystic fibrosis. Taurine concentrations are reduced in the plasma and in platelets of type 1 diabetic patients. Oral taurine supplementation to these patients restored the levels of taurine and normalized the amount of arachidonic acid required for platelet aggregation (Fraconi et al., 1995). Inconclusive evidence exists that protein supplementation during pregnancy is beneficial for pregnancy-induced hypertension and preeclampsia (Roberts et al., 1974; Williams et al., 1981). Although exogenous nucleotides are thought to influence favourably the function of the rapidly dividing tissues of infants, studies on human infants have not conclusively confirmed findings about

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dietary nucleotides from experimental models (Carver, 1994; Kulkarni et al., 1994; Gil and Uauy, 1989; Sanchez-Pozo et al., 1994; Navarro et al., 1996). Both n-3 and n-6 polyunsaturated fatty acids are essential for the organism, especially during development, and contrary to widespread belief n-6 polyunsaturated fatty acids per se are not detrimental to our health. The important factor seems to be a proper balance between n-6 and n-3 polyunsaturated fatty acids, especially during growth and development (Galli and Marangoni, 1997; Simopoulos, 1991). Unfortunately, the intake of n-3 polyunsaturated fatty acids has decreased with lower fish consumption and increased industrial production of animal feeds rich in n-6 polyunsaturated fatty acids. This has led to meats, eggs and cultured fish rich in n-6 polyunsaturated fatty acids. Even cultivated vegetables contain fewer n-3 polyunsaturated fatty acids than those in the wild (Simopoulos, 2000). This shift in n-6 to n-3 ratio has led to an increase in cardiovascular disease, type 2 diabetes, etc., and supplementation of the diet aimed at improving this ratio has been beneficial in this respect (Simopoulos, 2000). Although a double-blind, placebo-controlled trial of the benefit of supplementing the diet of pregnant women with fish-oil, vitamins and minerals was expected to reduce the frequency of preeclampsia, it did not, however, reduce the incidence of pregnancy-induced hypertension (Onwude et al., 1995). Recommendations on proper intakes may be found in Simopoulos (2000). The recommendations for pregnant and lactating mothers are the same as for the rest of the adult population, whereas recommendations for milk formula suggest simultaneous reduction of n-6 and increase of n-3 polyunsaturated fatty acids. Saturated fatty acids are also commonly considered to impact negatively on health. Intakes of short- and medium-length saturated fatty acids are not associated with increased risk of cardiovascular heart disease, however, nor are longer-chain saturated fatty acids significantly detrimental in this respect. Again, it is the ratio of polyunsaturated to saturated fatty acids that strongly and inversely correlates with the risk for cardiovascular disease (Hu et al., 1999a). Trans fatty acids are unsaturated fatty acids with at least one double bond in the trans configuration that leads to a rigid molecule close to a saturated fatty acid. They appear in animal fat and hydrogenated oils; margarines and bakery shortenings contain relatively high levels of trans fatty acids. They can be incorporated into tissues and although their transfer across the placenta remains controversial, trans isomers have been inversely correlated with birth weight. They are incorporated into infant tissues from breast milk. While blood and liver are vulnerable, the brain seems to be protected from trans fatty acid accumulation in experimental animals, but human data have not yet been reported (Larque et al., 2001). A recent study has demonstrated that maternal supplementation with very long-chain n-3 fatty acids coming from cod liver oil during gestation and lactation augments children's IQ at four years of age (Helland et al., 2003). This beneficial effect was not obtained with n-6 fatty acids. On the other hand, supplementation with 10% of fish oil versus olive oil during pregnancy and

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lactation in rats inhibits the synthesis of arachidonic acid and delays postnatal development of the offspring (Amusquivar et al., 2000). Although the dietary requirements for micronutrients during development are small, adequate quantities are essential for the short- and long-term health of the embryo, foetus and neonate. Severe micronutrient deficiencies during pregnancy, lead to foetal malformation and death, but even more moderate insufficiencies compromise pregnancy outcomes (Ashworth and Antipatis, 2001). Supplementation with calcium or zinc, but not magnesium during pregnancy, has been positive for the offspring's health in the case of malnourished mothers, whereas pregnancy outcomes were not affected by calcium or zinc supplementations when the mothers' intakes were sufficient (Prentice, 2003). Dietary intakes of minerals, whether sufficient or not, during lactation did not influence the mother's health or breast milk mineral secretion (Prentice, 2003). Water-soluble vitamins reach the foetus by active transport, whereas fatsoluble vitamins are transferred by facilitated diffusion across the placenta and do not achieve the same degree of storage in the developing foetus (Malone, 1975). Supplementation with vitamin D before or after birth has led to positive outcomes in malnourished populations (Salle et al., 2000), but this same treatment might not be optimal for well-nourished mothers and infants, because excessive administration of the lipophilic vitamins A and D has been described to be detrimental (Malone, 1975). Finally, the vitamin D status of the child is more influenced by the mother's status during pregnancy than during lactation (Prentice, 2003). From a recent survey of the results of systematic reviews of randomized trials of nutritional interventions during pregnancy, it appears that no specific nutrient supplementation was identified for reducing preterm delivery. However, iron and folate supplementation reduce anaemia and therefore should be included in antenatal care programmes. Calcium supplementation appears promising for women with low calcium intakes who are at high risk for preeclampsia and hypertension. Fish oil and vitamin E and C are promising also for prevention of preeclampsia (Villar et al., 2003; Roberts et al., 2003).

2.4 The role of functional foods: nutrition during pregnancy and infancy Nutrition is truly functional during pregnancy and lactation, since it exerts prenatal and early postnatal influences on the developing baby: maternal nutrition impacts on the intra-uterine development of the baby and determines the quality of the breast milk needed to support adequate growth and gut flora composition. There are two approaches with respect to functional foods. One approach makes use of specific foods with a high or low content of a certain component, whereas the other concerns designed foods where ingredients have been added or removed. Functional foods should be addressed differently according to their intended applications. These may be either prophylactic or

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therapeutic. Healthy population groups with adequate food supplies should perceive functional foods as a prophylactic means to maintain their health and opt for a balanced diet that provides the widest range of different nutrients of as many different origins possible. Some examples of natural functional foods are cited in Milner (2000), Puupponen-Pimia et al. (2001) and StacewiczSapuntzakis et al. (2001). Only a few examples that seem particularly relevant in the context of pregnant and lactating women and infants will be discussed in more detail in this review. Eggs are a relatively cheap, low-calorie source of protein, vitamin K, choline, carotenoids, riboflavin, selenium and polyunsaturated fatty acids (Hasler, 2000; Hu et al., 1999b; Simopoulos, 2000). Egg yolk is also a rich source of the antioxidant carotenoids lutein and zeaxanthin that have been linked with reduced risk of age-related blindness (Hasler, 2000). Such a variety of different nutrients and the presence of high quantities of the essential nutrient choline makes eggs particularly valuable for the foetus and newborn because it benefits cognitive function, especially when present during early brain development (Blusztajn, 1998; Zeisel, 2000). Diabetes in the pregnant mother affects choline metabolism and slows the maturation of her child's lungs. Choline in eggs might counteract the reduced production of the lung surfactant phosphatidylcholine and the reduced maturation of the lungs of children born to mothers with poorly controlled diabetes in humans (Tyden et al., 1986) and rats (Nijjar et al., 1984). Experiments in rats have indeed shown that hyperglycemia decreased phosphatidylcholine secretion in vitro (Gewolb and O'Brien, 1997). Choline supplementation in mid-pregnancy increased choline incorporation into the foetuses, and increased phosphatidylcholine production and secretion (Garner et al., 1995). Adequate consumption of choline in the shape of eggs might, therefore, conceivably alleviate the consequences of maternal diabetes on an unborn child. Unfortunately, this has not yet been investigated. Eggs are a truly functional food, despite the widespread belief that egg consumption is unhealthy due to its cholesterol content. Recent epidemiological studies have shown that dietary cholesterol had little impact on plasma cholesterol levels (Howell et al., 1997) and that regular consumption of eggs did not have a substantial impact on the risk of coronary heart disease (Hu et al., 1999b). On the contrary, individuals eating more than four eggs a week had significantly lower serum cholesterol than those consuming one or fewer eggs per week (Song and Kerver, 2000). Prunes have traditionally been consumed for their laxative and antimicrobial effects (Stacewicz-Sapuntzakis et al., 2001), but their consumption has other beneficial effects on health. Prunes have particularly high contents of minerals, vitamins, fibre and phenolics, but contain fewer amino acids. The main amino acids are asparagine, taurine, proline and GABA (Stacewicz-Sapunzakis et al., 2001). Despite providing substantial amounts of energy, prunes only slowly elevate blood sugar, their glycaemic index being only moderate. Encouraging the consumption of nutrients with a low glycaemic index (i.e. that produce a low glycaemic response) such as prunes may be useful not only for healthy pregnant

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women, but also for children and adults suffering from type 1 or type 2 diabetes. Long-term consumption of prunes reduced plasma insulin and insulin secretion in healthy subjects (Stacewicz-Sapunzakis et al., 2001). Hence, the inclusion of prunes in the diet of pregnant women and type 1 diabetic individuals may counteract a tendency for weight gain and generally improve the well-being of type 1 diabetic patients, including children. Whole rice is also a natural functional food due to the presence of rice-bran, a rich source of fibre, myo-inositol, inositol hexaphosphate and antioxidants. Inositol hexaphosphate, as found for instance in rice bran, is the main dietary supplier of cell membrane phosphatidylinositol needed for the maintenance of membrane integrity (Jariwalla, 2001). Since inositol hexaphosphate is also a strong chelator, whole rice might increase the bioavailability of metal ions and thus reduce the risk of micronutrient insufficiencies. Rice-bran represents about 10% of whole rice, and some of its components have also been linked with reduced risk for cancer and cardiovascular disease (Jariwalla, 2001). It might, therefore, be useful to encourage pregnant and lactating mothers to consume whole rice instead of polished rice; this would help combat micronutrient deficiencies, one of the nutritional major risks of these population groups (Dijkhuizen et al., 2001; WHO, 2000). High circulating levels of phytoestrogens during critical periods of development have arisen as a consequence of replacing milk with soy products. Data on developmental, behavioural and physiological effects of soy products containing soy isoflavones (phytoestrogens) during pregnancy and infancy are scarce. Animal data suggest that they affected some endocrine functions: they altered the development of the reproductive system (Wisniewski et al., 2003), delayed puberty, reduced 17±beta estradiol concentrations and blunted 17± estradiol responses (Badger et al., 2001). Although in humans phytoestrogens have been suggested as estrogen replacement therapy (Setchell and Cassidy, 1999), and soy in general is said to reduce the risk for some cancers, including breast cancer (Adlercreutz, 2003; Goodman et al., 1997, Messina, 2003), the possibility has been raised that phytoestrogen supplements or a high soy product consumption during pregnancy may actually favour estrogen-dependent breast cancers (Hilakivi-Clarke et al., 2001) and alter the time of onset of puberty (Teilmann et al., 2002). It should therefore be clarified whether such products are safe for pregnant women and their unborn children and whether soy-milk is healthy for infants. The other, and more commonly used approach to functional foods, involves designed foods in which ingredients have been added or removed. Only the former category will be considered here. Different types of designed foods are classified as functional foods: pre-, pro- and synbiotics, vitamins and minerals, bioactive molecules, and fatty acids. Probiotics are life microbial food ingredients (bacteria, yeasts, microalgae) that are beneficial for health (Roberfroid, 2000; Kay, 1991). They are mostly lactobacilli and bifidobacteria and either used as freeze-dried cultures (in capsules) or to prepare fermented dairy products (yoghurt or sour milk).

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Probiotics added to food products must meet several criteria such as a beneficial effect on health, survival during transit through the gastrointestinal tract, adhesion (permanently or temporarily) to the intestinal epithelial cell lining, production of antimicrobial substances towards pathogens or stabilization of the intestinal microflora. Over-the-counter supplements, however, often do not fulfil these criteria and do not even survive in the gastrointestinal tract (Kopp-Hoolihan, 2001). The health benefits of probiotics include the improvement of intestinal function, immune function and thus reduction of atopic disease as well as reduction of hypertension, hypercholesterolemia and certain cancers (KoppHoolihan, 2001; Takano, 2002; Agerholm-Larsen et al., 2000). With particular relevance for the subject on hand are several trials with either pregnant women, lactating mothers and their babies, or with children, that have demonstrated several beneficial effects of probiotics. These include the maturation and health of the intestinal tract (Schiffrin and Blum, 2002) and the immune system (Cunningham-Rundles et al., 2000), the reduction of lactose intolerance and allergy prevalence (Shermak et al., 1995; Rautava et al., 2002), the reduction of the risk of microorganism-induced diarrhoea (Saavedra et al., 1994; Guandalini and Dincer, 1998) or the enhancement of nutrient bioavailability (Branca and Rossi, 2002). Not only are probiotics therefore promising functional foods for pregnant women and infants, but they can be considered for prophylactic as well as therapeutic uses. Prophylactic administration of probiotics to women during the last trimester of pregnancy and through childbirth, for instance, permanently colonized the gastrointestinal tracts of their infants (Vanderhoof, 2001). It is not yet known, whether the immune-boosting properties of these probiotics require periodic pulse dosing rather than continuous administration (Vanderhoof, 2001). Probiotic administration to pregnant and lactating mothers increased the immuno-protective potential of breast milk and reduced the incidence of atopic eczema during the first two years of life in their children (Rautava et al., 2002). Another study showed that in addition to allergy occurrence, the number of infections and the need for antibiotics due to preventive probiotic treatment after birth were reduced even ten years later (Lodinova-Zadnikova et al., 2003). Preventive feeding of fermented milk also increased the absorption (i.e. the bioavailability) of iron due to the liberation of lactic acid and other organic acids during fermentation (Branca and Rossi, 2002). The authors even suggested that consumption of fermented milk during meals might also have a positive effect on the absorption of iron from other foods. Based on such findings and the fact that even temporary colonization of a baby's intestines with probiotic bacteria prevents colonization with less beneficial bacteria, probiotic supplementation of milk formula has been proposed. Used therapeutically, probiotics effectively treated diarrhoea and reduced the incidence of respiratory disease in infants (Rio et al., 2002; Vanderhoof, 2001). The incidence of milk allergy in toddlers was reduced if they had received Lactobacillus GG pre- and postnatally (Kalliomaki et al., 2001). Treatment of milk-allergic toddlers with Lactobacillus GG improved both the extent and

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severity of allergic eczema (Isolauri et al., 2000). Congenitally HIV-infected children treated with lactobacilli showed an increased immune response as well as an improvement in health and development (Cunningham-Rundles et al., 2000). An eight-week trial of daily consumption of fermented milk products containing different types of probiotic strains improved cardiovascular parameters such as LDL-cholesterol or systolic blood pressure in adult overweight or obese subjects (Agerholm-Larsen et al., 2000). It might therefore be interesting to observe these parameters in adult subjects that had received probiotic supplementation in early childhood in order to verify whether early probiotic supplementation confers long-term protection against cardiovascular or other degenerative diseases. In the case of undernutrition, the beneficial effects of probiotic supplementation were also described for parameters such as incidence and severity of fever and diarrhoea, weight gain and growth rate (Oberhelman et al., 1999; Saran et al., 2002; Solis et al., 2002). Nevertheless, as a consequence of the malnutritioninduced reduction of immunocompetence, the effectiveness of combating respiratory disease after probiotic administration was decreased in undernourished babies and toddlers compared to the adequately nourished group (Rio et al., 2002). Prebiotic foods are `non-digestible food ingredients that beneficially affect the host by selectively stimulating the growth and/or the activity of one or a limited number of bacteria in the colon' (Roberfroid, 2000). They may be plant and/or animal polysaccharides or oligosaccharides. They are often defined as starch- and non-starch polysaccharides, their definition being different from one country to another (Prosky, 2000). Prebiotic oligosaccharides from different origins have been used as ingredients of functional foods. They may be inulin, lactulose, fructo-, galacto-, isomalto- or xylo-oligosaccharides. According to their chemical nature they support higher populations of individual bacterial species in the gut flora (Rastall and Maitin, 2002). The largest increase in lactobacilli was seen with xylo-oligosaccharides and lactulose. Although fructooligosaccharides promoted a large increase in lactobacilli, they also supported higher populations of streptococci than did galacto-oligosaccharides. The latter supported higher populations of bifidobacteria and higher levels of lactate than fructo-oligosaccharides. Galacto-oligosaccharides were also the most effective in reducing clostridia (Rastall and Maitin, 2002). Lactulose, xylo- and galactooligosaccharides thus stimulate the growth of bacteria found in the colon of breast-fed infants. Formula-fed infants, on the other hand, have a more diverse and adult microflora and tend to suffer more from microbial infections than breast-fed infants (Harmsen et al., 2000). This means that lactulose, xylo- and galacto-oligosaccharides are the prebiotic oligosaccharides of choice for functional foods aimed at infants. Supplementing milk formula with these oligosaccharides should therefore circumvent the problem of aberrant colon colonization in formula-fed infants. However, prebiotic functional foods will be effective only where there is a real need, since responses to prebiotics depend on the numbers of bacteria colonizing the colon. Individuals with low

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bifidobacterial counts displayed much higher responses to prebiotics than individuals with higher bacterial counts (Rastall and Maitin, 2002). Prebiotics positively affect the absorption of various minerals (Scholz-Ahrens et al., 2001) as well as mineral contents in bones (Roberfroid et al., 2002). The risk of osteoporosis is higher in formula-fed children than in breast-fed children born at term (Jones et al., 2000), even though milk formula has a higher calcium content than breast milk (Bosscher et al., 2003). Prebiotic supplementation of milk formula might thus contribute to a reduction of the risk of osteoporosis in formula-fed children born at term. This finding does not apply, however, to children born pre-term, where the source of milk does not seem to influence bone mass later on (Fewtrell et al., 1999). Nevertheless, it might be worthwhile to follow these term and pre-term children, into adult age in order to check for possible long-term protection against osteoporosis due to early prebiotic supplementation. Synbiotics are a combination of a probiotic and a prebiotic. One of the main benefits of synbiotics is believed to be the increased persistence of the probiotic in the gastrointestinal tract (Rastall and Maitin, 2002). There is a complete lack of information about synbiotics in pregnant or lactating women and only one case report of synbiotic therapy in children. Synbiotic therapy in the shape of orally administered living B. breve and L. casei together with galactooligosaccharides was successful in improving the intestinal function of a prematurely born baby with laryngo-tracheo-esophagal cleft (Kanamori et al., 2002). The little girl born with esophagal atresia did not grow satisfactorily because she could not tolerate enteral feeding. She did not have bowel movements, nor were bifidobacteria or lactobacilli detected in her faeces. Once the synbiotic therapy was started, bowel movement was restored within a day. B. breve and L. casei were detected in the faeces. Within a month, short-chain fatty acids in faeces increased, all of them signs of intestinal colonization. The presence of such short-chain fatty acids also affects the motility of the intestinal tract and increases intestinal blood flow. These short-chain fatty acids were mainly produced by B. breve and used by intestinal epithelial cells as energetic substrates. Normal fermentation was also restored. Milk feeding became possible and the child's body weight doubled during the course of the 11 months of therapy (Kanamori et al., 2002). Further examples of pro- or prebiotic trials in pediatrics are reported by Van Den Driessche and Veereman-Wauters (2002). Functional foods have also been designed to regulate fat metabolism. Margarines were among the first functional lipid foods designed in Western societies to reduce the risk of excessive consumption of cholesterol and saturated fatty acids. Another type of functional lipid food aimed at reducing the consumption of cholesterol and saturated fatty acids are fat substitutes. By reducing the total fat intake it is intended to help to diminish calorie intake. However, it is not clear if it is the case. These fat substitutes, unlike other functional food additives, make up a substantial proportion of the total diet. Further problems arising from the consumption of these substitutes are a reduced bioavailability of other nutrients, and adverse effects on gastrointestinal tract

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function or on the intestinal flora (Vanderveen and Glinsmann, 1992). Fat substitutes from natural sources may be carbohydrates, proteins or a combination of both. With regard to small children as well as the unborn child consumption of these artificial food ingredients may actually be more harmful than beneficial (Lifshitz and Tarim, 1996). Except for the inactive, overweight (or obese) child with excessive fat and sugar consumption, concern about children's fat intake is rather inappropriate, because fat is essential for a nutritionally balanced diet in childhood. Children are indeed not `little adults'. A low fat intake in early childhood (18±43 months) may be associated with suboptimal intake of essential nutrients such as calcium, zinc, iron or vitamin C (Rogers et al., 2001). In addition, increasing the fat supply of pregnant and lactating women by supplementing their diet with long-chain polyunsaturated fatty acid favours neurocognitive function in their babies and toddlers (Helland et al., 2003). The use of protein-derived fat substitutes may create a high-protein diet that may be particularly unhealthy to small children's immature kidneys and programme degenerative disease. In addition, since nutrients are transferred from the pregnant mother to her child and it is not known how regular consumption of fat substitutes affects the unborn child, it may be prudent to discourage the use of fat substitutes in pregnant women. It is also unclear how they would affect the quality of breast milk. Last, but not least, a further category of designed functional foods are bioengineered foods like GMOs enriched in vitamin A (f. ex. `golden rice') or iron (King, 2002) that are used in parts of the world such as Asia where nutritional deficiencies of vitamin and mineral intake are common, as in the case of pregnant Indonesian women (Hartini et al., 2003).

2.5

Safety concerns of functional foods

Functional foods are open to many different interpretations. Moreover, legally, functional foods do not appear as a specific category. Current legislation defines and regulates foods, foods for special dietary use, medical foods, dietary supplements and drugs, but not a single regulation addresses functional foods. In the United States, for instance, functional foods are more or less strictly regulated depending on the name they are given; they are sold either as `foods', `dietary supplements', `drugs', `medical foods', `foods for special dietary use', etc., depending on the claims that are made as well as the ingredients that are used. This lack of specific laws for functional foods and the difficulty of defining the borderline between all these differently defined food categories, leaves large loopholes for marketing so-called functional foods with unsubstantiated or exaggerated health claims, such as fortified junk foods (Heller, 2001; Silverglade and Heller, 1997). Different countries have started to deal with this problem through different approaches (Silverglade and Heller, 1997; Halsted, 2003; Richardson, 1996;

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Sanders and Huis in't Veld, 1999; Stanton et al., 2001). Efforts are currently also being made by the European Commission who recently published a proposal for regulating nutrition and health claims in the European Union (European Commission, 2003). This proposal was presented to the European Parliament and the European Council, and it is hoped that it will be implemented by 2005 after approval by the European Parliament and the European Council. Unfortunately, this proposal does not once consider the possibility that the new legislation should take into account the particular requirements of different population groups, especially pregnant women and infants, and that a one-sizefits-all approach to designed functional foods must by all means be prevented. This new legislation should therefore also question over-the-counter sale of certain supplements and entail regulations concerning labelling. Finally, the proposal for the new rules does not, but should, address the prophylactic or therapeutic applications of designed functional foods, because one and the same supplement may have different consequences on the healthy population, and individuals at risk or already affected. More information on the current status of functional food science in the European Union may be found in the Consensus Document published by ILSI (1999). There is in fact a real need for regulating nutrition and health claims, because claims about the health benefits of many functional foods are often based only on `emerging evidence' or `preliminary data', and are the basis for their marketing (Hasler, 2002). Furthermore, many products are sold with vague and not verifiable claims (European Commission press release IP/03/1022 of 16/07/ 2003). Clinical data supporting benefits of functional foods are scarce, except for a small number of claims approved by the relevant national authorities in different countries (Hasler et al., 2001; Arai, 2000). It should be verified, whether these claims apply to all population groups, and in particular pregnant and lactating women and infants. It is therefore not surprising that there exists some controversy regarding the concept of using functional foods to reduce the incidence of degenerative diseases and that it has both proponents and opponents. Proponents state that functional foods reduce health care expenditure, whereas opponents argue that the total diet is important, and not some `magic bullets' and point out the risk of going into a `marketing hyperbole' (Lawrence and Rayner, 1998). There is also a real risk that the distinction between foods and drugs will be blurred. This is of particular concern, since any food supplement used to prevent or ease a disease must be considered as a drug. In addition, some dietary supplements such as herbal products contain potentially toxic components, particularly in relation to interactions with other drugs (Halsted, 2003). Some components of functional foods do indeed interact with certain medications for cancer, heart disease and birth control, which raises the question of safety of these foods. Detrimental interactions between food additives and drugs have been described for St John's wort and oral contraceptives, medications for heart disease or cancer, for Ginko biloba and anticoagulants or aspirin, etc. (Hasler et al., 2001; Kruger and Mann 2003).

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The perception of the healthiness of food in general, and consequently also of functional foods, is influenced by a variety of factors, such as type and processing of raw materials, origin, conservation methods and additives (Bech-Larsen and Grunert, 2003). A recent study showed that, compared to attitudes to genetically modified foods, the attitudes towards functional foods were only slightly associated with cultural values, and that the consumers' perception of the healthiness of functional foods was more dependent on their perception of the nutritional qualities of the base-product than on any type of health claim (Bech-Larsen and Grunert, 2003). The fact that public perception of the risk associated with a given food category is not necessarily scientifically justified tends to lead to erroneous nutritional behaviours. One example already mentioned is the fear of fat overconsumption that leads to parents undersupplying their infants with fats (Bech-Larsen and Grunert, 2003). A potential risk of selling some foods labelled with health claims is that consumers will perceive those nutriments with health claims as being better for their health than those without and this might then lead to overconsumption of such foods. Combined with the fact that consumers, more than ever, are taking charge of their own health and are buying food with the purpose of reducing health risks (Hasler, 2002), this may lead to overconsumption of nutriments such as gingko (a blood thinner), soy isoflavones (proposed in estrogen-replacement therapy, in cancer and cardiovascular disease risk reduction), beta-carotene supplements (cancer risk reduction) in spite of being either ineffective or potentially harmful (Hasler et al., 2001). Functional foods that address a specific health claim will be effective when eaten in the right context and amount, and incorporated into an overall balanced and healthy diet. Otherwise, new malnutrition situations will be created. Since functional foods are consumed with the purpose of producing an effect on health, their inappropriate or unnecessary use may do harm rather than good, as happens with inappropriately used drugs. This important issue is particularly relevant considering that age-related differences in response to drugs are already known to occur, arising not so much from pharmacodynamic, but rather from pharmacokinetic differences (Kruger and Mann, 2003). Together with the knowledge that pregnant women and their young offspring have particular and very specific nutritional needs and that age, sex, health, genetic makeup and lifestyle all influence these nutritional needs, the `one-size-fits-all' basis on which functional foods are sold and consumed should be abandoned.

2.6

Future trends

There is a clear need for the identification of biomarkers of nutritional status and the development of standardized bioassays to estimate this nutritional status as well as the nutritional requirements at different periods of life. More research on the metabolic fate of essential nutrients and on the interactions between individual

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nutrients is also required. Equally lacking are data on nutrient impact, on gene expression and body functions, and on intellectual or physical performance. This is particularly the case for pregnant and lactating women and their infants. Recommended daily allowances of particular nutrients should be fine-tuned to fit individual population groups with specific needs. In addition, it might be necessary to reassess recommendations of the daily intakes of nutrients, considering, for example that pregnant women in one trial consumed only 85% of the recommended daily allowances for pregnant women (Swensen et al., 2001). Recommended daily allowances are intake recommendations that have been calculated based on population requirements and the average bioavailability of a nutrient in `normal' food. Since very little information exists about the bioavailability of the supplements in functional foods, this will need to be verified and the recommended daily allowances corrected for specific functional foods. For instance, one clinical trial reported extremely low bioavailability of iron that was part of a vitamin supplement (Dawson et al., 1998). Investigation of the chemical `shape' in which the supplements are to be administered to ensure maximum bioavailability and minimum side effects need to be carried out, especially in the case of functional foods destined for infants with their notoriously immature body functions. The establishment of state-wide nutrition programmes will be a great challenge, because they will have to simultaneously combat apparently opposite nutritional problems, i.e. under- vs. over-nutrition, and malnutrition vs. nutritional imbalance. These programmes will have to take into account that the social representations of new foods such as functional foods vary according to age gender, education level, etc., (BaÈckstroÈm et al., 2003). `One-size-fits-all' approaches may not only fail to achieve their aim of changing dietary habits, but also be detrimental, rather than beneficial, for selected population groups such as pregnant and lactating mothers and their infants. Since women are more concerned about food and eating habits than men (BaÈckstroÈm et al., 2003), and women are the usual medium through which children acquire their eating habits and dietary education, a good starting point for getting the message across may be to address women, especially mothers. With changing lifestyles, i.e., more women working, changing household structures, more people living alone, more active lifestyles and thus less time or willingness for preparing food or eating meals in a family setting at regular hours, the tendency of consuming snacks between and instead of meals will increase even further, as will the number of meals eaten away from home. Most of the increase in energy intake in US children and adolescents is already accounted for by snacks and/or evening meals (Gleason and Suiter, 2002), fastfood meals providing most of the total and saturated fat consumed (Lin et al., 2001). With regard to the nutrition of pregnant women and children this will increase the risk of nutritional imbalances due to skipped meals and uncontrolled food consumption in these particularly vulnerable population groups.

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2.7

43

Sources of further information and advice

· Flair Flow Europe (http://www.flair-flow.com) FLAIR-FLOW EUROPE disseminates information from food R&D and nutrition projects funded by the European Union and is geared towards informing the food industry, consumers and health professionals. · World Health Organisation (http://www.who.int/nut/). This site of the WHO presents a series of documents concerning nutrition (and malnutrition) from a public health perspective. · ILSI (http://www.ilsi.org) The International Life Science Institute has regional branches and aims at advancing the understanding of scientific issues of nutrition, toxicology, food safety and the environment. · Nutrition Reviews (1996), volume 54 (11 Pt 2) features a series of reviews of the functional food policies in different countries around the world. · The dietetic associations as well as the relevant government authorities of individual countries all present guidelines and national regulations on healthy dieting and different concerns of individual population groups. Their sites are available through the internet. · The FDA Centre for food safety and applied nutrition (http:// vm.cfsan.fda.gov) presents guidelines on healthy eating and addresses different issues such as women's health, children, infant formula, foodborne illnesses and dietary supplements. · The Nutrition navigator (http://navigator.tufts.edu).

2.8

References

(2003) Phytoestrogens and breast cancer. J. Steroid Biochem. Mol. Biol. 83, 113±118. AGERHOLM-LARSEN L, RABEN A, HAULRIK N, HANSEN AS, MANDERS M, ASTRUP A (2000) Effect of 8 week intake of probiotic milk products on risk factors for cardiovascular diseases. Eur. J. Clin. Nutr. 54, 288±297. AL MD, HORNSTRA G, VAN DER SCHOUW YT, BULSTRA-RAMAKERS MT, HUISJES HJ (1990) Biochemical EFA status of mothers and their neonates after normal pregnancy. Early Hum. Dev. 24, 239±248. ADLERCREUTZ H

AL MD, VAN HOUWELINGEN AC, KESTER ADM, HASAART THM, DE JONG AEP, HORNSTRA G

(1995) Maternal essential fatty acid patterns during normal pregnancy and their relationship with the essential fatty acid status. Br. J. Nutr. 74, 55±68. ALLEN LH (2002) Iron supplements: Scientific issues concerning efficacy and implications for research and programs. J. Nutr. 132, 813S±819S. AMUSQUIVAR E, RUPEÂREZ FJ, BARBAS C, HERRERA E (2000) Low arachidonic acid rather than alpha-tocopherol is responsible for the delayed postnatal development in offspring of rats fed fish oil instead of olive oil during pregnancy and lactation J. Nutr. 130, 2855±2865. ANDERSON B, PORRAS O, HANSON LA, LAGERGARD T, SVANBORG-EDEÂN C (1986) Inhibition of Streptococcus pneumonia and Hemophilus influenzae by human milk and receptor oligosaccharides. J. Infect. Dis. 153, 232±237.

44

Functional foods, ageing and degenerative disease

(1993) Intrauterine food restriction is associated with obesity in young rats. J. Nutr. 123, 1421±1428. ANTRAS-FERRY J, ROBIN P, ROBIN D, FOREST C (1995) Fatty acid and fibrate are potent inducers of transcription of the phosphoenolpyruvate carboxykinase gene in adipocytes. Eur. J. Biochem. 234: 390±396. ARAI S (2000) Functional food science in Japan. Biofactors 12, 13±16. ASHWORTH CJ, ANTIPATIS C (2001) Micronutrient programming of development throughout gestation. Reproduction 122, 527±535. È CKSTRO È M A, PIRTTILA È -BACKMAN A-M, TUORILA H (2003) dimensions of novelty: a social BA representation approach to new foods. Appetite 40, 2999±3007. BADGER TM, RONIS MJ, HAKKAK R (2001) Developmental effects and health aspects of soy protein isolate, casein and whey in male and female rats. Int. J. Toxicol. 20, 165± 174. BAILEY LB, RAMPERSAUD GC, KAUWELL GPA (2003) Folic acid supplements and fortification affects the risk for neural tube defects, vascular disease and cancer: evolving science. J. Nutr. 133; 1961S±1968S. BARKER DJ (1994) Mothers, babies and disease in later life. London BMJ publishing. BARKER DJ, OSMOND C (1986) Infant mortality, childhood nutrition, and ischaemic heart disease in England and Wales. Lancet 1, 1077±1081. BARKER DJ, WINTER PD, OSMOND C, MARGETTS B, SIMOONDS SJ (1989) Weight in infants and death from heart disease. Lancet 2, 577±580. BARKER DJ, HALES CN, FALL CH, OSMOND C, PHILLIPS K, CLARK PM (1993) Type 2 (noninsulin dependent) diabetes mellitus, hypertension and hyperlipidaemia (syndrome X): relation to reduced fetal growth. Diabetologia 36, 62±67. ANGUITA RM, SIGULIM DM, SAWADA AL

BAVDEKAR A, YAJNIK CS, FALL CHD, BAPAT S, PANDR AN, DESHPANDE V, BHAVE S, KELLINGRAY SD, JOGLEKAR C (1999) Insulin resistance syndrome in 8±year-old Indian children. Small at birth, big at 8 years or both? Diabetes 48, 2422±2429. BECH-LARSEN T, GRUNERT KB (2003) The perceived healthiness of functional foods. A conjoint study of Danish, Finnish and American consumers' perception of functional foods. Appetite 40, 9±14. BENNIS-TALEB N, REMACLE C, HOET JJ, REUSENS B (1999) A low protein isocaloric diet during gestation affects brain development and alters permanently cerebral cortex blood vessels in rat offspring. J. Nutr. 129, 1613±1619. BERTRAM CE, HANSON MA (2001) Animal models and programming of the metabolic syndrome. Br. Med. Bull. 60, 103±121. BLUSZTAJN JK (1998) Choline, a vital amine. Science 281, 794±795. BOSSCHER D, VAN CAILLIE-BERTRAND M, VAN CAUWENBERGH R, DEELSTREA H (2003) Availabilities of calcium, iron, and zinc from dairy infant formulas is affected by soluble dietary fibers and modified starch fractions. Nutr. 19: 641±645. BOUJENDAR S, ARANY E, HILL D, REMACLE C, REUSENS B (2003) Taurine Supplementation of a Low Protein Diet Fed to Rat Dams Normalizes the Vascularization of the Fetal Endocrine Pancreas. J Nutr. 133, 2820±2825. BOUJENDAR S, REUSENS B., MEREZAK S., AHN MT, ARANY E, HILL D, REMACLE C (2002) Taurine supplementation to the low protein diet during foetal and early postnatal life restores a normal proliferation and apoptosis of rat pancreatic islets. Diabetologia 45, 856±866. BRANCA F, ROSSI L (2002) The role of fermented milk in complementary feeding of young children: lessons from transition countries. Eur. J. Clin. Nutr. 56 (Suppl. 4), S16± S20.

Diet and the prevention of degenerative disease

45

(1995) Idiopathische InfertilitaÈt and SterilitaÈt ± spielt FolsaÈure eine SchluÈsselrolle? (Idiopathic infertility and sterility: does folic acid play a key role?) Journal fuÈr FertilitaÈt und Reproduktion 2, 21±24. BURTON GJ, WATSON AL, HEMPSTOCK J, SKEPPER JN, JAUNIAUX E (2002) Uterine glands provide histiotrophic nutrition for the human foetus during the first trimester of pregnancy. J. Clin. Endocrinol. Metabol. 87: 2954±2959. CAMPBELL DM, HALL MH, BARKER DJ, CROSS J, SHIELL AW, GODFREY KM (1996) Diet in pregnancy and the offspring's blood pressure 40 years later. Br. J. Obst. Gynecol. 103, 273±280. CARLSON SE, COOKE RJ, WERKMAN SH, TOLLEY EA (1992) First year growth of preterm infants fed standard compared to marine oil n-3 supplemented formula. Lipids 27, 901±907. CARVER JD (1994) Dietary nucleotides: cellular immune, intestinal and hepatic system effects. J. Nutr. 124, 144S±148S. CATALANO PM, KIRWAN JP (2001) Maternal factors that determine neonatal size and body fat. Curr. Diab. Rep. 1, 71±77. CHANDRA RK (2002) Nutrition and the immune system from birth to old age. Eur. J. Clin. Nutr. 56 (Suppl. 3), S73±S76. CHERIF H, REUSENS B, AHN MT, HOET JJ, REMACLE C (1998) Effect of taurine on the insulin secretion of rat fetal islets from dams fed a low protein diet. J. Endocrinol. 159, 341±348. CLAPP JF (2002) Maternal carbohydrate intake and pregnancy outcome. Proc. Nutr. Soc. 61, 45±50. COHEN A, BARANKIEWICZ J, GELFAND EW (1984) roles of alternative synthetic and catabolic purine pathways in T lymphocyte differentiation. Proc. Natl. Acad. Sci. USA 81, 26±33. CORDERO L, LANDON MB (1993) Infant of the diabetic mother. Clin. Perinatol. 20, 635±647. Â N H, RUIZ J, DEL VEDOVO S, NEESER JR (1991) Inhibition of CRAVIOTO A, TELLO A, VILLAFRA localized adhesion of enteropathogenic Escherichia coli to Hep-2 cells by immunoglobulin and oligosaccharide fractions of human colostrum and breast milk. J. Infect. Dis. 163, 1247±1255. BUNG P, PRIETL G, WILRODT A, KREBS D, PRINZ-LANGENOHL R, PIETRZIK K

CUNNINGHAM-RUNDLES S, AHRNEÂ S, BENGMARK S, JOHANN-LIANG R, MARSHALL F, METAKIS L,

(2000) Probiotics and immune response. Am. J. Gastroenterol. 95 (Suppl), S22±S25. DABELEA D, KNOWLER WC, PETTITT DJ (2000) Effect of diabetes in pregnancy on offspring: follow-up research in the Pima Indians. J. Matern. Fetal Med. 9, 83±88. DAHRI S, REUSENS B, REMACLE C, HOET JJ (1995) Nutritional influences on pancreatic development and potential links with non-insulin dependent diabetes. Proc. Nutr. Soc., 54, 345±356. DANDRIFOSSE G, PEULEN O, EL KHEFIF N, DELOYER P, DANDRIFOSSE AC, GRANDFILS C (2000) Are milk polyamines preventive agents against food allergy? Proc. Nutr. Soc. 59, 81±86. DAWSON EB, DAWSON R, BEHRENS J, DEVORA MA, MCGANITY WJ (1998) Iron in prenatal multivitamin/multimineral supplements. Bioavailability. J. Reprod. Med. 43, 133± 140. DE ONIS M, VILLAR J, GULMEZUGLU M (1998) Nutritional intervention to prevent intrauterine growth retardation: evidence from randomized controlled trials. Eur. J. Clin. Nutr. 52: S83±S93. CALIFANO C, DUNN A-M, GRASSEY C, HINDS G, CERVIA J

46

Functional foods, ageing and degenerative disease

(1995) Adult glucose and lipid metabolism may be programmed during fetal life. Biochem. Soc. Trans. 23, 331± 335. DIJKHUIZEN M, WIERINGA FT, WEST CE, MUHERDIYANTININGSIH, MUHILAL (2001) Concurrent micronutrient deficiencies in lactating mothers and their infants in Indonesia. Am. J. Clin. Nutr. 73, 786±791. DOBBLING J, SANDS J (1971) Vulnerability of developing brain. IX. The effect of nutritional growth retardation on the timing of the brain growth-spurt. Biol. Neonate 19, 363±378. DUGGLEBY SC, JACKSON AA (2002) Protein amino acid and nitrogen metabolism during pregnancy: how might the mother meet the needs of her fetus? Curr. Opin. Clin. Nutr. Metab. Care 5, 503±509. DUPONT C (2003) Protein requirements during the first year of life. Am. J. Clin. Nutr. 77, 1544S±1549S. EGGE H, DELL A, VON NICOLAI H (1983) Fructose containing oligosaccharides from human milk. 1. Separation and identification of new constituents. Arch. Biochem. Biophys. 224, 235±253. ENDRES S (1996) n-3 Polyunsaturated fatty acids and human cytokine synthesis. Lipids 31, S239±S242. EUROPEAN COMMISSION (2003) Proposal for a regulation of the European Parliament and of the Council on nutrition and health claims made on foods presented by the Commission. 2003/0165 (COD). FARQUHARSON J, COCKBURN F, PATRICK WA, JAMIESON EC, LOGAN RW (1992) Infant cerebral cortex phospholipid fatty-acid composition and diet. Lancet 340, 810±813. DESAI M, CROWTHER NJ, OZANNE SE, LUCAS A, HALES CN

FEWTRELL MS, PRENTICE A, JONES SC, BISHOP NJ, STIRLING D, BUFFENSTEIN R, LUNT M, COLE

TJ, LUCAS A (1999) Bone mineralization and turnover in preterm infants at 8±12 years of age: the effect of early diet. J. Bone Miner. Res. 14: 810±820. FORSDAHL A (1977) Are poor living conditions in childhood and adolescence an important factor for arteriosclerotic heart disease. Br. J. Prev. Soc. Med. 31, 91±95. FORSEN T, ERIKSSON JG, TUOMILEHTO J, OSMOND C, BARKER DJP (1999) Growth in utero and during childhood among women who develop coronary heart disease: longitudinal study. Brit. Med. J. 319, 1403±1407. FOUFELLE F, GOUHOT B, PEÂGORIER JP, PERDEREAU D, GIRARD J, FERREÂ P (1992) Glucose stimulation of lipogenic enzyme gene expression in white adipose tissue. J. Biol. Chem. 267: 20543±20546. FRACONI F, BENNARDINI F, MATTANA A, MICELI M, CIUTI M, MIAN M, GIRONI A, ANICHINI R,

(1995) Plasma and platelet taurine are reduced in subjects with insulindependent diabetes mellitus: effects of taurine supplementation. Am. J. Clin. Nutr. 61, 1115±1119. GAITHER K, QURAISHI AN, ILLSLEY NP (1999) diabetes alters the expression and activity of the human placental GLUT1 glucose transporter. J. Clin. Endocrinol. Metabol. 84, 695±701. GALLI C, MARANGONI F (1997) Recent advances in the biology of n-6 fatty acids. Nutr. 13, 978±985. GARNER SC, MAR MH, ZEISEL SH (1995) Choline distribution and metabolism in pregnant rats and fetuses are influenced by the choline content of the maternal diet. J. Nutr. 125: 2851±2858. GAROFANO A, CZERNICHOW P, BREANT B (1997) In utero undernutrition impairs rat beta cell development. Diabetologia 40, 1231±1234. SEGHIERI G

Diet and the prevention of degenerative disease

47

(1999) Effect of ageing on beta-cell mass and function in rats malnourished during the perinatal period. Diabetologia 42, 711±718. GAULL GE, RASSIN DK, RAIHA NCR, HEINONEN K (1977) Milk protein quantity and quality in low-birth weight infants: III. Effects of sulfur amino acids in plasma and urine. J. Pediatr. 90, 348±355. GAULL GE, STURMAN JA, WEN GY, WINIEWSKI HM (1982) Brain and nutrition: the role of taurine. Prog. Clin Biol. Res 91, 349±355. GEORGE L, MILLS JL, JOHANSSON AL, OLANDER B, GRANATH F, CNATTINGUIS S (2002) Plasma folate levels and risk for spontaneous abortions. JAMA 288: 1867±1873. GEWOLB IH, O'BRIEN J (1997). Surfactant secretion by type II peumocytes is inhibited by high glucose concentrations. Exp. Lung Res. 23: 245±255. GIL A, UAUY R (1989) Dietary nucleotides in infant nutrition. J. Nutr. Gastroenterol. 4, 145±153. GIL A, UAUY R (1995) Nucleotides and related compounds in human and bovine milks. In Handbook of milk composition, pp 436±464 (RG Jensen, ed.). San Diego, Ca: Academic Press. GIRARD J, FERREÂ P, PEÂGORIER JP, DUEÂE PH (1992) Adaptations of glucose and fatty acid metabolism during perinatal period and suckling-weaning transition. Physiol. Rev. 72, 507±562. GLEASON P, SUITER CW (2002) Changes in children's diets: 1989±1991 to 1994±1996. US Department of Agriculture, Food and Nutrition Service. January. Available at http://www.fns.usda.gov/oane/MENU/Published/CNP/CNP.HTM. GODFREY K, ROBINSON S, BARKER DJP, OSMOND C, COX V (1996) Maternal nutrition in early and late pregnancy in relation to placental and foetal growth. Br. Med. J. 312, 410±414. GOODMAN MT, WILKENS LR, HANKIN JH, LYU LC, KOLONEL LN (1997) Association of soy and fiber consumption with the risk of endometrial cancer. Am. J. Epidemiol. 146, 294± 306. Á res GUANDALINI S, DINCER AP (1998) Nutritional management in diarrheal disease. Baillie Clin. Gastroenterol. 12, 697±717. HAGGARTY P, ASHTON J, JOYNSON M, ABRAMOVICH DR, PAGE K (1999) Effect of maternal polyunsaturated fatty acid concentration on transport by the human placenta. Biol. Neonate 75, 350±359. HAGUE WN (2003) Homocysteine and pregnancy. Best Pract. Res. Clin. Obstet. Gynaecol. 17, 459±469. HALES CN, BARKER DJP, CLARK PMS, COX LJ, FALL C, OSMOND C, WINTER PD (1991) Fetal and infant growth and impaired glucose tolerance at age 64. Br. Med. J. 303, 1019±1022. HALES CN, DESAI M, OZANNE SE, CROWTHER NJ (1996) Fishing in the stream of diabetes: from measuring insulin to the control of fetal organogenesis. Biochem. Soc. Trans. 24, 341±350. HALSTED CH (2003) Dietary supplements and functional foods: two sides of a coin? Am. J. Clin. Nutr. 77 (4 Suppl.): 1001S±1007S. GAROFANO A, CZERNICHOW P, BREANT B

HARMSEN HJM, WILDEBOER-VELOO ACM, RAANGS GS, WAGENDORP AA, KLIJN N, BINDELS JG,

(2000) Analysis of intestinal flora development in breast-fed and formula-fed infants by using molecular identification and detection methods. J. Pediatr. Gastroenterol. 30, 61±67.

WELLING GW

HARTINI TN, WINKVIST A, LINDHOLM L, STENLUND H, PERSSON V, NURDIA DS, SURJONO A

(2003) Nutrient intake and iron status of urban poor and rural poor without access to rice fields are affected by the emerging economic crisis: the case of pregnant Indonesian women. Eur. J. Clin. Nutr. 57: 654±666.

48

Functional foods, ageing and degenerative disease

HASLER CM (2000) The changing face of functional foods. J. Am. Coll. Nutr. 19, 499S±506S.

(2002) Functional foods: benefits, concerns and challenges a position paper from the American Council of Science and Health. J. Nutr. 132, 3772± 3781. HASLER C, MOAG-STAHLBERG A, WEBB D, HUDNALL M (2001) How to evaluate the safety, efficacy, and quality of functional foods and their ingredients. J. Am. Diet. Assoc. 1001, 733±736. HAY WW (1994) Placental transport of nutrients to the fetus. Horm. Res. 42, 215±222. HELLAND IB, SMITH LS, SAAREM K, SAUGSTAD OD, DREVON CA (2003) Maternal supplementation with very long chain n-3 fatty acids during pregnancy and lactation augments children's IQ at 4 years of age. Pediatrics 111, e39±e44. HELLER IR (2001) Functional Foods: regulatory and marketing developments. Food Drug Law J. 56, 197±225. HASLER CM

HELLERSTEIN MK, WU K, MCGRATH M, FAIX D, GEORGE D, SHACKLETON CH, HORN W, HOH R, NEESE RA (1996) Effects of dietary n-3 fatty acid supplementation in men with weight loss associated with the acquired immune deficiency syndrome: relation to indices of cytokine production. J. Acquir. Immune Defic. Syndr. Hum. Retrovirol. 11, 258±270. HERRERA E (2002) Implications of dietary fatty acids during pregnancy on placental, fetal and postnatal development. Placenta 23 (Supplement A, Trophoblast Research) 16, S9±S19. HILAKIVI-CLARKE L, CHO E, DEASSIS S, OLIVO S, BOUKER KB, WELCH JN, KHAN G, CLARKE R,

CABANES A (2001) Maternal and prepubertal diet, mammary development and breast cancer risk. J. Nutr. 154S±157S. HOD M, MERLOB P, FRIEDMAN S, SCHOENFELD A, OVADIA J (1991) Gestational diabetes mellitus. A survey of perinatal complications in the 1980s. Diabetes 40 (Suppl. 2), 74±78. HOWELL WH, MCNAMARA DJ, TOSCA MA, SMITH BT, GAINES JA (1997) Plasma lipid and lipoprotein responses to dietary fat and cholesterol ± a meta-analysis. Am. J. Clin. Nutr. 65, 1747±1764. HU FB, STAMPFER MJ, MANSON JE, ASCHERIO A, COLDITZ GA, SPEIZER FE, HENNEKENS CH,

(1999a) Dietary saturated fats and their food sources in relation to the risk of coronary heart disease in women. Am. J. Clin. Nutr. 70, 1001±1008.

WILLETT WC

HU FB, STAMPFER MJ, RIMM EB, MANSON JE, ASCHERIO A, COLDITZ GA, ROSNER BA,

(1999b) A prospective study of egg consumption and risk of cardiovascular disease in men and women. J. Am. Med. Assoc. 281, 1387±1394. HUBEL C, LYALL F, WEISSFELD L, GANDLEY RE, ROBERTS JM (1998) Small low-density lipoproteins and vascular cell adhesion molecule-1 are increased in association with hyperlipidemia in preeclampsia. Metabolism 47, 1281±1288. ILSI (1999) (International Life Science Institute). Scientific concepts of functional foods in Europe. Consensus document. Br. J. Nutr. 81: S1±S27. INSTITUTE OF MEDICINE (1998) DRI Dietary Reference Intakes for Thiamin, Riboflavin, Niacin, Vitamin B6, Folate, Vitamin B12, Pantothenic Acid, Biotin, and Choline, Washington DC: National Academy Press. INSTITUTE OF MEDICINE (2002) DRI Dietary Reference Intakes for Energy, Carbohydrates Fiber, Fat, Protein and Amino-acids (Macronutriments). Washington, DC: National Academy Press. È TAS Y, MOILANEN E, SALMINEN S (2000) Probiotics in the ISOLAURI E, ARVOLA T, SU SPIEGELMAN D, SPEIZER FE, SACKS FM, HENNEKENS CH, WILLET WC

Diet and the prevention of degenerative disease

49

management of atopic eczema. Clin. Exp. Allergy 30, 1604±1610.. (1988) Vegetarian children: appropriate and inappropriate diets. J. Am. Coll. Nutr. 18, 811±818. JAMES DC (2001) Eating disorders, fertility, and pregnancy: relationships and complications. J. Perinatal Neonatal Nurs. 15: 36±48. JARIWALLA RJ (2001) Rice-bran products: phytonutrients with potential applications in preventive and clinical medicine. Drugs Exp. Clin. Res. 27, 17±26. JAUNIAUX E, GULBIS B, GERLO E, RODECK C (1998) Free amino acid distribution inside the first trimester human gestational sac. Early Human Development 51, 159± 169. JAUNIAUX E, WATSON AL, BURTON GJ (2001) Evaluation of respiratory gases and acid-base gradients in fetal fluids and uteroplacental tissue between 7 and 16 weeks. Am. J. Obstet. Gynecol. 184: 998±1003. JONES CJ (1997) The life and death of the embryonic yolk sac. In: Jauniaux E, Barnea ER, Edwards RG, eds. Embryonic medicine and therapy. Oxford: Oxford University Press; 180±186. JONES AP, FRIEDMAN MI (1982) Obesity and adipocyte abnormalities in offspring of rats undernourished during pregnancy. Science. 215, 1518±1519. JONES G, RILEY M, DWYER T (2000) Breastfeeding in early life and bone mass in prepubertal children: a longitudinal study. Osteoporos. Int. 11: 146±152. KALLIOMAKI M, SALMINEN S, ARVILOMMI H, KERO P, KOSKINEN P, ISOLAURI E (2001) Probiotics in primary prevention of atopic disease: a randomised placebocontrolled trial. Lancet 357, 1076±1079. KANAMORI Y, HASHIZUME K, SUGIYAMA M, MOROTOMI M, YUKI N, TANAKA R (2002) A novel symbiotic therapy dramatically improved the intestinal function of a pediatric patient with laryngotracheo-esophagal cleft (LTEC) in the intensive care unit. Clin. Nutr. 21, 527±530. KARASOV WH, SOLBERG DH, CHANG SD, HUGHES M, STEIN ED, DIAMOND JM (1985) Is intestinal transport of sugars and amino acids subject to critical-period programming? Am. J. Physiol. 249 (Gastrointestinal and Liver Physiology 12), G770±G785. KAY RA (1991) Microalgae as food and supplement. Crit. Rev. Food Sci. Nutr. 30, 555± 573. KELLEY DS, ERICKSON KL (2003) Modulation of body composition and immune cell functions by conjugated linoleic acid in humans and animal models: benefits vs. risks. Lipids 38, 377±386. KHWAJA SS, UDUMAN SA, AL-SIBAI H, AL-SULEIMAN SA (1986) The oversized infant. A study of 86 cases. Aust. NZ. J. Obstet. Gynaecol. 26, 22±25. KING JC (2002) Biotechnology: a solution for improving nutrient bioavailability. Int. J. Vitam. Nutr. Res. 72: 7±12. KOLDOVSKY O, STRBAK V (1995) Hormones and growth factors in human milk. In Handbook of milk composition, pp 428±436 (RG Jensen, ed.) San Diego, CA: Academic Press. KOLDOVSKY O, THORNBURG W (1987) Hormones in human milk: a review. J. Pediatr. Gastroenterol. and Nutr. 6, 172±196. KOLETZKO B (1992) Fats for brains. Eur. J. Clin. Nutr. 46 (Suppl. 1), S51±S62. JACOBS C, DWYER JT

KOLETZKO B, AGGETT PJ, BINDELS JG, BUNG P, FERREÂ P, GIL A, LENTZE MJ, ROBERFROID M, STROBEL S (1998) Growth, development and differentiation: a functional food science approach. Br. J. Nutr. 80 (Suppl. 1) S5±S45.

50

Functional foods, ageing and degenerative disease

(1991) Arachidonic acid and early human growth: is there a relation? Ann. Nutr. Metabol. 35, 128±131. KOPP-HOOLIHAN L (2001) Prophylactic and therapeutic uses of probiotics: a review. J. Am. Diet. Assoc. 101, 229±238. KRAMER MS (2002) Balanced protein/energy supplementation in pregnancy (Cochrane Review). In: The Cochrane Library, issue: CD 000032. Update Software, Oxford. KRUGER CL, MANN SW (2003) Safety evaluation of functional ingredients. Food Chem. Toxicol. 41, 793±805. KULKARNI AD, RUDOLPH FB, VAN BUREN CT (1994) The role of dietary sources of nucleotides in immune function: a review. J. Nutr. 124, 1442S±1446S. KUNZ C, RUDLOFF S, BAIER W, KLEIN N, STROBEL S (2000) Oligosaccharides in human milk: structural, functional and metabolic aspects. Annu. Rev. Nutr. 20, 699±722. KWONG WY, WILD AE, ROBERTS P, WILLIS AC, FLEMING TP (2000) Maternal undernutrition during the preimplantation period of rat development causes blastocyst abnormalities and programming of postnatal hypertension. Development 127, 4195±4202. LACEY JM, WILMORE DW (1990) Is glutamine a conditionally essential amino acid? Nutr. Rev. 48, 297±309. LANGLEY-EVANS SC (2000) Critical differences between two low protein diet protocols in the programming of hypertension in the rat. Int. J. Food. Sci. Nutr. 51, 11±17. LARQUE E, ZAMORA S, GIL A. (2001) dietary trans fatty acids in early life: a review. Early Hum. Dev. 65 (Suppl), S31±S41. LAWRENCE M, RAYNER M (1998) Functional foods and health claims: a public health policy perspective. Pub. Health Nutr. 1, 75±82. LELEIKO NS, WALSH MJ, ABRAHAM S (1995) Gene expression in the intestine: effect of dietary nucleotides. Adv. Pediatr. 42, 145±169. LEON D (1998) Fetal growth and adult disease. Eur. J. Clin. Nutr. 52 (suppl 1), S72±S82. LEWIS DS, BERTRAND HA, MCMAHAN CA, MCGILL HC, CAREY KD, MASARO EJ (1986) Preweaning food intake influences the adiposity of young adult baboons. J. Clin. Invest. 78, 899±905. LIFSHITZ F, TARIM O (1996) Considerations about dietary fat restrictions for children. J. Nutr. 126 (Suppl): 1031S±1041S. LIN B, GUTHRIE J, FRAZAO E (2001) America's children's diets not making the grade. Food Review 24, 8±17. LODINOVA-ZADNIKOVA R, CUKROWSKA B, TLASKALOVA-HOGENOVA H (2003) Oral administration of probiotic Escherichia coli after birth reduces frequency of allergies and repeated infections later in life (after 10 and 20 years). Int. Arch. Allergy Immunol. 131, 209±211. È NNERDAL B (2003). Nutritional and physiologic significance of human milk proteins LO Am. J. Clin. Nutr. 77, 1537S±1543S. LUCAS A (1991) Programming by early nutrition in men. In: Bock, G.R., Whelan J. (eds), The childhood environment and adult disease (CIBA Foundation symposium 156). Wiley, Chichester, UK, pp. 38±55. LUCAS A (1998) Programming by early nutrition : an experimental approach. J. Nutr. 128, 401S±406S. MAKRIDES M, NEUMANN MA, BYARD RW, SIMMER K, GIBSON RA (1994) Fatty acid composition of brain, retina, and erythrocytes in breast- and formula-fed infants. Am. J. Clin. Nutr. 60, 189±194. KOLETZKO B, BRAUN M

Diet and the prevention of degenerative disease

51

(1975) Vitamin passage across the placenta. Clin. Perinatol. 2, 295±307. (2001) Intrauterine low protein diet increases fetal û cell sensitivity to NO and IL-1±û: the protective role of taurine. J. Endocrinol. 171, 299±308. MERLET-BENICHOU C, GILBERT T, MUFFAT-JOLY M, LELIEÁVRE-PEÂGORIER M, LEROY B (1994) Intrauterine growth retardation leads to permanent nephron deficit in the rat. Pediatr. Nephrol. 8, 175±180. MESSINA MJ (2003) Emerging evidence on the role of soy in reducing prostate cancer risk. Nutr. Rev. 61, 117±31. MALONE JI

MEREZAK S, HARDIKAR A, YAJNICK CS, REMACLE C, REUSENS B

MICHELS KB, TRICHOPOULOS D, ROBINS JM, ROSNER BA, MANSON JE, HUNTER DJ, COLDITZ GA,

HANKINSON SE, SPEIZER FE, WILLETT WC (1996) Birthweight as a risk factor for breast cancer. Lancet 348, 1542±1546. MIKHAIL LN, WALKER CK, MITTENDORF R (2002) Association between maternal obesity and fetal cardiac malformations in African Americans. J. Natl. Med. Assoc. 94, 695± 700. MILNER JA (2000) functional foods: the US perspective. Am. J. Clin. Nutr. 71 (Suppl) 1654S±1659S. Â L, ROBLES R, GGIL A, SA Â NCHEZ-POZO A (1994) Lipoprotein changes in MORILLAS J, MOLTO small-for-gestational-age infants fed nucleotide-supplemented milk formula. Acta Paediatr. 83, 481±485. NAVARRO J, RUIZBRAVO A, JIMEÂNEZVALERA M, GIL A (1996) Modulation of antibodyforming cell and mitogen-driven lymphoproliferative responses by dietary nucleotides in mice. Immunol. Lett. 53, 141±145. NEWSHOLME EA, CARRIEÂ AL (1994) Quantitative aspects of glucose and glutamine metabolism by intestinal cells. Gut 35 (Suppl. 1), S13±S17. NIJJAR MS, KHANGURA BS, JURAVSKY LI (1984) The effect of maternal diabetes on the synthesis and secretion of phosphatidylcholine in fetal and maternal rat lungs in vitro. Diabetologia 27: 219±224. OBERHELMAN RA, GILMAN RH, SHEEN P, TAYLOR DN, BLACK RE, CABRERA L, LESCANO AG, MEZA R, MADICO G (1999) A placebo-controlled trial of Llactobacillus GG to prevent diarrhea in undernourished Peruvian children. J. Pediatr. 134, 15±20. OLSEN SF, HANSEN HS, SOERENSEN TI, JENSEN B, SECHER NJ, SOMMER S, KNUDSEN LB (1986) Intake of marine fat, rich in (n-3) polyunsaturated fatty acids, may increase birth weight by prolonging gestation. Lancet 2, 367±369. OLSEN SF, SOERENSEN JD, SECHER NJ, HEDEGAARD M, HENRIKSEN TB, HANSEN HS, GRANT A

(1992) Randomized controlled trial of the effect of fish-oil supplementation on pregancy duration. Lancet 339, 1003±1007. ONWUDE JL, LILFORD RJ, HJARTARDOTTIR H, STAINES A, TUFFNELL D (1995) A randomised, double blind placebo controlled trial of the effect of fish-oil in high risk pregnancy. Brit. J. Obst. Gynecol. 102, 95±100. OSMOND C, BARKER DJ (2000) Fetal, infant and childhood growth are predictors of coronary heart disease, diabetes and hypertension in adult men and women. Environ. Health Perspect. 108 (suppl. 3), 545±553. OZANNE SE, HALES CN (2002) Early programming of glucose-insulin metabolism. Trends Endocrinol. Metabol. 13, 368±373. PATTON S, GENDLER S, SPICER AP (1995) The epithelial mucin, MUC1, of milk mammary gland and other tissues. Biochim. Biophys. Acta 1241, 407±424. PEARSE WH, SORNSON H (1969) Free amino acids of normal and abnormal human placenta. Am. J. Obstet. Gynecol. 105, 696±700.

52

Functional foods, ageing and degenerative disease

(1999) A low protein diet alters the balance of islet cell replication and apoptosis in the fetal and neonatal rat, and is associated with a reduced pancreatic expression of insulin-like growth factors-II. Endocrinology, 140, 4861±4873. PICCIANO MF (2003) Pregnancy and lactation: physiological adjustments nutritional requirements and the role of dietary supplements. J. Nutr. 133, 1997S±2002S. PIETRZIK K, PRINZ R, REUSCH K, BUNG P, MALLMANN P, CHRONIDES A (1992) Folate status and pregnancy outcome. Ann. NY. Acad. Sci. 669, 371±373. PRENTICE A (2003) Micronutrients and the bone mineral content of the mother, fetus and newborn. J. Nutr. 133, 1693S±1699S. PRENTICE A, GOLDBERG G (1996) Maternal obesity increases congenital malformations. Nutr. Rev. 54, 146±150. PREZIOSI P, PRUAL A, GALAN P, DAONDA H, BOUREIMA H, HERCBERG S (1997) Effect of iron supplementation on the iron status of pregnant women: consequences for newborns Am. J. Clin, Nutr. 66, 1178±1182. PROSKY L (2000) When is dietary fiber considered a functional food? BioFactors 12, 289± 297. PETRIK J, REUSENS B, ARANY E, REMACLE C, COEHLO C, HOET JJ , HILL DJ

È R, NOHYNEK L, MEIER C, KA È HKO È NEN M, HEINONEN M, HOPIA A OKSMANPUUPPONEN-PIMIA

(2001) Antimicrobial properties of phenolic compounds from berries. J. Appl. Microbiol. 90, 494±507. RASMUSSEN KM, WALLACE MH, GOURNIS E (2001) A low-fat diet but not food restriction improves lactational performance in obese rats. Adv. Exp. Med. Biol. 501, 101± 106. RASSIN DK, STURMA, JA, GALL GE (1978) Taurine and other amino acid in milk of man and other mammals, Early Hum. Dev., 2: 1±13. RASTALL RA, MAITIN V (2002) Prebiotics and synbiotics: towards the next generation. Curr. Op. Biotechnol. 13, 490±496. RAUTAVA S, KALLIOMAKI M, ISOLAURI E (2002) Probiotics during pregnancy and breastfeeding might confer immunomodulatory protection against atopic disease in the infant. J. Allergy Clin. Immunol. 109, 119±121. RAVELLI GP, STEIN ZA, SUSSER MW (1976) Obesity in young men after famine exposure in utero and early infancy. New Eng. J. Med. 295; 349±353. RAVELLI AC, VAN DER MEULEN JH, OSMOND C, BARKER DJ, BLEKER OP (1999) Obesity at the age of 50 y in men and women exposed to famine prenatally Am. J. Clin. Nutr. 70, 811±816. REUSENS B, REMACLE C (2001) Intergenerational effects of adverse intrauterine environment on perturbation of glucose metabolism. Twin Res. 4, 406±411. RICHARDSON D (1996) Functional foods-shades of grey: an industry perspective. Nutr. Rev. 54: S174±S185. CALDENTEY K-M

RICH-EDWARDS JW, STAMPFER MJ, MANSON JE, ROSNER B, HANKINSON SE, COLDITZ GA,

(1997) Birthweight and risk of cardiovascular disease in a cohort of women followed up since 1976. Brit. Med. J. 315, 396±400. RIO ME, ZAGO BL, GARCIA H, WINTER L (2002) The nutritional status changes the effectiveness of a dietary supplement of lactic bacteria on the emerging of respiratory tract diseases in children. Arch. Latinoam. Nutr. 52, 29±34. ROBERFROID MB (2000) Prebiotics and probiotics: are they functional foods? Am. J. Clin. Nutr. 71 (Suppl) 1682S±1687S. ROBERFROID MB, CUMPS J, DEVOGELAER JP (2002) Dietary chicory inulin increases wholebody bone mineral density in growing male rats. J. Nutr. 132, 3599±3602. WILLETT WC, HENNEKENS CH

Diet and the prevention of degenerative disease

53

(1974) Maternal protein deprivation and toxemia of pregnancy studies in the rhesus monkey (Macacca mulatta) Am. J. Obst. Gynecol. 118, 14. ROBERTS JM, BALK JL, BODNAR LM, BELIZAN JM, BERGEL E, MARTINEZ A (2003) Nutrient involvement in preeclampsia. J. Nutr. 133, 1684S±1692S. RODESCH F, SIMON P, DONNER C, JAUNIAUX E (1992) Oxygen measurements in endometrial and trophoblastic tissues during early pregnancy. Obstet. Gynecol. 80: 283±285. ROGERS IS, EMMETT PM and ALSPAC STUDY TEAM (2001) Fat content of the diet among preschool children in southwest Britain: II. Relationship with growth, blood lipids and iron status. Pediatr. 108, E49. ROBERTS JA, HILL GW, PROPELLE AJ

ROSENBOOM TJ, VAN DER MEULEN JHP, OSMOND C, BARKER DJP, RAVELLI ACJ, BLEKER OP

(2000) Plasma lipid profile after prenatal exposure to the Dutch famine. Am. J. Clin. Nutr. RUSH D (2001) Maternal nutrition and perinatal survival. Nutr. Rev. 59: 315±326. SAAVEDRA JM, BAUMAN NA, OUNG I, PERMAN JA, YOLKEN RH (1994) Feeding of Bifidobacterium bifidum and Streptococcus thermophilus to infants in hospital for prevention of diarrhoea and shedding of rotavirus. Lancet 344, 1046±1049. SALAFIA C, SHIVERICK K (1999) Cigarette smoking and pregnancy II: vascular effects. Placenta (1999) 20: 273±279. SALLE BL, DELVIN EE, LAPILLONNE A, BOSHOP NJ, GLORIEUX FH (2000) Perinatal metabolism of vitamin D. Am. J. Clin. Nutr. 71 (Suppl), 1317S±1324S. SALLE H (1989) Ramadan fasting among pregnant women in Muar district, Malaysia and its association to health outcomes Malys J. Reprod. Health 71, 69±83. Â NCHEZ-POZO D, MORILLAS J, MOLTO Â L, ROBLES R, GIL A (1994) Dietary nucleotides SA influence lipoprotein metabolism in newborn infants. Pediatr. Res. 35, 112±116. SANDERS ME, HUIS IN'T VELD J (1999) Bringing a probiotic-containing functional food to the market: microbiological, product, regulatory and labeling issues. Antonie van Leeuwenhoek 76: 293±315. SANDERSON M, WILLIAMS MA, MALONE KE, STANFORD JL, EMANUEL I, WHITE E, DALING UJR

(1996) Perinatal factors and risk of breast cancer. Epidemiology 7, 34±37. (2002) Use of fermented foods to combat stunting and failure to thrive, Nutr. 18, 393±396. SCHIFFRIN EJ, BLUM S (2002) Interactions between the microbiota and the intestinal mucosa. Eur J. Clin. Nutr. 56 (Suppl. 3), S60±S64. SCHOLZ-AHRENS KE, SCHAAFSMA G, VAN HEUVEL EGHM, SCHREZENMEIR J (2001) Effects of probiotics on mineral metabolism. Am. J. Clin. Nutr. 73, 459S±464S. SARON S, GOPALAN S, KRISHNA TP

SEBIRE NJ, JOLLY M, HARRIS JP, WADSWORTH J, JOFFE M, BEARD RW, REGAN L AND ROBINSON S

(2001) Maternal obesity and pregnancy outcome: a study of 287,213 pregnancies in London. Int. J. Obes. Relat. Metab. Disord. 25, 1175±1182. SECHER NJ, OLSEN SF (1990) Fish oil and preeclampsia. Br. J. Obst. Gynecol. 97, 1077± 1079. SETCHELL KDR, CASSIDY A (1999) Dietary flavones: biological effects and relevance to human health. J. Nutr. 129, 758S±767S. SHAH DM (2001) Perinatal implications of maternal hypertension. Semin. Pediatr. Neurol. 8: 108±119. SHERMAK MA, SAAVEDRA JM, JACKSON TL, HUANG SS, BAYLESS TM, PERMAN JA (1995) Effect of yoghurt on symptoms and kinetics of hydrogen production in lactosemalabsorbing children. Am. J. Clin. Nutr. 62, 1003±1006.

54

Functional foods, ageing and degenerative disease

(1999) Cigarette smoking and pregnancy I: ovarian, uterine and placental effects. Placenta 20: 265±272. SIBLEY CP, BOYD RDH (1992) Mechanisms of transfer across the human placenta. In: Polin RA, Fox WW, editors. Fetal and Neonatal Physiology, vol 1. Philadelphia: Saunders, 62±74. SILVERGLADE BA, HELLER AR (1997) Are functional foods the solution to dysfunctional diets? A review of US regulatory requirements and lessons from abroad. Food Drug Law J. 52, 313±321. SIMOPOULOS AP (1991) Omega-3 fatty acids in health and disease and in growth and development. Am. J. Clin. Nutr. 54, 438±463. SIMOPOULOS AP (2000) Symposium: role of poultry products in enriching the human diet with n-3 PUFA. Poultry Science 79, 961±970. SMART J (1986) Undernutrition learning and memory: review of experimental studies. In: Proceedings of XII International Congress of Nutrition (Taylor TG and Jenkins NK eds), pp 74±78, John Libbey, London, UK. SNOECK A, REMACLE C, REUSENS-BILLEN B, HOET JJ (1990) Effect of a low protein diet during pregnancy on the fetal rat endocrine pancreas. Biol. Neonate, 57, 107±118. Â MEZ S, NOVA E, DE LA ROSA B, MARCOS A (2002) Probiotics as a help SOLIS B, SAMARTIN S, GO in children suffering from malnutrition and diarrhoea. Eur. J. Clin. Nutr. 56 (Suppl. 3), S57±S59. SONG WO, KERVER JM (2000) Nutritional contribution of eggs to American diets. J. Am. Coll. Nutr. 19 (Suppl. 5), 556S±562S. SHIVERICK K, SALAFIA C

STACEWICZ-SAPUNTZAKIS M, BOWEN PE, HUSSAIN EA, DAMAYANTI-WOOD BI, FARNSWORTH NR

(2001) Chemical composition and potential health effects of prunes: a functional food? Crit. Rev. Food Sci. Nutr. 41, 251±286. STANTON C, GARDINER G, MEEHAN H, COLLINS K, FITZGERALD G, LYNCH PB, ROSS RP (2001) Market potential for probiotics. Am. J. Clin. Nutr. 73 (suppl): 476S±483S. STEIN CE, FALL CHD, KUMARAN K, OSMOND C, COX V, BARKER DJP (1996) Fetal growth and coronary heart disease in South India. Lancet 348, 1269±1273. SUAREZ A, FAUS MJ, GIL A (1996a) Dietary long-chain polyunsaturated fatty acids modify heart, kidney, and lung fatty acid composition of weanling rats. Lipids 31, 345± 348. SUAREZ A, FAUS MJ, GIL A (1996b) Dietary supplementation with long-chain polyunsaturated fatty acids increases the susceptibility of weanling rat tissue lipids to in vitro lipid peroxidation. J. Nutr. Biochem. 7, 252±260. SWENSEN AR, HARNACK LJ, ROSS JA (2001) Nutritional assessment of pregnant women enrolled in the Special Supplemental Program for Women, Infants and Children (WIC). J. Am. Diet. Assoc. 101, 903±908. TAKANO T (2002) Anti-hypertensive activity of fermented dairy products containing biogenic peptides. Antonie van Leeuwenhoek 82, 333±340. TANAKA M, LEE K, MARTIÂNEZ-AUGUSTIN, HE Y, SANDERSON IA, WALKER W (1996) Exogenous nucleotides alter the proliferation, differentiation and apoptosis of human small intestinal cells epithelium. J. Nutr. 126, 424±433. TEILMANN G, JUUL A, SKAKKEBAEK NE, TOPPARI J. (2002) Putative effects of endocrine disrupters on pubertal development in the human. Best Pract. Res. Clin. Endocrinol. Metabol. 16, 105±121. TYDEN O, ERIKSSON UJ, BERNE C (1986) Fetal lung maturation in diabetic pregnancy. Acta Endocrinol. Suppl. (Copenh.) 277: 101±106. UDIPI SA, ANTHONY U (2000) Nutrition in pregnancy and lactation. J. Indian Med. Assoc.

Diet and the prevention of degenerative disease 987, 548±557. VALAZQUEZ A, ROSADO A, BERNAL A

Biol. Neonate 29, 28±40.

55

(1976) Amino acid pools in the foet-maternal system.

(2002) Functional foods in pediatrics. Acta Gastro-Enterol Belg. 65: 45±51. VANDERHOOF JA (2001) Probiotics: future directions. Am. J. Clin. Nutr. 73 (Suppl) 1152S1155S. VANDERVEEN JE, GLINSMANN WH (1992) Fat substitutes: a regulatory perspective. Ann. Rev. Nutr. 12, 473±487. VAN DEN DRIESSCHE M, VEEREMAN-WAUTERS G

VATTEN LJ, MAEHLE BO, LUND NILSEN TI, TRETLI S, HSIEH CC, TRICHOPOULOS D, STUVER SO

(2002) Birthweight as a predictor of breast cancer: a case-control study in Norway. Br. J. Cancer 7, 89±91. VICKERS MH, REDDY S, IKENASIO BA, BREIER BH (2001) Dysregulation of the adipoinsular axis a mechanism for the pathogenesis of hyperleptinemia and adipogenic diabetes induced by fetal programming. J. Endocrinol. 170, 323±332. VILLAR J, CARROLI G (1996) Methodological issues of randomized controlled trials for the evaluation of reproductive health interventions. Prev. Med. 25, 365±375. È LMEZOGLU M, ABALOS E, CARROLI G, KULIER R, ONI M (2003) VILLAR J, MERIALDI M, GU Nutritional interventions during pregnancy for prevention or treatment of maternal morbidity and preterm delivery: an overview of randomized controlled trials. J. Nutr. 133, 1606S±1625S. WANG X, SATO R, BROWN MS, HUA X, GOLDSTEIN JL (1994) SREBP-1, a membrane bound transcription factor released by sterol-regulated proteolysis. Cell 77, 53±62. WATANABE S, SAKAI N, YASUI Y, KIMURA Y, KOBAYASHI T, MIZUTANI T, OKUYAMA H (1994) A high alpha-linolenate diet suppresses antigen-induced immunoglobulin E response anaphylactic shock in mice. J. Nutr. 124, 1566±1573. WELPLY JK, KEENE JL, SCHMUKE JJ, HOWARD SC (1994) Selectins as potential targets of therapeutic intervention in inflammatory diseases. Biochim. Biophys. Acta 1197, 215±226. WELSH AO (1993) Uterine cell death during implantation and early placentation. Micro. Res. Tech. 25, 223±245. WERLER MM, LOUIK C, SHAPIRO S, MITCHELL AA (1996) Prepregnant weight in relation to risk of neural tube defects. JAMA 275, 1089±1092. WILLIAMS C, HIGHLY W, MA EH, LEWIS J, TOLBERT B, WOULLARD D, KIRMANI S, CHUNG RA

(1981) Protein, amino acid and caloric intakes in selected pregnant women. J. Am. Diet. Assoc. 78, 28±35. WINICK M (1970) Cellular growth in intrauterine malnutrition. Pediatr. Clin. North Am. 17, 969±977. WISNIEWSKI AB, KLEIN SL, LAKSHMANAN Y, GEARHART JP (2003) Exposure to genistein during gestation and lactation demasculinizes the reproductive system in rats. J. Urol. 169, 1582±1586. WORLD HEATH ORGANISATION (1995) Maternal antropometric and pregnancy outcomes: a WHO Collaborative Study. Bull. W.H.O. 73. WORLD HEALTH ORGANISATION (2000) Turning the tide of malnutrition: Responding to the challenge of the 21st century. Geneva, World Health Organization (Document WHO/NHD/00.7). YOKOGOSHI H, ODA H (2002) Dietary taurine enhances cholesterol degradation and reduces serum and liver cholesterol concentrations in rats fed a high-cholesterol diet. Amino Acids 23, 433±439.

56

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(2000) Choline: needed for normal development of memory. J. Am. Coll. Nutr. 19, 528S±531S. ZEISEL SH, MAR M-H, ZHOU Z-W, DA COSTA K-A (1995) Pregnancy and lactation are associated with diminished concentrations of choline and its metabolites in rat liver. J. Nutr. 125, 3049±3054. ZEISEL SH

3 New functional foods for age-related diseases D. Rivera, University of Murcia and C. OboÂn, University Miguel HernaÂndez, Spain

3.1

Introduction: the Mediterranean diet and healthy living

3.1.1 The Mediterranean idea of food as medicine The idea of developing different foods according to their secondary effect on health is becoming widely accepted by the food industry at the beginning of the 21st Century (Mazza, 1998). This idea is not novel: some two thousand years ago the Greeks and Romans formulated the direct relationship between lifestyle, diet and health status (Lonie, 1977; Longrigg, 1998; Smith, 1982). In the Medieval Islamic Mediterranean cultures, the borderline between food and medicine appears extremely diffuse. Huici (1966) recorded many recipes of `functional foods', viz. dishes especially recommended for pregnant women, persons with liver complaints, etc. Complex mixtures of herbs and spices in Palestine and Syria are still consumed as a kind of tea or beverage for improving health (Crowfoot and Baldensperger, 1932; Carmona et al., submitted). The traditional folk medicine in rural areas of the western Mediterranean region uses a high percentage of food plant species (between 25 and 30% of total species). Examples of foods that are reported by local people as healthy and medicinal cover a wide range, from citrus, tomato, carrots, onions, and garlic to rosemary, thyme and sage (Rivera and OboÂn, 1996).

3.1.2 Determinants of healthy ageing in the Mediterranean: genetics, lifestyle, climate and food In the 1960s Keys and collaborators showed that, though we are all one species, major cultural differences exist in the distribution of risk characteristics and risk

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behaviours, including diet, and in the geographic and time distribution of the major causes of death. Keys demonstrated that traits, heretofore considered irrevocable and constitutional, such as body type, blood fat levels (cholesterol), blood pressure, heart rate, and responses to stress, were, in fact, largely modifiable by simple changes in the composition and quantity of diet and physical activity (Blackburn, 2003; Keys et al., 1963; Keys, 1980). The beneficial health effects of Mediterranean diet have been shown in studies with different cultural and ethnic groups outside the Mediterranean region. In Sweden a modified Cretan Mediterranean diet reduced signs and symptoms of a 26-patient group with rheumatoid arthritis (age 33±73) while no changes were seen in the control diet group (n = 25, age 35±75) in the dietary intervention study. Those that adopted the Cretan Mediterranean diet obtained a reduction in disease activity, improved physical function, and improved vitality (Hagfors, 2003). Itsiopoulos et al. (2003) have shown with Australian-born patients that the traditional Cretan Mediterranean diet can be successfully implemented in the treatment of diabetes with modest benefits in metabolic control. Recent studies comparing men aged 80 and over, survivors of two cohorts of the Seven Countries Study, from Crete (Greece) and Zutphen (Netherlands), have shown that elderly men from Crete are exposed to less oxidative stress than elderly men from Zutphen as measured by serum hydroperoxydes and serum iron and ferritin (Buijsse et al., 2003), Cretan elderly have significantly higher levels of serum folate and lower levels of serum vitamin B12 probably due to dietary differences (Jansen et al., 2003). Although most of the Mediterranean dietary items come from plants, fish is another relevant component. Yahiaa et al. (2003) have shown that administration of fish protein to spontaneously hypertensive rats has a favourable influence on blood pressure, plasma angiotensin II and cholesterol concentration as compared to casein.

3.1.3 The Mediterranean diet and healthy lifestyle The term `Mediterranean diet' was coined in the book written by Ancel and Margaret Keys (Keys and Keys, 1959). The nutritionist Ancel Keys, who stayed in the 1950s in Rome, Naples and Madrid, found that the Naples firemen and Madrid poor had a significantly lower blood cholesterol levels than Americans, and fat represented a smaller percentage of their daily diets. But 50 professional men in Madrid (Spain), all of whom had diets comparable to diets in the United States, had cholesterol levels comparable to those of their American counterparts (Hoffman, 2003). Naska (2003) (modifying Trichopoulou et al., 1995) defined nine components of Mediterranean diet: 1. 2.

High consumption of olive oil and low consumption of lipids of animal origin. Expressed by Trichopoulou et al. (1995) in terms of a high ratio of monunsaturated to saturated fat. High consumption of vegetables.

New functional foods for age-related diseases 3. 4. 5. 6. 7. 8. 9.

59

High consumption of legumes. High consumption of cereals (including bread). High consumption of fruit. Moderate to high consumption of fish. Low to moderate consumption of milk and dairy products. Low consumption of meat and meat products. Moderate wine (alcohol) consumption.

In fact there are many Mediterranean diets in the different countries with coasts on the Mediterranean sea (Noah and Truswell, 2001; Simopoulos, 2001; Simopoulos and Visioli, 2001). Fischler (1996) has discussed the subjective nature of the `Mediterranean diet' idea. It is difficult to define precisely a Mediterranean type diet. In fact Food Composition Databases (FCDB) are defined at strictly national level and their nutrient values are not truly comparable. The need for a standardised FCDB at European level to derive comparable nutrient values across countries, to increase understanding of nutritional epidemiology and the relationship between nutrition and health has been recently proposed (Charrondiere et al., 2002). Measurement errors in nutrient intake between population groups may be one of the reasons for many contradictory results on the relations between diet and chronic disease (Riboli, 1989). Therefore we say `Mediterranean diet' because the different case studies fall within the Mediterranean area and show a more or less common pattern, but not because there is a clear-cut definition of what is and what is not a Mediterranean food or a Mediterranean diet. The Crete cohort is the only cohort among the Seven Country Study associated with a striking protective effect of both coronary heart disease and mortality from all causes (Renaud, 2003). The traditional Greek diet of Crete is therefore assumed to be the prototype of Mediterranean diet (Trichopoulou et al., 2000). The Mediterranean-type diet is assumed to be rich in vegetables, legumes, fruits and olive oil. Different trials have shown that this type of diet is associated with a pronounced decline in coronary disease morbidity and mortality, not only in the Mediterranean but also in the non-Western population of India or Israel (Renaud, 2003; Singh et al., 2002). Unfortunately, despite the worldwide acceptance of the beneficial effects of the Mediterranean diet, people in the region have progressively moved from their long-standing tradition, specially concerning the increase in meat and cheese and the decrease in fruit availability (De Lorgeril et al., 2002; Gerber and Lairon, 2003; Oikonomou et al., 2003; SaÂnchez et al., 2002). The European Prospective Investigation into Cancer and Nutrition (EPIC) is a network of prospective studies involving about 500,000 subjects from ten western European countries. The countries participating in EPIC are characterised by specific dietary patterns (studied with a single 24hour diet recall from a representative sample of study subjects). Overall, Italy and Greece have a dietary pattern characterised by plant foods (except potatoes), and a lower consumption of animal and processed food compared to the other EPIC countries. France and particularly Spain present more heterogeneous

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dietary patterns (Slimani, 2003). According to Kromhout (2003) different Mediterranean diets are associated with different mortality rates from coronary heart disease and the lowest rate was observed in Crete. The nutrient content of the Mediterranean diet of Crete has been shown by Kafatos et al. (2000). Physical exercise is essential for successful ageing. It helps to keep cardiovascular fitness reduces risks of osteoporosis and increases the sense of equilibrium. Sarri et al. (2003) have found that high physical activity reduces the levels of triglycerides and LDL-C while endurance capacity is inversely related to obesity, even in children. Most cases of Parkinson's disease are not due to a genetic defect but are caused by other factors that are probably environmental (exposure to chemicals such as pesticides and herbicides, diet and smoking). Genetic variation may modify the effect of life style and diet on health and longevity, and some of the variation in life style and diet may be due to genetic differences (Vollset, 2003b). The outstanding life expectancy of the Crete cohort in the Seven Country Study could be due to the exceptional climate, the absence of stress and pollution, the after-lunch siesta or other factors yet to be discovered (Yarnell and Evans, 2000) although diet and particularly -linolenic acid plays a key role in that protective effect (Renaud, 2003). Trichopoulou et al. (2003) assessed the relationships between adherence to a Mediterranean Diet and survival in a Greek population (involving 22,043 adults from all regions of Greece). They concluded that a greater adherence to traditional Mediterranean diet is directly associated with a significant reduction in total mortality and inversely with both death due to coronary heart disease and death due to cancer. Associations between individual food groups contributing to the Mediterranean-diet score and total mortality were generally not significant.

3.2

Mediterranean foods and their functional properties

3.2.1 Legumes Legumes, specifically lentils, chickpeas and Pinto beans, are an excellent source of folate, which in addition to being an essential nutrient is thought to reduce the risk of neural tube defects (Messina, 1999). Isoflavonoids are particularly prevalent in the Leguminosae, in which they are widely distributed. They function as preformed or inducible antimicrobial or anti-insect compounds to protect plants, as inducers of the nodulation genes of symbiotic bacteria, or as allelopathic agents. Isoflavones exhibit estrogenic, antiangiogenic, antioxidant, and anticancer activities. Major sources of isoflavones for humans are seed products of soybean (daidzein and genistein) and chickpea (biochanin A) (Dixon and Summer, 2003). Dietary supplements and ingredients such as RimostilTM or ClovoneTM contain isoflavones such as genistein and biochanin A and are supposed to provide cardiovascular benefits and assist in maintaining bone health. Genistein is an isoflavonoid present in soy products but also in Mediterranean and American Leguminosae like Phaseolus vulgaris L. (Durango et al., 2002).

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Andlauer et al. (2000) have shown the favourable uptake of genistein in isolated rat small intestine. Genistein exhibits a potent antiandrogenic activity in addition to its estrogenic activity. The therapeutic potential of this compound in prostate cancer patients may be related to its combined estrogenic and antiandrogenic properties (Zand et al., 2000). However a randomised isoflavone intervention among premenopausal women over a one±year period does not support the hypothesis that isoflavones affect the ovulatory cycles (Maskarinec et al., 2002). Kim et al. (2001) indicate that genistein, and probably other related phytoestrogens, have pleiotropic actions, some of which may involve transforming factor activity. Although genistein inhibits cancer cell growth in vitro, it is unlikely that the plasma concentration required to inhibit cancer cell growth in vivo can be achieved from a dietary dosage of genistein (Santell et al., 2000). Broad bean (Vicia faba L.), a widely cultivated and consumed legume of the Mediterranean region which is a natural source of l-Dopa, prolongs `on' periods in patients with Parkinson's disease who have `on-off' fluctuations (Apaydin et al., 2000). 3.2.2 Vegetables The results based on the first five years of follow-up of the EPIC subjects support the view that fruits and vegetables are strongly associated with reduced risk of cancer of the upper aero-digestive tract, but more weakly associated with a reduction of other cancers (Riboli and Norat, 2003). The current evidence points towards a role of folate in carcinogenesis and neoplasic development that is complex and interacts with genetic background, diet, and types and subtypes of neoplasia and stages of carcinogenesis (Vollset, 2003a). Gebhardt and Fausel (1997), and Gebhardt (1997), have shown that a variety of artichoke (Cynara scolymus L.) specific compounds, like cynarin and luteolin-7-O-glucoside as well as several more abundant polyphenolic compounds such as caffeic acid and chlorogenic acid, may contribute to the antioxidative and hepatoprotective potential of artichoke extracts. Shimoda et al. (2003) reported anti-hyperlipidemic sesquiterpenes from artichoke. Humans consume isothiocyanates (ITC) through eating cruciferous vegetables such as arugula (Miyazawa et al., 2002), watercress, broccoli and cabbage. These compounds exhibit activities against chemical-induced carcinogenesis in various animal models, which coincide with results of epidemiological studies. The extensive evaluation and development of some ITCs as chemopreventive agents in clinical trials has been presented as a practical alternative to dietary sources (Chung, 2002). Chard (Beta vulgaris L. var. cicla) extract may reduce serum urea and creatinine levels and confer a protective effect on the kidney of diabetic rats (Yanardag et al., 2002). Dietary fruits, vegetables, and their products appear to provide some protection against the direct impairment in endothelial function produced by high-fat foods, including olive oil in terms of their postprandial effect (Vogel et al., 2000). Cayenne pepper and hot chili peppers (Capsicum spp.), although American in origin, have become common components of many Mediterranean recipes.

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Capsaicin, the vanilloid pungent compound from Cayenne pepper, is successfully used in peripheral analgesia, as an agent with block transduction processes (Ness, 2001). In persons with osteoarthritis of the hand or knee who have mild to moderate pain, capsaicin is used as topical analgesic in the form of a thin film applied several times daily (Felson et al., 2000). Recently, it has been shown that capsaicin makes tumour cells commit suicide (death of these cells by apoptose) but with seemingly low selectivity in front of the nonmalignat cells (Surh, 2002). Notwithstanding, Capsicum vainilloids are presented in registered supplements for cancer therapy as Capsibiol-TÕ. Lycopene is present in tomatoes (Lycopersicon esculentum Mill.) and some citrus fruits. It is one of the most potent antioxidants among dietary carotenoids. Dietary intake of tomatoes and tomato products containing lycopene have been shown to be associated with a decreased risk of chronic diseases, such as cancer and cardiovascular disease. Mechanisms of action of lycopene are not only restricted to its antioxidant properties (Agarwal and Rao, 2000). Other reported or potential health benefits of lycopene from tomatoes are treatment of male infertility (Gupta, 2003), synergistic reduction of the risk of prostate cancer (Kucuk, 2003) and breast cancer (Levy, 2003), recovery of osteoblasts (Rao, 2003) and prevention of eye diseases (Khachick, 2003). 3.2.3 Cereals The results based on the first five years of follow-up of the EPIC subjects are strongly supportive of a protective effect of fibre-rich foods (cereal, fruit and vegetables) against colorectal cancer (Riboli and Norat, 2003) although doubts are cast on the paper on fibre in colorectal cancer prevention (Schatzkin, 2003). Wheat germ oil is a rich source of , , and -tocopherol. Tocopherols are absorbed in the same path as other nonpolar lipids. Tocopherols act as antioxidants, but other activities are investigated as anti-inflammatory, lowering the risk of cancer or preventing Alzheimer's disease (Papas, 2002). 3.2.4 Fruits and nuts It appears probable that fruit intake may reduce the risk of Benign Prostatic Hyperplasia (BPH) according to the case-control study in Athens. Butter and margarine were shown as probably increasing the risk of BPH (Lagiou et al., 1999). The EPIC study has shown a significant protective effect of increased nut intake on colon cancer in women, with no effects on rectal cancer for either gender (Jenab et al., 2003). Nuts are very high in protein and have an unusually high arginine content. That becomes important in relation to nitric oxide and nitric oxide synthase, which are important not only in cardiovascular disease, but also in the immune system (Davis, 2003). In one study of prelesional markers, ACFs (aberrant crypt foci) of colorectal cancer in rats, the whole almond diet was the only one that significantly reduced the number of ACFs even though it was very high in fat (Davis, 2003).

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Walnuts are different from most nuts in their fatty acid composition. Walnut fat is primarily composed of polyunsaturated fatty acids. They provide appreciable amounts of -linolenic acid (ALA), which has shown in the Nurses Health Study, an inverse relationship with risk of myocardial infarction (SabateÂ, 2003). Nuts are an excellent source of tocopherols. Almonds and hazelnuts are high in -tocopherol while walnuts are high in -tocopherol. The content of tocopherol has been shown to determine the oxidative resistance of LDL particles, which may influence its atherogenic potential (SabateÂ, 2003). 3.2.5 Grapes and wine De Lorgeril and Salen (1999) have shown that the low rate of coronary heart disease (CHD) in France, despite high risk factors the so-called French paradox, may be explained in several ways with respect to the role of platelets and thrombosis in CHD. The populations of the south of France may be protected because they follow a Mediterranean diet, while the rate of CHD may be reduced for those in the north because of the positive effect of wine ethanol in platelet aggregation (inverse relationship). The polyphenols present in grape and wine possess antioxidant activity and may potentially modify certain risk factors associated with atherogenesis and cardiovascular diseases (Dubick, 2002). Grapevine resveratrol has been shown to be a natural antioxidant that is incorporated in the diet through consuming wine, unfiltered juice and whole grape berries and especially products made with pomace pureÂe (Murcia and MartõÂnez, 2001). Grape skin extracts contain antioxidants such as resveratrol, catechin, myricetin, caffeic acid, rutin, etc. (Schwarz et al., 2001) that in part are extracted in wine. But although both red wine and white wine drinking resulted in significant increases of the initial antioxidative capacity of human plasma, this plasmatic increase did not correspond either to the antioxidant capacity of the wines or to the total phenolic content, or to the concentration of selected polyphenols in plasma. The relative capacity of raising the antioxidant capacity of white and red wines depended on the type of test used (Bitsch et al., 2003). 3.2.6 Local Mediterranean foods and their functional products Olive cultivars There is good evidence that olive oil is protective in cardiovascular diseases. Its mechanism of action may involve effects on blood lipids, but other mechanisms, including effects on immune function, endothelial function and the coagulation pathways remain possible. The effects of olive oil in obesity and cancer are less clear. Many questions still remain about the potential health effects of the many non-lipid components of olive oil (Harwood and Yaqoob, 2002). Manna et al. (1997) as a result of the study of induced cytotoxicity in Caco-2 cells, suggested that dietary intake of olive oil polyphenols may lower the risk of reactive oxygen metabolite-mediated diseases such as some gastrointestinal diseases and atherosclerosis.

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The presence of biophenols in table olives, with recognised antioxidant activity, is strictly linked by Bastoni et al. (2001) to the texture and organoleptic characteristics of the food product, giving a functional value to this Mediterranean food. In fact, the cultivars examined with HPLC and NMR techniques were `Hojiblanca' (Spain), `Douro' (Portugal), `Conservolia' and `Thasos' (Greece), `Taggiasca' and `Cassanese' (Italy). They detected contents between 115 and 220 mg of oleuropein, and 143 to 186 mg of cornoside, per 100 g of fresh olives, which is, respectively a 19±28% and 21±26% of total biophenols content. The pattern of glucosidic and free biophenolics fraction is similar among the cultivars. The total BP fraction and the free and alkalihydrolisable BP fraction displayed a higher variability between samples. Gikas et al. (2003) have found significant variation of active substances (oleuropein, tyroxol, hydroxytyroxol) in different Greek olive cultivars. Citrus cultivars The Mediterranean area is a secondary centre of citrus diversity. Citrus fruits were used to cure scurvy as early as the 16th century. Portuguese, Spanish, Arab and Dutch sailors planted citrus trees along trade routes to prevent scurvy. Citrus is a good source of vitamin C. Citrus fruits are alternative sources of folic acid. Frequent consumption of folate-rich foods, such as oranges and orange juice, tends to increase plasma folate levels and, thus, lower homocysteine levels, and reduce the risk of cardiovascular disease (Economos and Clay, 2003). On the contrary, the EPIC-Heart group have shown that no association appears between consumption of citrus fruits and coronary heart disease mortality (Saracci, 2003). Bitter limonoids (limonin, nomilin) have been shown to inhibit tumour formation in animals. Other studies show the compounds may reduce formation of LDL cholesterol in rabbits. Linomoid glucosides are present in citrus fruits at greater concentrations than vitamin C. They are also abundant in citrus juice processing by-products such as seeds, pulp and peel (Stelljes, 2003). Synephrine is a sympathomimetic agent with vasoconstrictor and bronchiectatic activities that is a constituent of peel of citrus fruit, e.g., Bitter or Seville orange (Citrus aurantium L). The structure of synephrine is similar to that of ephedrine (Takei et al., 1999). Bitter orange peel preparations or Seville orange juice have been marketed for weight loss, as a nasal decongestant and to reduce swelling of the eyelids. Consumption of these preparations is not advised to individuals with severe hypertension or those persons taking decongestantcontaining cold preparations (Brooks et al., 2003; Penzak et al., 2001). Other local fruit cultivars The Mediterranean region is a primary centre of diversity for fig trees (Ficus carica L.). Figs provide more fibre than other common fruits. Figs have a high overall content of minerals, specially calcium (Ca). Other active compounds are polyphenols, benzaldehyde, coumarins and potassium (Vinson, 1999).

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3.3 The functional properties of Mediterranean herbs, spices and wild greens 3.3.1 Sage, rosemary and thyme Rosemary (Rosmarinus officinalis L.) extracts, containing carnosol, carnosic acid and rosmarinic acid have shown differences according to the extraction procedure. The correlation coefficient of the total phenol concentration vs. major antioxidant concentration for rosemary extracts was clearly lower than for coffee extracts. By contrast, high correlation was found for galvinoxyl and DPPH (,-diphenyl- -picrylhydrazyl) assays ± efficiencies as scavengers of stable free radicals vs. antioxidant concentration in rosemary extracts. Similar correlation was found with the lipid oxidation assays based on thermal acceleration (formation of conjugated dienes in methyl linol and Rancimat test with lard). Rosemary extracts were among the most potent extracts for protection of processed foods against lipid peroxidation (Schwarz et al., 2001). ProvencËal herb mixtures containing sage (Salvia officinalis L.), rosemary (Rosmarinus officinalis L.), thyme (Thymus vulgaris L.) and origano (Origanum vulgare L.) contained as principal antioxidants carvacrol, thymol, carnosol and carnosic acid (Aruoma et al., 1996; Schwarz et al., 2001). MartõÂnez et al. (2001) have shown that Mediterranean spices like oregano, rosemary and saffron exhibit good antioxidant activity as scavengers of several reactive oxygen species. 3.3.2 Garlic Although originating in Central Asia, garlic (Allium sativum L.) is a typical Mediterranean condiment. Garlic has been used as a food and herbal medicine for thousands of years. Recent studies have demonstrated that garlic contains several medically active substances that possess many favourable effects, such as a decrease in low-density lipoprotein (LDL), antioxidant, anti-thrombosis and suppression of platelet aggregation. Oxidative modification of DNA is related to ageing and diseases such as cardiovascular diseases, neurodegenerative diseases and even cancer. Garlic extract exhibits antioxidant action by increasing the levels of cellular antioxidant enzymes, such as superoxide dismutase, catalase and glutathione peroxidase, and scavenging reactive oxygen species (Borek, 2001; Qi and Wang, 2003). Recent studies have demonstrated that garlic extract can inhibit LDL oxidation in vitro, reduce plasma LDL in vivo and retard atherogenesis in rabbits. It is therefore reasonable to suggest that garlic extract might be useful in the prevention of the progression of atherosclerosis in humans (Qi and Wang, 2003). Ajoene, a natural compound present in garlic cloves, has been shown to induce apoptosis in leukemic cells in addition to other blood cells of leukemic patients. Ajoene induces apoptosis in human leukemic cells via stimulation of peroxide production and activation of nuclear factor B (Dirsch, 1998). Allicin, another major compound of garlic extract, inhibited proliferation of human

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mammary, endometrial and colon cancer cells. The inhibition of growth was accompanied by an accumulation of the cell in different phases of the cell cycle, but not by a significant increase in cell death. This result implies that allicin is responsible for the antiproliferative effect of garlic extract (Hirsch, 2000). Taken together, the beneficial effects of garlic extract make it useful in health care but the exact biological mechanism of garlic extract still remains unclear (Qi and Wang, 2003). 3.3.3 Wild greens and fruits Wild edible greens are frequently consumed throughout the Mediterranean countries. Wild greens and vegetables together with snails have been reported as relevant sources of -linolenic acid (ALA) of the traditional Cretan diet (Lanzmann and Renaud, 2003). Some wild green extracts such as those of Urtica dioica L. or Arctium minus L. have shown in vitro immunomodulatory activities (Basaran et al, 1997). Hypotensive flavonoids have been isolated from Chenopodium spp. leaves (Gohar and Elmazar, 1997) that are occasionally consumed as food. Trichopoulou et al. (2000) analysed seven wild greens and traditional Cretan green pies for their nutritional composition and flavonoid content, in particular flavonols and flavones. A high nutritional value and a low energy value characterised the wild greens. These wild greens and herbs (Foeniculum vulgare Mill., Allium schoenoprassum L., Sonchus oleraceus L., Papaver rhoeas L., Rumex obtusifolius L., Tordilium apulum L. and wild Daucus carota L.) have a very high flavonol content when compared with regular fresh vegetables, fruits and beverages commonly consumed in Europe. Accordingly wild greens are potentially a very rich source of antioxidant flavonols and flavones in the Greek diet. Oh et al. (2000) have shown the antifungal activity of Portulaca oleracea, a wild green consumed in salads. Capers are the flower-buds of Capparis spp. They are brined and widely consumed in salads and traditional Mediterranean dishes, tender shoots and fruits are also locally brined and have been consumed since times of antiquity (Rivera et al., 2002). Inocencio et al. (2000) have shown that capers have a high rutin content. Capers are employed in Ayurvedic-based supplement formulations used in the treatment of various liver disorders and lipid management such as LiverCareÕ (Liv.52Õ).

3.4

Diet and age-related diseases

Target activities are related with the widespread pathologies associated with ageing, as preventive, curative or palliative. Some noteworthy problems are cardiovascular diseases, cancer, Alzheimer's, Parkinson's, osteoporosis, rheumatoid arthritis, behavioural diseases and immunodeficiencies. Regular consumption of fruit and vegetables is associated with reduced risks of cancer,

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cardiovascular disease, stroke, Alzheimer's disease, cataracts and some of the functional declines associated with ageing. Alzheimer's disease is characterised by the presence of intracellular and extracellular plaques composed of a protein fragment called beta amyloid. The exact role of these plaques in the disease process is not yet known but it is possible that they play an active role in neuron degeneration, in the loss of cellular connections with other neurons and provoke neuronal death. Recent findings suggest a possible role of diet in age-related cognitive decline, and cognitive impairment of both degenerative (Alzheimer's disease), or vascular origin. In particular, in an older population of Southern Italy with a typical Mediterranean diet, high monounsaturated fatty acid (MUFA) from olive oil appeared to be associated with a high protection against cognitive decline. Whether this protective effect of olive oil is attributable exclusively to the high MUFA intake or to the presence of antioxidant compounds, or both, remains to be elucidated. A style of diet based on complex carbohydrates, fibres, cereals, red wine, fresh fruit and vegetables, and non-animal fat appears to protect against age-related cognitive decline and cognitive decline of vascular or degenerative origin (Solfrizzi et al., 2003). Elevated homocysteine levels are associated with cognitive dysfunction in the elderly. Older subjects with greater intakes of fruits and vegetables, and the corresponding nutrients vitamin C and folate, have been shown to perform better on cognitive tests (Economos and Clay, 2003; Ortega et al., 1997). Oxidation of the eye's lens plays a central role in the formation of age-related cataracts. Lower cataract risk has been shown in individuals with high blood concentrations or intakes of vitamin C and carotenoids (Economos and Clay, 2003; Jacques et al., 2003). The unbalance in the dietary supply of sugars, proteins, and lipids may initiate major health problems including obesity, coronary heart disease, cancer, diabetes mellitus, high blood pressure, stroke, gout, and gall bladder disease. In old people a lack of vitamins causes vitamin deficiency. Osteoporosis is preventable. A diet rich in calcium and vitamin D and a lifestyle that includes regular weight-bearing exercise are the best ways to prevent osteoporosis. Osteoporosis can be treated by hormone replacement therapy (HRT). This treatment should be administered carefully because all side effects and risk of long-term use are not perfectly understood. Long-term intake of various foods may be important to bone health. Vitamin C intake has been associated with bone mineral density, but more work in this area is necessary to understand the mechanism of interaction (Economos and Clay, 2003, New et al., 1997). Failure to neutralise fixed acidity leads to low-grade metabolic acidosis, with long-term deleterious effects on bone Ca status, since Ca is mobilised to neutralise excess acidity. Providing a sufficient supply of K organic anions through potatoes, fruit and vegetable intake should be recommended (Remesy and Demigne, 2003). Cancer predisposition is genetic but could also be influenced by exposure to thousands of carcinogenic factors. The incidence of lung cancer, prostate and

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breast cancer increases with age. Case-control studies suggest that a diet rich in vegetables and fruit provides protection against epithelial cancers. The evidence is less convincing and consistent for vegetables than for fruit, and in regard to epithelial cancers, mainly those of the upper respiratory and digestive tracts. The possible mechanism of action of micronutrients and other constituents of vegetables and fruit, including antioxidants, and their interactions with risk factors and other aspects of diet require further elucidation (La Vecchia et al., 2001). Special attention must be paid to the presence of genotoxic substances or contaminants in foodstuffs (Sugimura, 2002). Simopoulos (2001) suggested that there is enough evidence that should serve as a strong incentive for the initiation of intervention trials that will test the effect of specific dietary patterns in the prevention and management of patients with cancer. Normally functioning older adults are at no greater risk of depression than younger adults. However, personal dependence, medical histories, disabilities and ill-health may increase the possibility of developing depression or committing suicide. Panagiotakos et al. (2002) have shown the association of Mediterranean diet with a lower risk of acute coronary syndromes in hypertensive subjects. An experimental study of a Mediterranean diet intervention for patients with rheumatoid arthritis (RA) in Sweden has shown that patients with RA, by adjusting to a Mediterranean diet, obtained a reduction in inflammatory activity, an increase in physical function, and improved vitality (Hagfors, 2003; Skoldstam et al., 2003).

3.5

Methods of identifying and analysing plant extracts

3.5.1 Different extraction approaches: lipophilic vs. hydrophilic Modern methods of bioseparation utilise principles of extraction that are based on polarity (relative solubility in organic solvents), solubility in water and various alterational solubilities based on salts and pH (Kaufman et al., 1999). Stobiecki et al. (1999) have shown the applicability of HPLC combined with electrospray ionisation MS (LC-ESI-MS) for the simultaneous analysis of secondary metabolites of different polarities (free aglycones and their glycosides). 3.5.2 Detection of functional compounds Traditional analytical methods like thin-layer chromatography (TLC) and reverse-phase high-performance liquid chromatography (RP-HPLC) present constraints concerning either poor reproductibility, resolution, or cost. Sevcikova et al. (2002) reported the successful use of Micellar Electrokinetic Capillary Chromatography for the analysis of artichoke extract. The ability to reliably detect and quantify every metabolite in a plant extract is unlikely to be attained by any single analytical method available at present. Most technology

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for metabolomics is based on mass spectrometry (MS). Gas chromatography ± MS and HPLC-photodiode array ± MS remain the method of choice for quantitative and qualitative metabolite profiling (Hall et al., 2002). The use of rapid scanning time-of-flight (TOF) coupled with gas chromatography separation and integrated with peak deconvolution software increased the number of metabolites detectable by gas chromatography-MS in crude plant extracts from 500 to 1000. However, the dynamic range of TOF detectors is still restrictive when faced with mixtures containing compounds with concentration differences of several orders of magnitude (Hall et al., 2002). The mobile Fourier transform spectrometer, equipped with a diamond ATR with an integrated video microscope, combined with a specially designed sample press, allows the performance of analyses with very small amounts of sample. The IR spectra determined in the wavelength range of 650 to 4000 cmÿ1 provide a high level of specific information of the ingredients with a maximum spectral resolution of 2 cmÿ1, which can at least partly be interpreted qualitatively without the use of chemometric methods. For further analysis it is necessary to use a multivariate statistical spectral program. The ATR-IR technique provides a high level of information on focal points of concentration of selected components. This technique is promising for quality control because it is quick and economical, and can be used for determining optimal harvesting time for increasing the yield of desirable compounds (Schulz and Quilitzsch, 2003). NMR analysis of crude extracts and direct examination of crude extracts by mass spectrometry are two recent technological approaches not involving chromatography of metabolites (Hall et al., 2002). Proton NMR of crude plant extracts, followed by multivariate analysis, is used to cluster data sets to highlight differences. It gives a comprehensive summation fingerprint of all (hydrogen-containing) metabolites extracted and it is suitable for highthroughput, rapid, first-pass screening. Subtraction of data sets generates virtual NMR spectra and important structural data on compounds contributing to differences between samples. Post-sample collection techniques such as noise reduction, deconvolution, profile alignment, reference to internal standards, and peak labelling using spectral libraries are needed (Hall et al., 2002). 3.5.3 Metabolomics and Mediterranean plant extracts Metabolomics is the term coined for essentially comprehensive, nonbiased, high-throughput analyses of complex metabolite mixtures typical of plant extracts. It is part of the `postgenomic' movement toward the functional characterisation of sequenced genomes and comprehensive investigations of biological systems in response to external stimuli (Hall et al., 2002). The need is evident for whole-process data capture integrated with a laboratory information management system and followed through well-structured archives and databases. The volume of data generated by one research group working at industrial-scale is in the order of 10 gigabytes per day and the volume of data

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produced will continue to increase (Hall et al., 2002). Metabolic profiling aims at a quantitative assessment of a predefined number of target plant metabolites. Often, such profiles are restricted to certain pathways or compound classes. Metabolomic analysis attempts to avoid bias against certain compound classes and to allow for the analysis of every metabolite individually. This aim has not yet been reached. The metabolomic approach is opening new ways for understanding the wide range of plant metabolites consumed in a Mediterranean type diet. Also it can help to improve breeding programmes in order to optimise the phytochemical profile of active substances present in food-plant cultivars.

3.6 Developing supplements for healthy ageing and other future trends 3.6.1 Simplifying dosage and processes There is a strong tendency to reduce the time spent in processing food at home, especially in Western countries. This has caused commercially viable initiatives that tend to replace traditional food by nutritional complements that are supposed to be at least as effective as the former. For instance OmegacoeurÕ, a mixture of different natural oils enriched with ! 3, ! 6, and ! 9 fatty acids, is sold as a `Mediterranean nutritional complement' (Maixent et al., 2003) and presumably as an alternative to olive oil. The development of food products with `functional' properties or health benefits can be done in the form of dietary supplements or simply foods. Particular ingredients provide the health benefits. The food or ingredient conferring health properties may consist of the plants themselves, extracts thereof, or more purified components (Schilter et al., 2003). Since the linolenic acid (ALA) supply is found to be too low in a French population study, Combe et al. (2003) recommended food enrichment to increase the ALA intake. Vasiliopoulou and Trichopoulou (2003) suggested an alternative approach: registering and standardising traditional foods could provide an incentive for their reinstatement into the daily diet. The `organic' approach is more time consuming but it offers interesting alternatives. The use of sprouts grown from various seeds including legumes, broccoli and sunflower seeds is presented by Goodwin (2003) as a positive contribution to health, by suggesting they are produced at home. Humans can alter plant metabolism to favour the synthesis of a particular metabolite of medicinal value (Cseke and Kaufman, 1999). For instance, there is an interest in genetically engineering crops for herbicide resistance, by transferring genes for this purpose but similar techniques can be applied to metabolic engineering of secondary pathways for valuable phytochemicals in food plants (Bais and Ravishankar, 2001). For instance, isoflavonoid biosynthesis in nonlegumes is intended to expand the delivery of dietary isoflavones and to develop new sources for the more complex bioactive isoflavonoids (Dixon and Summer, 2003).

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3.6.2 Future trends Synergy research and synergic foods In synergy the whole is better than the sum of its parts. Duke and Bogenschutz (1999) warmly reviewed several examples of synergy in Citrus fruit and cancer prevention, garlic health benefits, celery and arthritis, and sedative Melissa, among others. In the SerranõÂa de Cuenca (Fajardo et al., 2004) Berberis leaves and fruits are consumed as food. Recently, Stermitz et al. (2000 a and b) have shown that the antimicrobial action of berberine alkaloids is potentiated by 50 methoxyhydnocarpin, a multidrug pump inhibitor (MDR inhibitor). Both substances are found in Berberis species used in native American medicine but berberine is also present in European Berberis species. The presence of small amounts of berberine alkaloids together with MDR inhibitors in the consumed Berberis fruits and leaves (berberine is mainly accumulated in roots) could confer some advantage to the occasional consumer against microbial infections. Tegos et al. (2002) generalised the finding suggesting that plant antimicrobials might be developed into effective, broad-spectrum antibiotics in combination with inhibitors of MDRs. The failure of single-nutrient supplementation to prevent disease in intervention studies underlines the necessity of alternative approaches. Gerber et al. (2000) developed a holistic view model of food intake with a diet quality index and identified biomarkers of multidimensional dietary behaviour. In this study, in Mediterranean southern France, subjects with beta carotene levels greater than 1 micromol/l, vitamin E greater than 30 micromol/l and eicosapentanoic acid (EPA) greater than 0.65 % and docosahexanoic acid (DHA) greater than 4% of fatty acids in erythrocytes were likely to have a healthy diet (Gerber et al., 2000). Ruidavets et al. (2000) have shown that the highest plasma concentration of (+)-catechin was observed in subjects consuming fruit, vegetable and wine, and its antioxidant and antiaggregant activity could partly explain the relative protection against coronary heart disease (CHD). Liu (2003) has proposed that the additive and synergistic effects of phytochemicals in fruit and vegetables are responsible for their potent antioxidant and anticancer activities, and that the benefit of a diet rich in fruit and vegetables is attributed to the complex mixture of phytochemicals present in whole foods. Therefore antioxidants are best acquired through whole-food consumption, not as a pill or an extract. Ethnomics or ethnobotanomics: from traditional food knowledge to genomics It has been found that most major diseases (cardiovascular disease, diabetes, obesity, cancers, etc.) result from the interaction between genetic traits (susceptibility) and environmental factors, especially diet. The study of interactions between particular single nucleotid polymorphisms and metabolic responses to diets could help to improve dietary recommendations by taking into account known genetic variability within a given population (Lairon et al., 2003).

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Functional foods, ageing and degenerative disease

Sources of further information and advice

Comparative data between countries on the availability of foods in nationally representative samples of European households can be retrieved from the DAFNE databank (Naska, 2003). Ethnobotanical studies in different countries bordering the Mediterranean have shown that many minor items of plant origin (local cultivars, wild greens and fruits) and mushrooms are also a component of the present-day diets in rural areas of the Mediterranean and were much more relevant in past times (Arcidiacono et al., 2003; Ertug, 2000; Pieroni, 2001; Pieroni et al., 2002; Rivera and OboÂn, 1991). This opens new areas for research of functional compounds heretofore overlooked. Trichopoulou et al. (2000) show an example of the possibilities still open. The project `Local Mediterranean food plants: potential new nutraceuticals and their current role in the Mediterranean diet' led by Prof. M. Heinrich of The School of Pharmacy (University of London) and financed by the European Commission, is developed by a consortium of seven European universities and research centres. The consortium studies local knowledge on traditional foods ethnobotanically. Nearly 150 taxa of local Mediterranean food plants are under evaluation in a variety of primary in vitro assays. Active samples are studied in mechanistic in vitro/in vivo models focusing on the CNS and the cardiovascular system. Results are expected to bridge, in part, the gaps in our present knowledge concerning minor but relevant items of the Mediterranean diet, to promote the use of these almost forgotten local foods and, last but not least, lead to the discovery of new promising functional compounds. Because the wide use of flavonoids as dietary supplements for the prevention and cure of diseases (Havsteen, 2002; Middleton et al., 2000), for instance, the high doses of quercetin recommended for the treatment of allergies, Skibola and Smith (2000) reviewed the risk of excessive flavonoid intake. This was especially critical for the unborn foetus, since flavonoid readily crosses the placenta. Safety in botanical preparations for use in food and food supplements is crucial. Health hazards may arise from inherent toxicity or contaminants of the plant material. Risk assessment should address all the available hazard characterisation data in relation to the potential or predicted human intake, both the daily intake and the duration of intake (Schilter et al., 2003).

3.8

Acknowledgement

The authors wish to thank Mr R. Llorach for skilful assistance.

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73

References

(2000), Tomato lycopene and its role in human health and chronic diseases. Canadian Medical Association J, 163(6), 739±44. ANDLAUER W et al. (2000), Absorption and Metabolism of Genistein in Isolated Rat Small Intestine. Journal of Nutrition, 130, 843±6. È ZEKMEKCËI S (2000), Broad Bean (Vicia faba L.) ± A Natural Source APAYDIN H, ERTAN S, O of L-Dopa, Prolongs `On' Periods in Patients With Parkinson's Disease Who Have `On-Off' Fluctuations. Movement Disorders, 15(1), 164±6. ARCIDIACONO S, PAVONE P, SALMERI C (2003), Le Erbe commestibili dell Etna. http:// www.unict.it/dipartimenti/biologia_animale/webnatur/pavone... ARUOMA O I et al. (1996), An Evaluation of the Antioxidant and Antiviral Action of Extracts of Rosemary and ProvencËal Herbs. Food and Chemical Toxicology, 34, 449±56. BAIS H, RAVISHANKAR G (2001), Cichorium intybus L. ± cultivation, processing, utility, value addition and biotechnology, with an emphasis on current status and future prospects. Journal of the Science of Food and Agriculture, 81, 467±84. BASARAN A et al. (1997), Immunomodulatory Activities of Some Turkish Medicinal Plants. Phytotherapy Research, 11, 609±11. BASTONI L et al. (2001), Biophenolic profile in olives by nuclear magnetic resonance. Food Chemistry, 73, 145±51. BITSCH R et al. (2003), Effects of German red or white wine drinking on the antioxidative capacity of human plasma, in Barnat S, International Conference on Health benefits of Mediterranean Diet Abstract Book, Aghia Pelagia, Egea, 93. BLACKBURN H (2003), Ancel Keys. http://mbbnet.umn.edu/first/blackburn_h.html BOREK C (2001), Antioxidant health effects of aged garlic extract. J. Nutr., 131, 1010±15. BROOKS J, NAGELIN E, ZUCKERMAN D (2003), Examining the Safety of Natural Supplements, CPR for Women and Families Issue Brief, May 2003, 1±3. BUIJSSE B et al. (2003), Indicators of oxidative stress in elderly men aged 80 and over: differences between Crete and Zutphen (NL), in Barnat S, International Conference on Health benefits of Mediterranean Diet Abstract Book, Aghia Pelagia, Egea, 94. CARMONA M D et al. (submitted), The `zahraa' a Unani multicomponent herbal tea widely consumed in Syria: study of the crude drug mixtures and alleged medicinal properties. Journal of Ethnopharmacology. CHARRONDIERE U R (2002), The European Nutrient Database (ENDB) for Nutritional Epidemiology. Journal of Food Composition and Analysis, 15, 435±51. CHUNG F L (2002), Dietary isothiocyanates: roles in cancer prevention and metabolism in rodents and humans, in Meskin M et al., Phytochemicals in Nutrition and Health, Boca Raton, CRC Press, 87±104. COMBE N et al. (2003), Intake of Linoleic and alpha-linoleic acids, and dietary origins in a French population, reactions with plasma and adipose contents, in Barnat S, International Conference on Health benefits of Mediterranean Diet Abstract Book, Aghia Pelagia, Egea, 81. CROWFOOT G, BALDENSPERGER L (1932), From Cedar to Hyssop. A Study in the Folklore of Plants in Palestine. London, The Sheldon Press. CSEKE L, KAUFMAN P (1999), Regulation of Metabolite Synthesis in Plants, in Kaufman et al., Natural Products from Plants, Boca Raton, CRC Press, 91±121. DAVIS P (2003), The Role of Nuts in Cancer, in Sansalone W, Storey M, Forshee R, Making AGARWAL S, RAO A V

74

Functional foods, ageing and degenerative disease

the Claim for Nuts. Alexandria, Center for Food and Nutrition Policy, 11±16. (1999), Wine ethanol, platelets, and Mediterranean diet. The Lancet, 353, 1067. DE LORGERIL M et al. (2002), Mediterranean diet and the French paradox: Two distinct biogeographic concepts for one consolidated scientific theory on the role of nutrition in coronary heart disease. Cardiovascular Research, 54, 503±15. DIRSCH V M (1998), Ajoene, a compound of garlic, induces apopotosis in human promyeloleukemic cells, accompanied by generation of reactive oxygen species and activation of nuclear factor B. Mol. Pharmacol., 53, 402±7. DIXON R A, SUMMER L (2003), Legume Natural Products: Understanding and Manipulating Complex Pathways for Human and Animal Health. Plant Physiology, 131, 878±85. DUBICK M (2002), Wine Polyphenols and Protection from Atherosclerosis and Ischemic Heart Disease: Drink To Your Health? in Meskin M et al., Phytochemicals in Nutrition and Health, Boca Raton, CRC Press, 41±60. DUKE J A, BOGENSCHUTZ M (1999), The Synergy Principle at Work in Plants, Pathogens, Insects, Herbivores, and Humans, in Kaufman P et al., Natural Products from Plants, Boca Raton, CRC Press, 183±205. DURANGO D et al. (2002), Phytoalexin Accumulation in Colombian Bean Varieties and Aminosugars as Elicitors. Molecules, 7, 817±32. ECONOMOS C, CLAY W D (2003), Nutritional and health benefits of citrus fruits. http:// www.fao.org/docrep/x2650T/x2650t03.htm ERTUG F (2000), An Ethnobotanical Study in Central Anatolia (Turkey), Economic Botany, 54(2), 155±82. Â nica de la SerranõÂa de Cuenca, Cuenca, DiputacioÂn FAJARDO J et al. (2004). Etnobota Provincial. FELSON D et al. (2000), Osteoarthritis: New Insights, Annals of Internal Medicine, 133(9), 726±37. FISCHLER C (1996), PenseÂe magique et utopie dans la science. Les Cahiers de l'OCHA, 5, 111±27. GEBHARDT R (1997), Antioxidative and Protective properties of Extracts from Leaves of the Artichoke (Cynara scolymus L.) against Hydroperoxide-Induced Oxidative Stress in Cultured Rat Hepatocytes. Toxicology and Applied Pharmacology, 144, 279±86. GEBHARDT R, FAUSEL M (1997), Antioxidant and Hepatoprotective Effects of Artichoke Extracts and Constituents in Cultured Rat Hepatocytes. Toxicology in Vitro, 11, 669±72. GERBER M, LAIRON D (2003), The Med-Rivage Intervention Study on Mediterranean diet and risks for coronary heart disease, in Barnat S, International Conference on Health benefits of Mediterranean Diet Abstract Book, Aghia Pelagia, Egea, 59±60. GERBER M et al. (2000), Profiles of a healthful diet and its relationship to biomarkers in a population sample from Mediterranean southern France. Am Diet Assoc, 100, 1164±71. GIKAS E et al. (2003), Quantitation of olive oil bioactive components in Greek cultivars, in Barnat S, International Conference on Health benefits of Mediterranean Diet Abstract Book, Aghia Pelagia, Egea, 100. GOHAR A, ELMAZAR M (1997), Isolation of Hypotensive Flavonoids from Chenopodium Species Grown in Egypt. Phytotherapy Research, 11, 564±7. GOODWIN E L (2003), The nutritional value of sprouted seeds in the diet, in Barnat S, DE LORGERIL M, SALEN P

New functional foods for age-related diseases

75

International Conference on Health benefits of Mediterranean Diet Abstract Book, Aghia Pelagia, Egea, 83. GUPTA N (2003), Emerging Role of Lycopene in Male Infertility, in Storey M, Examining the Health Benefits of Lycopene from Tomatoes, Washington, CFNP, 5. HAGFORS L (2003), A Mediterranean dietary intervention study of patients with rheumatoid arthritis. Umea, Umea University. HALL R et al. (2002), Plant Metabolomics: The Missing Link in Functional Genomic Strategies. The Plant Cell, 1437±40. HARWOOD J, YAQOOB P (2002), Nutritional and health aspects of olive oil. Eur J Lipid Sci Technol, 104, 685±97. HAVSTEEN B (2002), The biochemistry and medical significance of the flavonoids. Pharmacology and Therapeutics, 96, 67±202. HIRSCH K (2000), Effect of purified allicin, the major ingredient of freshly crushed garlic, on cancer cell proliferation. Nutr. Cancer, 38, 254. HOFFMAN W (2003), Meet Monsieur Cholesterol. http://mbnet.umn.edu/hoff/hoff_ak.html  n espanÄola de un manuscrito anoÂnimo del Siglo XIII sobre la HUICI A (1966), Traduccio cocina Hispano-MagribõÂ. Madrid, Ayuntamiento de Valencia. INOCENCIO C et al. (2000), Flavonoid content of commercial capers (C. spinosa, C. sicula and C. orientalis) produced in Mediterranean countries. Eur Food Res Technol, 212, 70±4. ITSIOPOULOS C et al. (2003), The impact of the Traditional Cretan Mediterranean diet on the management of type 2 diabetes mellitus, in Barnat S, International Conference on Health benefits of Mediterranean Diet Abstract Book, Aghia Pelagia, Egea, 82. JACQUES P et al. (1997). Long-term vitamin C supplement use and prevalence of early age-related lens opacities. American Journal of Clinical Nutrition, 66, 911±16. JANSEN E et al. (2003), Folic acid, Vitamin B12 and Homocysteine status in elderly men aged 80 and over: differences between Crete and Zutphen (NL), in Barnat S, International Conference on Health benefits of Mediterranean Diet Abstract Book, Aghia Pelagia, Egea, 83. JENAB M et al. (2003), Nut intake in the EPIC study and colorectal cancer risk: gender and site protective effects, in Barnat S, International Conference on Health benefits of Mediterranean Diet Abstract Book, Aghia Pelagia, Egea, 83. KAFATOS A et al. (2000), Mediterranean diet of Crete: foods and nutrient content. Journal of the American Dietetic Association, 100, 1487±93. KAUFMAN P B, CSEKE L, OKUBO A (1999), Bioseparation of Compounds, in Kaufman et al., Natural Products from Plants, Boca Raton, CRC Press, 207±40. KEYS A (1980), Seven countries: a multivariate analysis of death and coronary heart disease. Cambridge, Harvard University Press. KEYS A, KEYS M (1959), How to eat well and stay well: the Mediterranean way, Doubledays, New York. KEYS A et al. (1963), Coronary heart disease among Minnesota business and professional men followed 15 years. Circulation, 28, 381±95. KHACHICK F (2003), Distribution, Metabolism, and the Role of Carotenoids in Disease Prevention, in Storey M, Examining the Health Benefits of Lycopene from Tomatoes, Washington, CFNP, 9±10. KIM H et al. (2001), Actions of the soy phytoestrogen genistein in models of human chronic disease: potential involvement of transforming growth factor . Biochemical Society Transactions, 29(2), 216±22. KROMHOUT D (2003), The Mediterranean diet and coronary heart disease, in Barnat S,

76

Functional foods, ageing and degenerative disease

International Conference on Health benefits of Mediterranean Diet Abstract Book, Aghia Pelagia, Egea, 39. KUCUK O (2003), Evidence for Reducing the Risk of Prostate Cancer ± A Clinical Trial, in Storey M, Examining the Health Benefits of Lycopene from Tomatoes, Washington, CFNP, 7. LA VECCHIA C, ALTIERI A, TAVANI A (2001). Vegetables, fruit, antioxidants and cancer: a review of Italian studies. Eur J Nutr, 40, 261±7. LAGIOU P et al. (1999), Diet and Benign Prostatic Hyperplasia: A study in Greece. Adult Urology, 54 (2), 284±90. LAIRON D, VINCENT S, PLANNELS R (2003), Diet, lipid metabolism and genes: interactions in the aetiology of cardiovascular disease, in Barnat S, International Conference on Health benefits of Mediterranean Diet Abstract Book, Aghia Pelagia, Egea, 69. LANZMANN D, RENAUD S (2003), Ventricular, atrial fibrillation and Cretan diet, in Barnat S, International Conference on Health benefits of Mediterranean Diet Abstract Book, Aghia Pelagia, Egea, 98. LEVY J (2003), The Role of Lycopene and Other Tomato Carotenoids in Breast and Endometrial Cancers, in Storey M, Examining the Health Benefits of Lycopene from Tomatoes, Washington, CFNP, 8. LIU, R H (2003), Health benefits of fruti and vegetables are from additive and synergistic combinations of phytochemicals. Am J Clin Nutr, 78 (suppl.), 517±20. LONGRIGG J (1998), Greek Medicine from the Heroic to the Hellenistic Age: a Source Book, London, Duckworth. LONIE I M (1977), A structural pattern in Greek dietetics and the early history of Greek medicine. Medical History, 21, 235±60. MAIXENT J M et al. (2003), Omegacoeur, a Mediterranean nutritional complement, inhibits monocyte adhesion to human endothelial cells, in Barnat S, International Conference on Health benefits of Mediterranean Diet Abstract Book, Aghia Pelagia, Egea, 98. MANNA C et al. (1997). The Protective Effect of the Olive Oil Polyphenol (3,4Dihydroxyphenil)-ethanol Counteracts Reactive Oxygen Metabolite-Induced Cytotoxicity in Caco-2 Cells. J Nutr, 127, 286±92. MARTIÂNEZ M et al. (2001), Antioxidant Properties of Mediterranean Spices Compared with Common Food Additives, Journal of Food Protection, 64(9), 1412±19. MASKARINEC G et al. (2002), A Randomized Isoflavone Intervention among Premenopausal Women. Cancer Epidemiology, Biomarkers & Prevention, 11, 195±201. MAZZA G. (1998), Functional Foods, Biochemical and Processing Aspects. Lancaster, Technomic Publishing Company, Inc. MESSINA M J (1999), Legumes and soybeans: overview of their nutritional profiles and health effects. American Journal of Clinical Nutrition, 70 (Suppl.), 439±50. MIDDLETON E, KANDASWAMI C, THEOHARIDES T (2000), The Effects of Plant Flavonoids on Mammalian Cells: Implications for Inflammation, Heart Disease, and Cancer. Pharmacol Rev, 52, 673±751. MIYAZAWA M, MAEHARA T, KUROSE K (2002), Composition of the essential oil from the leaves of Eruca sativa. Flavour and Fragrance Journal, 17, 187±90. MURCIA M A, MARTIÂNEZ M (2001), Antioxidant Activity of Resveratrol Compared with Common Food Additives, Journal of Food Protection, 64(3), 379±84. NASKA A (2003), Mediterranean diet at present, in Barnat S, International Conference on Health benefits of Mediterranean Diet Abstract Book, Aghia Pelagia, Egea, 21. NESS T J (2001), Pharmacology of Peripheral Analgesia. Pain Practice, 1(3), 243.

New functional foods for age-related diseases

77

NEW S et al. (2003), Nutritional influences on bone mineral density; a cross-sectional study

in premenopausal women. American Journal of Clinical Nutrition, 65, 1831±9. (2001). There are many Mediterranean diets. Asia Pacific J Clin Nutr, 10(1), 2±9. OH K B et al. (2000), Detection of Antifungal Activity in Portulaca oleracea by a Singlecell Bioassay System. Phytotherapy Research, 14, 329±32. OIKONOMOU E et al. (2003), Monitoring Food habits in the Mediterranean region through the Dafne Databank, in Barnat S, International Conference on Health benefits of Mediterranean Diet Abstract Book, Aghia Pelagia, Egea, 86. ORTEGA R et al. (1997), Dietary intake and cognitive function in a group of elderly people. American Journal of Clinical Nutrition, 66, 803±9. PANAGIOTAKOS et al. (2002) The association of Mediterranean diet with lower risk of acute coronary syndromes in hypertensive subjects. International Journal of Cardiology, 82, 141±7. PAPAS A M (2002), Beyond -Tocopherol: The Role of the Other Tocopherols and Tocotrienols, in Meskin M et al., Phytochemicals in Nutrition and Health, Boca Raton, CRC Press, 61±77. PENZAK S R et al. (2001), Seville (sour) orange juice: synephrine content and cardiovascular effects in normotensive adults. Journal of Clinical Pharmacology, 41, 1059±63. PIERONI A (2001), Evaluation of the cultural significance of wild food botanicals traditionally consumed in northwestern Tuscany, Italy. Journal of Ethnobiology, 21(1), 89±104. PIERONI A et al. (2002), Ethnopharmacology of liakra: traditional weedy vegetables of the ArbeÈresheÈ of the Vutlure area in southern Italy. Journal of Ethnopharmacology, 81, 165±85. QI R, WANG Z (2003), Pharmacological effects of garlic extract. Trends in Pharmacological Sciences, 24(2), 62±3. RAO L (2003), Lycopene, Tomatoes and Bone Health, in Storey M, Examining the Health Benefits of Lycopene from Tomatoes, Washington, CFNP, 8±9. REMESY C, DEMIGNE C (2003), Alkalinizing effects of fruits, vegetables and potatoes, in Barnat S, International Conference on Health benefits of Mediterranean Diet Abstract Book, Aghia Pelagia, Egea, 63. RENAUD S (2003), Cretan diet to prevent clinical manifestation of coronary heart disease, in Barnat S, International Conference on Health benefits of Mediterranean Diet Abstract Book, Aghia Pelagia, Egea, 41. RIBOLI E (1989), `Methodological issues in the investigation of diet and cancer in humans, in Miller A, Aetiology of Cancer, Heidelberg, Springer, 55±63. RIBOLI E, NORAT T (2003), Consumption of fruits and vegetables and prevention of cancers of the digestive tract: results from EPIC and other studies, in Barnat S, International Conference on Health benefits of Mediterranean Diet Abstract Book, Aghia Pelagia, Egea, 27.  tiles de la PenõÂnsula IbeÂrica  N C (1991), La GuõÂa de Incafo de las Plantas U RIVERA D, OBO y Baleares, Madrid, Incafo.  N, C (1996), Plant food as medicine in Mediterranean Spain, in Schro RIVERA D, OBO È der, E et al. Medicaments et aliments approche ethnopharmacologique. Paris, ORSTOM, 121±8. RIVERA D et al. (2002), Archaeobotany of capers (Capparis) (Capparaceae). Vegetation History and Archaeobotany, 11, 295±313. NOAH A, TRUSWELL A

78

Functional foods, ageing and degenerative disease

et al. (2000), Catechin in the Mediterranean diet: vegetable, fruit or wine? Atherosclerosis, 153, 107±17. SABATEÂ J (2003), The Role of Nuts in Heart Helath, in Sansalone W, Storey M, Forshee R, Making the Claim for Nuts. Alexandria, Center for Food and Nutrition Policy, 19± 24. Â NCHEZ A et al. (2002), Determinants of the adherence to an a priori defined SA Mediterranean dietary pattern. Eur J Nutr, 41, 249±57. SANTELL R, KIEU N, HLEFERICH W (2000), Genistein Inhibits Growth of EstrogenIndependent Human Breast Cancer Cells in Culture but not in Athymic Mice. Journal of Nutrition, 130, 1665±9. SARACCI R et al. (2003), Fruits and Vegetables and Fatal Myocardial Infarction, in Barnat S, International Conference on Health benefits of Mediterranean Diet Abstract Book, Aghia Pelagia, Egea, 49. SARRI K et al. (2003), Correlation of physical activity with diet and obesity in children, in Barnat S, International Conference on Health benefits of Mediterranean Diet Abstract Book, Aghia Pelagia, Egea, 87. SCHATZKIN A (2003), Fiber and colorrectal cancer prevention, in Barnat S, International Conference on Health benefits of Mediterranean Diet Abstract Book, Aghia Pelagia, Egea, 29. SCHILTER B et al. (2003), Guidance for safety assessment of botanicals and botanical preparations for use in food and food supplements. Food and Chemical Toxicology, 41, 1625±49. SCHULZ H, QUILITZSCH R (2003), Determination of active ingredients in vegetable drugs using Diamond ATR-IR Spectroscopy. GIT Laboratory Journal, 2, 86±8. SCHWARZ K et al. (2001), Investigation of plant extracts for the protection of processed foods against lipid oxidation. Comparison of antioxidant assays based on radical scavenging, lipid oxidation and analysis of the principal antioxidant compounds. Eur. Food Res. Technol, 212, 319±28. SEVCIKOVA P, GLATZ Z, SLANINA J (2002). Analysis of artichoke (Cynara cardunculus L.) extract by means of micellar electrokinetic capillary chromatography. Electrophoresis, 23, 249±52. SHIMODA H et al. (2003), Anti-Hyperlipidemic Sesquiterpenes and New Sesquiterpene Glycosides from the Leaves of Artichoke (Cynara scolymus L.): Structure Requirement and Mode of Action. Bioorganic and Medicinal Chemistry Letters, 13, 223±8. SIMOPOULOS A (2001), The Mediterranean Diets: What Is So Special about the Diet of Greece? The Scientific Evidence. J Nutr, 131, 3065±73. SIMOPOULOS A, VISIOLI F (2001), Mediterranean Diets, Basel, Karger Publications. SINGH R et al. (2002), Effect of an Indo-Mediterranean diet on progression of coronary artery disease in high risk patients (Indo-Mediterranean Diet Heart Study): a randomised single-blind trial. The Lancet, 360, 1455±61. SKIBOLA C, SMITH M (2000), Potential Health Impacts of Excessive Flavonoid Intake. Free Radical Biology and Medicine, 29(3±4), 375±83. SKOLDSTAM L, HAGFORS L, JOHANSSON G (2003), An experimental study of a Mediterranean diet intervention for patients with rheumatoid arthritis. Ann Rheum Dis, 62(3), 208±14. SLIMANI N (2003), Dietary patterns actually vary across the 10 western European populations participating to the EPIC study, in Barnat S, International Conference on Health benefits of Mediterranean Diet Abstract Book, Aghia Pelagia, Egea, 23. RUIDAVETS J B

New functional foods for age-related diseases

79

(1982), Erasistratus' dietetic medicine. Bulletin of the History of Medicine, 56, 398±409. SOLFRIZZI V, PANZA F, CAPURSO A (2003), The role of diet in cognitive decline. Journal of Neural Transmission, 110, 95±110. STELLJES K B (2003). Questions about Citrus Limonoids. http://www.ars.usda.gov/is/pr/ 1999/bg990323.htm STERMITZ F et al. (2000a), Synergy in a medicinal plant: Antimicrobial action of berberine potentiated by 50 -methoxyhydnocarpin, a multidrug pump inhibitor. PNAS, 97(4), 1433±7. STERMITZ F et al. (2000b), 50 -methoxyhydnocarpin-D, and pheophorbide A: Berberis species components that potentiate berberine growth inhibition of resistant Staphylococcus aureus. J. Nat. Prod., 63(8), 1146±9. STOBIECKI M et al. (1999), Detection of Isoflavonoids and their Glycosides by Liquid Cromatography / Electrospray Ionization Mass Spectrometry in Root extracts of Lupin (Lupinus albus). Phytochemical Analysis, 10, 198±207. SUGIMURA T (2002), Food and cancer. Toxicology, 181±182, 17±21. SURH Y J (2002), More Than Spice: Capsaicin in Hot Chili Peppers Makes Tumor Cells Commit Suicide. Journal of The National Cancer Institute, 94(7), 1263±5. TAKEI H, HIRABUKI M, YOSHIZAKI F (1999), Analysis of Synephrine in the Peel of Citrus Fruit, Immature Citrus Fruit and Decoctions of Chinese Medicinal Prescriptions Containing These Crude Drugs by Capillary Electrophoresis. Analytical Sciences, 15, 1017±20. TEGOS, G. et al. (2002), Multidrug Pump Inhibitors Uncover Remarkable Activity of Plant Antimicrobials. Antimicrobial Agents and Chemotherapy, 46(10), 3133±41. TRICHOPOULOU A et al. (1995), Diet and survival of elderly Greek: a link to the past. Am J Clin Nutr, 61, 1346±50. TRICHOPOULOU A et al. (2000), Nutritional composition and flavonoid content of edible wild greens and green pies: a potential rich source of antioxidant nutrients in the Mediterranean diet. Food Chemistry, 70, 319±23. TRICHOPOULOU A et al. (2003), Adherence to a Mediterranean Diet and Survival in a Greek Population. The New England Journal of Medicine, 348 (26), 2599±608. VASILIOPOULOU E, TRICHOPOULOU A (2003), Traditional foods: Health, Cultural and Standardization aspects, in Barnat S, International Conference on Health benefits of Mediterranean Diet Abstract Book, Aghia Pelagia, Egea, 89. VINSON J A (1999), The Functional Food Properties of Figs. Cereal Foods World, 44 (2), 82±7. VOGEL R, CORRETTI M, PLOTNICK G (2000), The Postprandial Effect of Components of the Mediterranean Diet on Endothelial Function. Journal of the American College of Cardiology, 36(5), 1455±60. VOLLSET S E (2003a), Folate and Cancer, in Barnat S, International Conference on Health benefits of Mediterranean Diet Abstract Book, Aghia Pelagia, Egea, 31. VOLLSET S E (2003b), Mediterranean diet: Genetics? Lifestyle factors? In Barnat S, International Conference on Health benefits of Mediterranean Diet Abstract Book, Aghia Pelagia, Egea, 67. YAHIAA D et al. (2003), Influence of fish protein on blood pressure, plasma, lipoprotein metabolism and on hepatic enzyme activities in spontaneously hypertensive rats, in Barnat S, International Conference on Health benefits of Mediterranean Diet Abstract Book, Aghia Pelagia, Egea, 92. YANARDAG R et al. (2002), The Effects of Chard (Beta vulgaris L. var. cicla) Extract on SMITH W D

80

Functional foods, ageing and degenerative disease

the Kidney Tissue, Serum Urea and Creatinine Levels of Diabetic Rats. Phytotherapy Research, 16, 758±61. YARNEL J, EVANS A (2000), The Mediterranean diet revisited ± towards resolving the (French) paradox. Q Journal Med, 93, 783±5. ZAND R, JENKINS D, DIAMANDIS E (2003), Genistein: A Potent Natural Antiandrogen. Clinical Chemistry, 46 (6), 887±8.

Part I Bone and oral health

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4 Diet and control of osteoporosis K. D. Cashman, University College Cork, Ireland

4.1

Introduction: definition and epidemiology of osteoporosis

Osteoporosis is a global health problem that will take on increasing significance as people live longer and the world's population continues to increase in number (European Commission, 1998). Thus, prevention of osteoporosis and its complications is an essential socioeconomic priority. There is an urgent need to develop and implement nutritional approaches and policies for the prevention and treatment of osteoporosis that could ± with time ± offer a foundation for population-based preventive strategies. However, to develop efficient and precocious strategies in the prevention of osteoporosis, it is important to determine which modifiable factors, especially nutritional factors, are able to improve bone health throughout life. The present review will firstly define the principal disease of bone mass (i.e. osteoporosis) as well as considering its epidemiology and risk factors. The review will then focus on the importance of dietary factors in bone health and conclude by considering how individual genetic variation influences the impact of diet on bone health.

4.1.1 Definition of osteoporosis and osteopenia Osteoporosis is defined as a systemic skeletal disease characterised by low bone mass and microarchitectural deterioration of bone tissue, with a consequent increase in bone fragility and susceptibility to fracture (Consensus Development Conference, 1993). Osteopenia is sometimes referred to as borderline low density because there is a loss of bone density, but less than is seen with osteoporosis. For the purposes of clinical diagnosis, a Working Party of the World Health Organization has redefined osteoporosis and osteopenia according

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to bone mass, at least for women. Their diagnostic criteria for osteoporosis and osteopenia, based on bone mineral content (BMC) or bone mineral density (BMD) include: normal, within 1 standard deviation (SD) of young adult reference mean for the population; osteopenia, between ÿ1 and ÿ2.5 SD of the young adult mean; osteoporosis, more than ÿ2.5 SD below the young adult mean, and established osteoporosis as the same mass definition but associated with a fragility fracture (World Health Organization, 1994). Fragility fractures are the hallmark of osteoporosis and are particularly common in the spine, hip and distal forearm, although they can occur throughout the skeleton. 4.1.2 Epidemiology of osteoporosis Osteoporotic fractures constitute a major public health problem. Currently, in the US alone, ten million individuals already have osteoporosis, and a further 18 million more have low bone mass, placing them at increased risk for this disorder (National Institutes of Health, 2000). One in eight European Union (EU) citizens over the age of 50 years will fracture their spine this year (European Commission, 1998). The estimated remaining lifetime risk of fractures in Caucasian women at age 50 years, based on incidence rates in North America is 17.5%, 15.6% and 16% for hip, spine and forearm respectively; the remaining lifetime risk for any fragility fracture approaches 40% in women and 13% in men (Melton et al., 1992). Similar rates have been reported from parts of Europe, although there is a marked variation in the incidence of fractures between countries and regions (Johnell et al., 1992) and even within countries (Elffors et al., 1994). Hip fractures in particular are associated with significant morbidity, necessitating hospital admission for an average of 20±30 days (Johnell et al., 1992). Osteoporosis patients currently occupy 500 000 hospital bed nights per year in the European Community (European Commission, 1998). Moreover, hip fracture patients have an overall mortality of 15±30% (Browner et al., 1996), the majority of excess deaths occurring within the first six months after the fracture. Vertebral fractures are also associated with reduced survival (Cooper et al., 1993), probably due to clustering of co-morbidity which predisposes independently to osteoporosis and premature death. Fractures can also have a profound impact on quality of life, as evidenced by the findings that 80% of women older than 75 years preferred death to a bad hip fracture resulting in nursing home placement (National Institutes of Health, 2000). Fear, anxiety, and depression are frequently reported in women with established osteoporosis and are likely to be under-addressed when considering the overall impact of this condition (National Institutes of Health, 2000). The incidence of vertebral and hip fractures increases exponentially with advancing age while that of wrist fractures levels off after the age of 60 years (Compston, 1993). This is of particular concern as it is projected that the number of elderly (80 years and older, in whom the incidence of osteoporotic fracture is greatest) in the EU population will grow from 8.9 million and 4.5 million women and men, respectively, in 1995 to 26.4 million and 17.4 million women

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and men, respectively, in the year 2050 (European Commission, 1998). Because of the increase in incidence rates of osteoporotic fractures with age, the above demographic changes and increasing life expectancy will have a huge impact on the number of fractures which can be expected to occur. For example, the number of hip fractures occurring each year in the EU alone is estimated to rise from current figures of 414,000 to 972,000 by the year 2050, representing an increase of 135% (European Commission, 1998). The increase in the number of vertebral fractures occurring each year is not expected to be of the same magnitude as for hip fractures; thus the estimated increase is from current figures of 237,000 to 373,000 by the year 2050, representing a rise of 57% (European Commission, 1998). From an economic perspective, the expenses of hospital care and rehabilitation associated with osteoporotic fractures are a considerable fiscal drain for the health care system, exceeding those of other highly prevalent pathologies of the elderly, such as myocardial infarction (Schurch et al., 1996). Osteoporosis costs national treasuries over ¨3500 million annually in hospital health care alone (European Commission, 1998).

4.2

Bone growth and factors affecting bone mass

Low bone mineral mass is the main factor underlying osteoporotic fracture (Prentice, 1997). Bone mass in later life depends on the peak bone mass (PBM) achieved during growth and the rate of subsequent age-related bone loss. Bone mineral is laid down throughout childhood, with the most rapid increase occurring during puberty. The deposition continues, at a slower rate, after growth in height has stopped (British Nutrition Foundation, 1989). PBM is achieved in early life (20±35 years), although the exact timing is not certain and may vary between different regions of the skeleton (Teegarden et al., 1995; Institute of Medicine, 1997). From the age of 20 years until approximately 40 years, bone mass is stable in both sexes (Reid and New, 1997). At greater ages, bone is gradually lost from the skeleton in both men and women (Prentice, 1997). For women, there is also a period of about 10±15 years when bone loss (especially at trabecular-rich sites such as the spine or wrist) is accelerated due to oestrogen withdrawal at the menopause, when more than one-third of bone is lost from the skeleton (Compston, 1993). This accelerated rate of loss seen in women, when associated with a low attainment of PBM, leads to excessive risk of future fracture (Reid and New, 1997). Bone is a living, dynamic tissue, and is constantly undergoing breakdown and formation as part of the natural process of renewal and repair (Prentice, 1997). For a more detailed review of this process of bone turnover, or bone remodelling, the interested reader is referred to a review by Cashman and Ginty (2003). The rate of bone formation and bone resorption, which together influence the rate of bone turnover, and thus bone mass, can be measured by biochemical markers, which are relatively non-invasive. Biochemical markers

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Table 4.1

Biochemical markers of bone turnover

Bone resorption Serum: Tartrate-resistant acid phosphatase (TRAP) Free -carboxy glutamic acid C-terminal pyridinoline cross-linked telopeptide of type 1 collagen (ICTP)

Bone formation Alkaline phosphatase (total, bone specific) Osteocalcin Amino-terminal procollagen extension Peptide (PINP) Carboxy-terminal procollagen extension Peptide (PICP)

Urine: Calcium Hydroxyproline (total, free) Pyridinium crosslinks of collagen: Deoxypyridinoline (Dpyr) (total, free) Pyridinoline (Pyr) (total, free) N-telopeptides of collagen (NTx) C-telopeptides of collagen (CTx) Hydroxylysine glycosides

that reflect the remodelling process and can be measured in blood or urine (see Table 4.1) fall into three categories: (a) enzymes or proteins that are excreted by cells involved in the remodelling process, (b) breakdown products generated in the resorption of old bone, and (c) byproducts produced during sythnesis of new bone (Watts, 1999). Increased bone turnover, as assessed by such biochemical markers, has been associated with increased risk of fracture (Cashman and Flynn, 2003). Development of maximal bone mass during growth and reduction of loss of bone later in life are the two main strategies for preventing osteoporosis (Weaver, 2000). Consequently, any factor that influences the development of PBM or the loss of bone in middle age will affect later fracture risk. Several factors are thought to influence bone mass. These can be broadly grouped into factors that cannot be modified, such as gender, age, body (frame) size, genetics and ethnicity, and those factors that can be modified, such as hormonal status (especially sex and calciotropic hormone status), lifestyle factors including physical activity levels, smoking and alcohol consumption patterns, and diet. The interaction of these genetic, hormonal, environmental and nutritional factors influences both the development of bone to PBM at maturity and its subsequent loss (see Fig. 4.1). It has been suggested that genetic factors probably account for up to 80% of the bone mass variation in the population (Morrison et al., 1994). While diet and lifestyle factors, such as physical activity, may have a smaller influence than genetics on bone mass, these factors are nonetheless important since they are modulators for the achievement of maximum genetic potential PBM as well as the subsequent

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Fig. 4.1 Model for pathogenesis of osteoporosis. The major cause of fractures in osteoporosis is a decrease in absolute bone mineral density (BMD). Factors contributing to low BMD are inadequate peak bone mass and increased bone loss. Adapted from Riggs (1988).

rate of bone loss and, unlike genotype, they can be modified (Cashman and Flynn, 1998). The remainder of this chapter will focus on the dietary components that may influence bone health as well as the impact of genetic variation on metabolic response of bone to diet.

4.3

Dietary strategies for preventing osteoporosis: minerals

Many of the nutrients and food components we consume as part of a Westernised diet can potentially have a positive or negative impact on bone health. They may influence bone by various mechanisms, including alteration of structural aspects of bone, the rate of bone metabolism, the endocrine and/or paracrine system, as well as homeostasis of calcium and possibly of other boneactive mineral elements. These dietary factors range from inorganic minerals, through vitamins, to macronutrients, such as protein and fatty acids. In addition, the relative proportions of these dietary factors derived from different types of diets (vegetarian versus omnivorous) may also affect bone health, and thus, osteoporosis risk. Formulation of dietary strategies for prevention of osteoporosis requires a thorough knowledge of the impact of these dietary factors, in the first instance individually, but also in combination, on bone. The following section will briefly review the influence of selected dietary components on bone health (other nutrients and bioactive food components which influence bone health have been reviewed in other chapters in this work, and where appropriate these have been indicated). Several minerals and trace

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elements have been implicated as having either beneficial or adverse effects on bone. These also will be overviewed in the following section. 4.3.1 Calcium The adult human body contains about 1,200 g of calcium, which amounts to about 1±2% of body weight. Of this, 99% is found in mineralised tissues, such as bones and teeth, where it is present as calcium phosphate (together with a small component of calcium carbonate), providing rigidity and structure (Nordin, 1997). The remaining 1%, found in blood, extracellular fluid, muscle, and other tissues, plays a role in mediating vascular contraction and vasodilation, muscle contraction, nerve transmission and glandular secretion (Institute of Medicine, 1997). Calcium is required for normal growth and development of the skeleton (National Research Council, 1989; Nordin, 1997). During skeletal growth and maturation, i.e., until the age of the early twenties in humans, calcium accumulates in the skeleton at an average rate of 150 mg per day. During maturity, the body ± and therefore ± the skeleton is more or less in calcium equilibrium. From the age of about 50 in men and from menopause in women, bone balance becomes negative and bone is lost from all skeletal sites. This bone loss is associated with a marked rise in fracture rates in both sexes, but particularly in women. Adequate calcium intake is critical to achieving optimal PBM and modifies the rate of bone loss associated with ageing (National Institutes of Health, 1994). In recent years, convincing evidence has emerged with respect to effects of dietary calcium on bone health in all age groups (European Commission, 1998; Cashman, 2002a). Intervention and cross-sectional studies have reported a positive effect of calcium on bone mass in children and adolescents (Kanders et al., 1988; Johnston et al., 1992; Dawson-Hughes, 1996; see also review by Cashman and Flynn, 1999). VaÈlimaÈki et al. (1994) reported that dietary calcium intake in childhood and adolescence was positively related to BMD in young women. A meta-analysis of 33 studies concluded that there was an overall association between calcium intake and bone mass in premenopausal women (Welten et al., 1995). There is considerable evidence that increasing calcium intake above that usually consumed in the diet may have benefits for the development and maintenance of bone, and may reduce the risk of osteoporosis in later life (Flynn and Cashman, 1999). The findings of many of these controlled calcium intervention trials have been reviewed (Dawson-Hughes, 1991; Institute of Medicine, 1997; Prentice, 1997; Department of Health, 1998; Cashman, 2002a). Despite the wealth of data from the various calcium intervention studies, there is still considerable debate on the meaning of these effects of calcium on bone. For example, some researchers argue that the increase in bone mass is due to a decrease in bone turnover and is transient and reversible (Department of Health, 1998). In the absence of longitudinal studies of sufficient duration it is

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not clear whether additional calcium consumed throughout early life results in increased PBM in adulthood. This question is of great significance since PBM in adulthood is predictive of bone mass, and therefore osteoporosis risk, in later life (Hansen et al., 1991). There is also still considerable debate on the significance of the reduction in the rate of bone loss observed in these calcium supplementation studies in elderly populations. A meta-analysis of calcium supplementation trials (Mackerras and Lumley, 1997) confirmed that calcium supplementation reduces postmenopausal bone loss, but the effects were significant only in the first year of supplementation. Although osteoporosis is usually defined in terms of reduced bone mass it is the end result, i.e., the greater tendency to sustain fractures, which is of major concern. There have been only a few studies on the effect of calcium supplementation on fracture rates in postmenopausal women. A reduction in vertebral fractures with calcium supplementation was observed in two studies in which habitual calcium intakes were low (450±620 mg) (Chevalley et al., 1994). Studies of combined supplementation with calcium and vitamin D for one and a half to three years have shown impressive reductions in hip-fracture incidence in elderly women (mean age 84 years) (Chapuy et al., 1992; 1994). More recently, Dawson-Hughes et al. (1997) showed that combined supplementation with calcium and vitamin D for three years significantly reduced non-vertebral fracture rates in men and women (mean age 71 years). Correction of poor vitamin D status and reduction in serum parathyroid hormone (PTH) levels, a mediator of bone resorption, appear to be central to the mechanism of this effect (Prentice, 1997). The dietary deficiency of calcium identified in some population groups as mentioned previously, may be addressed in a number of ways. This includes changing eating behaviour at the population level by increasing the consumption of foods which are naturally rich in calcium (e.g., milk and milk products), calcium fortification of foods consumed by target groups, or increasing calcium intakes from calcium supplements (Cashman, 2002a, and see Chapter 7 for more detailed discussion of these issues). These may be seen as complementary rather than alternative strategies and each has advantages and disadvantages (Flynn and Cashman, 1999). For example, it is notoriously difficult to achieve changes in the diet of entire populations, and thus persuading individuals to consume more dairy produce represents a considerable challenge. The use of calcium supplements can be effective in increasing calcium intake in individuals who consume them regularly, but it has limited effectiveness at the population level due to the poor compliance with supplement use (Flynn and Cashman, 1999). Calcium-fortified food products could provide additional choices for meeting calcium requirements; however, attention should be paid to the selection of products so that they reach the target groups (i.e., those population groups who have the greatest difficulty in meeting calcium requirements). Besides the amount of calcium in the diet, the absorption of dietary calcium in foods is also a critical factor in determining the availability of calcium for bone development and maintenance. Thus, there is a need to identify food

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components and/or functional food ingredients that may positively influence calcium absorption in order to ensure that calcium bioavailability from foods can be optimised (Cashman, 2002a). This approach may be of particular value in individuals who fail to achieve the dietary recommended level of calcium. A number of food constituents have been suggested as potential enhancers of calcium absorption. Individual milk components, such as lactose, lactulose and casein phosphopeptides have attracted considerable attention and these have been reviewed recently by Flynn and Cashman (1999). In addition, emerging evidence has shown that non-digestible oligosaccharides can improve calcium absorption in adolescents and adults and these have been extensively reviewed recently (Cashman, 2002b, 2003). 4.3.2 Magnesium Magnesium deficiency has been identified as a possible risk factor for osteoporosis in humans (Institute of Medicine, 1997; Rude, 1998). Several studies have reported significant reductions in serum magnesium and bone magnesium content in postmenopausal women with osteoporosis (Reginster et al., 1989; Stendig-Lindberg et al., 1993). However, epidemiological studies relating magnesium intake to bone mass or rate of bone loss have been conflicting. Significant positive associations between magnesium intake and BMD or BMC have been seen at the lumbar spine in premenopausal women (New et al., 1997), at the forearm bone in premenopausal but not postmenopausal women (Angus et al., 1988), in the forearm for men but not postmenopausal women and at the hip for both men and postmenopausal women (Tucker et al., 1999). In another study, no correlation was found between BMC in pre- and postmenopausal women; however, magnesium intake was positively correlated with the rate of change in humerus and radius BMD in this same population (Freudenheim et al., 1986). Greater baseline magnesium intake was significantly associated with lower subsequent change in BMD at the hip in elderly men whereas there was no significant association between magnesium intake and longitudinal change in BMD in elderly women (Tucker et al., 1999). There are few studies available on the effect of magnesium supplementation on bone mass and bone metabolism. One study reported that magnesium supplementation (six months at 750 mg/d followed by 250 mg/d for 18 months) increased radial bone mass in 31 osteoporotic women after one year but that no further changes were seen by the end of the second year (Stendig-Lindberg et al., 1993). In 12 healthy young adult males (usual diet 310 mg Mg/d) 30 days of magnesium supplementation (365 mg/d) reduced serum-based biochemical markers of bone formation and bone resorption (Dimai et al., 1998). In contrast, a placebo-controlled, randomised crossover magnesium intervention trial showed that giving 26 healthy young adult females (usual magnesium intake, 267 mg/d) an additional 240 mg of magnesium for 28 days did not affect biochemical markers of bone turnover (Doyle et al., 1999). Thus, the effect of magnesium on bone health requires further investigation.

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4.3.3 Phosphorus There has been some controversy over the role of dietary phosphorus, and in particular, the dietary ratio of calcium to phosphorus, on bone health. The debate has been fuelled by relatively recent data to suggest that dietary phosphorus intakes have risen 10% to 15% over the past 20 years because of the increased use of phosphate salts in food additives and cola beverages (Institute of Medicine, 1997). Currently, dietary phosphorus intakes in adults in the US range between 1000 and 1500 mg/day, a level well above current recommendations of about 700 mg/day (Institute of Medicine, 1997). Although phosphorus is an essential nutrient, there is concern that excessive amounts may be detrimental to bone, especially when accompanied by low calcium consumption. For example, a rise in dietary phosphorus increases serum phosphorus concentration, producing a transient fall in serum ionised calcium resulting in elevated PTH secretion and potentially bone resorption. The hypothesis that excess dietary phosphorus (typically a ratio of approximately 4:1 phosphorus: calcium) is harmful to bone was tested in a number of relatively short-term studies. Two studies investigated this issue in young adults consuming controlled diets containing 1,660 mg phosphorus and 420 mg calcium. Within 24 hours, the diet resulted in elevated indexes of PTH action (Calvo et al., 1988) that persisted for four weeks (Calvo et al., 1990). Similar elevations in PTH were found in other investigations, including one of young adult males and females administered 2000 mg of phosphate orally for five days (Portale et al., 1986) and another of premenopausal women administered phosphate salts (Silverberg et al., 1986). These and other studies have been expertly reviewed by Calvo and Park (1996). While excess phosphorus appears to influence circulating PTH, an important mediator of bone turnover, its effect on bone per se is less clear. In one study, women given a low calcium diet, had significantly elevated PTH but in addition had changes in other bone biomarkers which were indicative of increased bone resorption (Barger-Lux and Heaney, 1993). However, human studies using calcium kinetic methodology showed no effect on bone turnover from doubling dietary phosphorus (Heaney and Recker, 1987), a conclusion supported by a nonisotopic study done in young men and women (Bizik et al., 1996; Silverberg et al., 1986). In fact, the study by Silverberg et al. (1986) actually showed that despite the elevated PTH, urinary hydroxyproline (a relatively crude measure of bone resorption) decreased on high phosphorus intakes, as does urinary calcium (Silverberg et al., 1986), suggesting a possible beneficial effect on bone rather than an adverse one (Institute of Medicine, 1997). In their excellent review of the area, Calvo and Park (1996) concluded that there was no clinical study that linked high phosphorus consumption, with or without adequate calcium intake to lower bone mass or higher rates of bone loss in humans. However, the same authors highlighted that this relationship has been demonstrated in animal models at concentrations of phosphorus and calcium comparable with current human intake (Calvo and Park, 1996). The Food and Nutrition Board of the US Institute of Medicine established the dietary reference values for phosphorus in 1997, and as part of their review of

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the data they evaluated the impact of high dietary phosphorus levels (including high consumption of phosphate additives) on bone. They concluded that `it is doubtful whether phosphorus intakes, within the range currently thought to be experienced by the US population and/associated with serum inorganic phosphorus values in the normal range, adversely affect bone health' (Institute of Medicine, 1997). This conclusion has recently been challenged (Sax, 2001) and debated (Anderson and Garner, 2001). Clearly, the ongoing debate over the role of dietary phosphorus on bone health will be clarified only as more human research is carried out on the long-term influence of a high-phosphorus, lowcalcium dietary pattern on the stimulation of PTH and the eventual reduction of bone mass and density. 4.3.4 Sodium Dietary salt (sodium chloride) has been considered potentially detrimental because increasing dietary salt increases urinary calcium excretion (also referred to as calciuria) (Shortt and Flynn, 1990; Massey and Whiting, 1996). The dependence of urinary calcium excretion on urinary sodium excretion has been attributed to the existence of linked or common re-absorption pathways for both ions in the convoluted portion of the proximal tubule and thick ascending loop of Henle (Shortt and Flynn, 1990). Therefore, when dietary sodium chloride is increased, the fractional reabsorption of sodium is decreased, leading to a parallel reduction in calcium reabsorption. Nordin et al. (1993) proposed that for every 100 mmol of sodium excreted there is approximately 1 mmol loss of urinary calcium in free-living, normocalciuric healthy populations. To place this calcium loss in the context of bone health, a net deficit of only 1 mmol per day of calcium would result in losing one third of the calcium contained in the typical adult skeleton in just over two decades unless a compensatory increase in intestinal calcium absorption occurred (Sellmeyer et al., 2002). While it has been shown that the sodium-induced calciuria in some adults, especially young adults, is compensated for by increased calcium absorption, mediated through serum PTH, this adaptive mechanism does not appear to function in all individuals (e.g., those with impaired parathyroid function, postmenopausal women with osteoporosis, as well as some healthy postmenopausal women (Shortt and Flynn, 1990). Even in those individuals who appear to adapt, the increase in net calcium absorption may not be sufficient to offset the increase in urinary calcium losses (Shortt and Flynn, 1990, Sellmeyer et al., 2002, and see review by Cashman and Flynn, 2003). While there is little doubt that increasing sodium chloride intake increases urinary calcium excretion, its effect on bone is less clear. The limited number of epidemiological studies which have investigated the association of dietary or urinary sodium with BMD and/or bone turnover in humans have produced conflicting results (Nordin and Polley, 1987; Dawson-Hughes et al., 1996; Greendale et al., 1994; Devine et al., 1995; Matkovic et al., 1995; Jones et al., 1997, 2001, and see review by Cashman and Flynn, 2003).

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Two controlled intervention studies have investigated the effects of dietary sodium on urinary pyridinium crosslinks of collagen in adult (premenopausal) women. Evans et al. (1997) reported that urinary excretion of deoxypyridinoline (Dpyr; see Table 4.1) was unaffected by increasing dietary sodium (i.e., from 50 to 300 mmol per day as salt supplements) for seven days in premenopausal women (mean age 32 years) with average daily calcium intake of 860 mg, although urinary calcium excretion increased. Similarly, Ginty et al. (1998) found that increasing sodium intake from 80 to 180 mmol per day for 14 days by dietary manipulation had no effect on urinary excretion of either pyridinoline (Pyr) or Dpyr in adult women (mean age 25 years) whose daily calcium intake was restricted to 500 mg, regardless of whether or not they were sodium-sensitive (i.e., showing a significant calciuric response to increased sodium intake). The lack of effect of sodium on urinary pyridinium crosslinks in the premenopausal women in these two studies suggests that the sodium-induced urinary calcium loss is compensated for by increased calcium absorption rather than increased bone resorption. Of particular note is that in the study by Ginty et al. (1998) the adaptive processes appeared to be adequate to protect bone, even though the calcium intake of the young women was restricted to 500 mg per day. Evidence for such adaptation is derived from the study of Breslau et al. (1982) which showed that sodium supplementation of young men and women (mean age, 27 years) increased fractional calcium absorption (26% on average). The capacity for such adaptation may, however, be related to age and menopausal status (Sellmeyer et al., 2002). Impaired adaptation may explain the increased urinary excretion of crosslinks of collagen in post-menopausal women in response to increased dietary sodium intake in some studies. For example, Evans et al. (1997) reported that, in postmenopausal women (mean age 57 years) with average daily calcium intake of 750 mg, urinary excretion of Dpyr was higher following seven days on a high sodium diet (300 mmol per day) than a low sodium diet (50 mmol per day). However, Leitz et al. (1997) did not observe this in a similar study of postmenopausal women (mean age 62 years) with average daily calcium intake of 800 mg using a lower sodium load (60±170 mmol per day). Recently, Sellmeyer et al. (2002) reported that when postmenopausal women, who received a daily supplement of 500 mg calcium in addition to their usual diet, and who were adapted to a low-salt diet (87 mmol sodium per day) for three weeks, were switched to a high-salt (225 mmol sodium per day) diet (achieved by sodium supplements and dietary manipulation) for a further four weeks, urinary N-telopeptides of collagen (NTx) levels, a sensitive and specific marker of bone resorption (see Table 4.1), were significantly increased. The above-mentioned studies suggest that increasing sodium intake within the usual dietary range can increase bone resorption in postmenopausal women, even when calcium intake is adequate, due to maladaption of calcium absorption to sodium induced calciuria. An increased rate of bone turnover in older adults contributes to faster bone loss and is recognised as a risk factor for fracture (Garnero et al., 1996a, 2000). However, it is worth noting that these studies were

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relatively short term and thus may not have allowed sufficient time for bone to reach a steady state after the sodium intervention. Larger studies with more long-term outcomes such as BMD and fracture will be needed to define the role of dietary sodium in postmenopausal bone loss and osteoporosis more completely. To date, there has not been any reported controlled intervention study of the effect of dietary sodium on BMD. 4.3.5 Potassium There has been increasing interest in the potential beneficial effects of potassium on bone. For example, alkaline salts of potassium (e.g., potassium bicarbonate) have been shown to significantly reduce urinary calcium excretion in healthy adults (Morris et al., 1999; Lemann et al., 1991), even in the setting of a high sodium intake (Morris et al., 1999). Of interest in the recent randomised, doubleblind, placebo-controlled study of Sellmeyer et al. (2002) (mentioned above), those postmenopausal women who were supplemented with 90 mmol of potassium citrate, displayed no calciuric response or elevation in urinary NTx levels during the four-week high-salt dietary period, relative to the low-salt dietary period, whereas the group of postmenopausal women who received a placebo during the high-salt dietary period had significantly elevated urinary calcium and NTx levels. Thus, the potassium citrate attenuated the negative effects of a high-salt diet. Various mechanisms have been proposed by which potassium may prevent the sodium-induced calciuria and increased rate of bone resorption. For example, alkaline salts of potassium are both natriuretic and chloruretic and as such may potentially reduce the extracellular volume expansion that occurs with increased salt intake. In addition, these salts reduce endogenous acid production and increase blood pH and plasma bicarbonate concentration. Through both of these mechanisms alkaline salts of potassium may reduce urinary calcium excretion. Potassium also appears to have a direct effect on the kidney to promote calcium reclamation. Sellmeyer et al. (2002) propose that the levels of alkaline salts of potassium, which prevented the adverse effects of sodium in their study, are achievable by consuming seven to eight servings of potassium-rich fruit and vegetables per day. This is of importance in informing the development of dietary guidelines for osteoporosis prevention. While consumption of the seven to eight servings of potassium-rich fruit and vegetables per day as suggested by Sellmeyer et al. (2002) is in line with the current dietary guidelines, the feasibility, however, of including these additional servings of fruit and vegetables per day, in addition to the usual fruit and vegetable content of a mixed diet, would need to be clarified (Harrington and Cashman, 2003). If this is not feasible then an alternative option is to fortify certain foods with potassium. In support of the contention that increased fruit and vegetable intake may positively influence bone metabolism and bone mass, a recent preliminary report of an ancillary study to the DASH (Dietary Approaches to Stop Hypertension)-

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Sodium trial has shown that in comparison to the DASH control diet (a typical Western-type diet), consumption of the DASH combination diet (high in fruit and vegetable and low-fat dairy produce) for 30 days reduced the levels of biochemical markers of bone formation (serum osteocalcin) and bone resorption (serum C-terminal telopeptide of type I collagen) (see Table 4.1) at each of three dietary sodium intake levels (low, intermediate and high) in adult men and women (Lin et al., 2003). While this diet contained various bone-active nutrients such as magnesium, calcium, phosphorus, vitamin K1 etc., it also had more than twice the usual potassium content of the diet. Furthermore, New et al. (2000) in a cross-sectional study of 62 healthy women, aged 45±55 years, identified potassium, as well as various other key nutrients (magnesium, fibre, -carotene, and vitamin C), and a high past intake of fruit as being important to bone health as assessed by axial and peripheral BMD and pyridinium crosslinks. 4.3.6 Trace elements A variety of trace elements are found in bone including, iron, copper, zinc, manganese, fluoride, strontium and boron, and although present in minute amounts, these may influence normal metabolic processes through interaction with, or incorporation into, proteins, particularly enzymes. The evidence for the involvement of each of these trace elements in bone metabolism has been reviewed elsewhere (Cashman and Flynn, 1998), and is beyond the scope of this present chapter.

4.4 Dietary strategies for preventing osteoporosis: vitamins, proteins and lipids Several of the vitamin components of the diet may also influence bone health. 4.4.1 Vitamin D Vitamin D (calciferol), which comprises a group of fat-soluble seco-sterols that are found in very few foods naturally, is photosynthesised in the skin by the action of solar ultraviolet (UV) B radiation (Holick, 1994). Vitamin D comes in many forms, but the two major physiologically relevant ones are vitamin D2 (ergocalciferol) and vitamin D3 (cholecalciferol). Vitamin D2 originates from the yeast and plant sterol, ergosterol; vitamin D3 originates from 7dehydrocholesterol, a precursor of cholesterol, when synthesised in the skin. Endogenous photoconversion of 7-dehydrocholesterol to vitamin D occurs when skin is exposed to UV B radiation. Any factor reducing the skin dosage of UV B photons reduces the production of vitamin D (Holick, 1995). In latitudes above 40ëN (and 40ëS) (Rome, for example, is at latitude 42ëN) the photoconversion of the precursor 7-dehydrocholesterol to vitamin D occurs little if at all during most of the three to four winter months, and this period is extended to six months in more northern latitudes (Webb et al., 1988). This means, in effect, that during

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the winter almost all of Europe is essentially `in the dark' with respect to solar radiation able to convert 7-dehydrocholesterol to vitamin D. As already mentioned above, dietary intake of vitamin D is problematic because only a few foods are naturally rich in vitamin D. Both of these issues will be dealt with in more detail in Chapter 6. Vitamin D is biologically inert, requiring two obligate hydroxylations (one at the 25±carbon position performed in the liver to form 25 hydroxyvitamin D (25 (OH)2D) and the second hydroxylation at the 1±carbon position to form 1,25 dihydroxyvitamin D (1,25 (OH)2D3) which occurs in the kidney) before it has biological activity as a hormone-like agent, i.e., 1,25 (OH)2D3 (Institute of Medicine, 1997). The major biologic function of vitamin D (as 1,25 (OH)2D3) in humans is to maintain serum calcium and phosphorus concentrations within the normal range by enhancing the efficiency of the small intestine to absorb these minerals from the diet (Institute of Medicine, 1997). When dietary calcium intake is inadequate to satisfy the body's calcium requirement, 1,25 (OH)2 D3 along with PTH, activates osteoclasts (the cells responsible for bone resorption or breakdown) which mobilises calcium stores from the bone, and in doing so buffers serum calcium levels. Vitamin D deficiency is characterised by inadequate mineralisation or demineralisation of the skeleton. In children, vitamin D deficiency results in inadequate mineralisation of the skeleton causing rickets, which is characterised by widening at the end of the long bones, rachitic rosary, and deformations in the skeleton, in particular the lower limbs (Institute of Medicine, 1997). In adults, vitamin D deficiency leads to a mineralisation defect in the skeleton causing osteomalacia. In addition, the secondary hyperparathyroidism associated with vitamin D deficiency enhances mobilisation of calcium from the skeleton. There is a considerable body of evidence that vitamin D deficiency is an important contributor to osteoporosis, through increased bone loss, muscle weakness and a weakened bone microstructure (Department of Health, 1998). Increasing vitamin D intake can significantly reduce risk of bone fracture in older people (Department of Health, 1998). Recent data from a European Commission 5th Framework Programme funded research project (Towards a Strategy for Optimal Vitamin D Fortification (OPTIFORD) (QLK1-2000-00623) ± see http://www.optiford.org) would suggest that adolescents and the elderly are two high-risk population groups for vitamin D deficiency within the general population (Andersen et al., 2003). In light of the high prevalence of sub-optimal vitamin D status among large population groups in Europe, strategies are needed to address this public health problem and these are discussed in more detail in Chapter 6. 4.4.2 Vitamin K The function of vitamin K is to serve as a cofactor for the vitamin K-dependent carboxylase, a micosomal enzyme that facilitates the post-translational conversion of glutamyl to -carboxyglutamyl (Gla) residues (Shearer, 2000).

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Its classic role in this respect involves the synthesis of several coagulation factors, including plasma prothrombin (coagulation factor II), plasma procoagulants (factors VII, IX and X) and anticoagulants (proteins C and S) (Shearer, 2000; Institute of Medicine, 2001). The maintenance of plasma prothrombin concentrations is the basis for the dietary reference value of 1 g/ kg/d (National Research Council, 1989; Dept. of Health, 1991). More recently, the identification of Gla-containing proteins in bone, notably osteocalcin and matrix Gla protein, has generated much interest in the role of vitamin K in bone metabolism and bone health (Binkley and Suttie, 1995; see reviews by Weber, 2001; Institute of Medicine, 2001). Furthermore, it has been suggested that dietary vitamin K levels which are sufficient to maintain normal blood coagulation may be sub-optimal for bone health (Vermeer et al,. 1996; Shearer, 2000). The circulating concentration of under- -carboxylated osteocalcin, a sensitive marker of vitamin K nutritional status (Sokoll and Sadowski, 1996), has been reported to be an indicator of hip fracture (Szulc et al., 1993; Vergnaud et al., 1997; Booth et al., 2000) and a predictor of BMD (Szulc et al., 1994; see reviews by Institute of Medicine, 2001; Weber, 2001). Moreover, the recent findings of two large, prospective cohort studies (the Nurses' Health Study and the Framingham Heart Study) support an association between relative risk of hip fracture and vitamin K intake (Feskanich et al., 1999; Booth et al., 2000). In the Nurses' Health Study, vitamin K1 intakes less than 109 g/d were associated with an increased risk of hip fracture in 72,327 women (Feskanich et al., 1999). In the Framingham Heart Study, elderly men and women in the highest quartile of vitamin K1 intake (median 254 g/d) had significantly lower adjusted relative risk of hip fracture than did those in the lowest quartile of intake (median 56 g/d) (Booth et al., 2000). Whether vitamin K intake is a significant aetiological component of osteoporosis is difficult to establish on the basis of the studies performed thus far (Institute of Medicine, 1997). However, clinical intervention studies presently being conducted, or near completion, in North America and in Europe (including a study as part of another European Commission 5th Framework Programme funded research project (Optimal Nutrition towards Osteoporosis Prevention: Impact of diet and gene-nutrient interactions in calcium and bone metabolism (OSTEODIET) (QLK1-1999-00752) ± see http:// osteodiet.ucc.ie)) will help elucidate this question within the next year or so. 4.4.3 Vitamin A Vitamin A (retinol) is present in food sources such as liver, kidney, and milk. Dairy foods are fortified with small amounts of vitamin A and D in many countries. The provitamin, -carotene is widely distributed in plants and is cleaved to form retinol. The liver stores retinol primarily as retinyl esters. Chronic vitamin A toxicity affects bone and mineral metabolism. Several groups of investigators have examined the possibility that excessive dietary intake of vitamin A is associated with decreased BMD and an increased risk of hip

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fracture. Melhus et al. (1998) showed in their multivariate analysis that, once adjusted for the body-mass index, energy intake, level of physical activity, smoking status, oestrogen status, and use of oestrogen, vitamin A intake was significantly associated with the BMD at various skeletal sites (lumbar spine, femoral neck, and trochanter, as well as total-body) in Swedish and Norwegian populations. The BMD was 10% lower in persons with a vitamin A intake that exceeded 1.5 mg per day than in those with an intake of 1.5 mg per day or less. The relative risk of hip fracture was 2.1 for persons with a vitamin A intake that exceeded 1.5 mg per day, as compared with those whose intake was less than 0.5 mg per day. These data were confirmed by a report from the Nurses' Health Study, in which a total vitamin A intake equal to or greater than 1.5 mg per day was associated with a relative risk of 1.64 for hip fracture, as compared with an intake of less than 0.5 mg per day (Feskanich et al., 2002). On the other hand, carotene intake had no significant influence on the risk of hip fracture (Feskanich et al., 2002). In the Rancho Bernardo Study, an inverse U-shaped association was found between vitamin A intake and BMD (Promislow et al., 2002). In that study, BMD was optimal when the vitamin A intake was 0.6 to 0.9 mg per day, indicating that both low and high intakes of vitamin A may compromise bone health (Promislow et al., 2002). Very recently, MichaeÈlsson et al. (2003) provided further data on the possible deleterious effects of vitamin A on bone, from a long-term, prospective study of 2,322 Swedish men in whom serum retinol and -carotene levels were measured at base line. During the 30±year follow-up period, fractures were reported in 266 men. The relative risk was 1.64 for any fracture and 2.47 for hip fracture among men in the highest quintile for serum retinol (more than 2.64 mol per litre), as compared with the middle quintile (2.17 to 2.36 mol per litre). The relative risk of any fracture was 7.14 among men with a serum retinol level that exceeded 3.60 mol per litre. carotene level was not associated with the risk of fracture. MichaeÈlsson et al. (2003) concluded that high serum retinol levels (above 3 mol per litre) may increase the risk of fracture and should thus be avoided. Thus, in light of such evidence, it might be wise for men or women in Western populations to desist from routinely using supplements containing vitamin A. Fortification of cereals with vitamin A for use by these populations is also an issue and may need to be carefully evaluated. 4.4.4 Vitamin C Vitamin C is an essential cofactor for key enzymes involved in procollagen formation, i.e., conversion of peptide-bound proline and lysine into their hydroxy forms, a major prerequisite for a controlled collagen protein synthesis. It also stimulates alkaline phosphatase activity, required for bone formation (Morton et al., 1997). Therefore, vitamin C is necessary for normal synthesis of collagen and thus is important to bone health. However, in contrast to studies investigating the influence of vitamins D, K and A on bone, there has been

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relatively little research emphasis on the effect of vitamin C on bone. Hall and Greendale, (1998) compared dietary vitamin C intakes with BMD measurements in 775 postmenopausal women who were participating in a study of hormone replacement therapy (HRT). They found a statistically significant positive association between vitamin C intake and BMD at the hip and a similar, though non-significant, relation for the spine. Morton et al. (1997) found that vitamin C supplement intake had beneficial effects on levels of BMD in older women (aged 50±98 years). 4.4.5 Protein An increase in protein consumption increases urinary calcium excretion over the entire range of protein intakes, from marginal to excess (Kerstetter et al., 2003). Protein increases urinary calcium excretion by its effects on both increasing glomerular filtration rate and production of acid (Kerstetter et al., 2003). The ammonium ions produced from the amino groups of the amino acids and sulfate generated from the S groups of cysteine and methionine influence blood pH and urinary acid excretion. Small decreases in blood pH have been shown to activate bone resorption (Barzel, 1995). The carbonate and citrate in bone are mobilised to neutralise these acids, so urinary calcium increases when dietary protein increases. Urinary calcium increases 0.04 mmol (1.6 mg) per gram of dietary protein (Massey, 2003). Frassetto et al. (2000) found the cross-cultural relationship between hip fracture rates and dietary protein was positively related to animal protein intake and inversely related to vegetable protein intake. The ratio of vegetable to animal protein was exponentially inversely related to hip fracture rate (Frassetto et al., 2000). Prospective epidemiological evidence is conflicting regarding the role of animal protein versus plant protein in bone loss. Several prospective studies examining the effect of dietary protein on bone health in older women have been published with conflicting results, and, overall, no pattern on the effect of animal versus plant protein seems to emerge from these studies (for a longer review on protein intake and bone health (including topical issues such as net renal acid excretion (NRAE), potential renal acid load (PRAL) and acid-base ratio, the interested reader is referred to an excellent review by Massey, 2003). In general, while the majority of studies of the impact of protein intake on bone health have focused on protein as the sole dietary factor, it is likely that the effect of protein in the diet on bone may be modified by other dietary constituents such as calcium, potassium, phosphorus, isoflavones, antioxidants, salt, oxalate, phytates and caffeine amongst others (Massey, 2003). 4.4.6 Lipids While dietary factors, such as those mentioned above have attracted considerable attention, the influence of dietary lipids on calcium metabolism and bone health has received much less research emphasis (Kruger and Horrobin, 1997). Kruger et al.

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(1998) reported the findings of their pilot controlled study in elderly women (mean age 79.5 years) with senile osteoporosis which showed that gamma linolenic acid and eicosapentonoic acid (together with calcium) have beneficial effects on bone turnover as well as on BMD of the lumbar spine and femur. On the other hand, van Dokkum et al. (1983) showed that increasing the linoleic acid level in the diet of young men participating in a mineral balance study significantly reduced faecal calcium, indicating stimulation of calcium absorption by n-6 essential fatty acids. Thus the effect of polyunsaturated fatty acids on bone health requires further investigation.

4.5 Preventing osteoporosis: the impact of genetic variation and diet Osteoporosis is a complex disease, which is mediated by an interaction between environmental factors (including nutrition, smoking and physical activity) and several different genes that individually have modest effects on BMD and other aspects of fracture risk (Gueguen et al., 1995). However, the notion of genetic determinants is of little value unless the specific genes that are involved can be identified, and moreover, that interactions between these genes and certain environmental factors, especially nutrition, which mediate expression of bonerelated phenotypes can be eludicated. 4.5.1 Candidate genes for osteoporosis There have been a staggering number of studies published over the last two decades which have reported associations, or lack thereof, between various candidate genes and bone turnover, BMD and/or fracture incidence, as well as other bone-related phenotypic characteristics, such as ultrasound properties of bone. These genes encode a wide range of proteins, including receptors for calciotrophic and steroid hormones, bone matrix proteins, and local regulators of bone metabolism, such as cytokines and growth factors (see Table 4.2) and the list is still expanding. The majority of association studies of BMD and candidate gene markers have investigated markers in the vitamin D receptor (VDR) gene (Wood and Fleet 1998). As mentioned already, 1,25(OH)2D3 has been shown to be an important hormonal regulator of bone and calcium metabolism (Norman, 1990) and the VDR mediates the biological actions of 1,25(OH)2D3. Thus, the prominent role of the VDR in calcium metabolism made the VDR gene a likely candidate gene in determining low BMD and hence, risk of osteoporosis. In 1994, a cardinal study by Morrison and colleagues reported a significant association between polymorphic sites situated between exon 8 and 9 at the 30 end of the VDR gene (detected using the Bsm 1, Taq 1, and Apa 1 restriction enzymes) and BMD in 250 Caucasian twins, aged 17±70 years, from Australia (Morrison et al., 1994). The study consisted of 70 monozygotic and 55 dizygotic adult twin pairs; with most subjects being female. They concluded that much of the genetic variation

Diet and control of osteoporosis Table 4.2

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Candidate genes for osteoporosis

Candidate gene

Physiological function

Vitamin D receptor

Calcium absorption; osteoblast/osteoclast activity Osteoblast/osteoclast activity Osteoblast/osteoclast activity Matrix component Osteoblast/osteoclast activity Osteoblast function Osteoclast activity Vitamin K transport Calcium homeostasis; osteoblast/osteoclast activity Osteoclast function Adipocyte differentiation

Oestrogen receptor  Oestrogen receptor Collagen I A 1 Transforming growth factor -1 Androgen receptor Interleukin 6 Apolipoprotein E Parathyroid hormone receptor Calcitonin receptor Perioxisome proliferator-activated receptor Osteocalcin Calcium-sensing receptor Methylenetetrahydrofolate reductase Metalloproteinase-1 gene

Matrix component Regulation of calcium homeostasis Homocysteine metabolism Matrix component

Modified from Cusack and Cashman (2003).

in BMD (up to 75%) could be explained on the basis of the Bsm 1 VDR genotype alone. Since the initial report by Morrison et al. (1994), many groups have investigated the relationship between VDR genotypes and BMD, calcium absorption, bone turnover (as measured by serum- and urinary-based biochemical markers) and sometimes, fracture incidence, either in twins or in general populations. In addition, this approach has also been used for other osteoporosis candidate genes, such as those encoding the oestrogen receptors, collagen type I, apolipoprotein E, methylenetetrahydrofolate reductase enzyme, amongst others (see Table 4.2). However, in general while some studies report positive associations between these genotypes and bone parameters, others fail to find such associations (see review by Cusack and Cashman, 2003). Some of the inconsistencies in the various studies performed to date may arise from modification of the effects of osteoporosis candidate genes on bone by dietary calcium, vitamins D, K and B-complex, caffeine and possibly the intake of other nutrients. These will be discussed briefly below. 4.5.2 Interaction of genotype and diet Understanding how inherited factors interact with environmental factors, especially nutrition, may hold the key to better prevention and treatment of osteoporosis. However, to date the number of studies which have investigated possible interactions between genotypes and nutrients/food components are limited. These will be reviewed in the following section.

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VDR genotype-calcium interactions Considering the important regulatory role of 1,25(OH)2 D3 on calcium homeostasis, which is mediated by the VDR, studies investigating the interaction between VDR genotype, calcium intake and bone integrity were among the first to test gene-nutrient interactions in bone health. Two longitudinal studies have investigated a relationship between VDR genotype, calcium intake and change in BMD (Krall et al., 1995; Ferrari et al., 1995). Krall et al. (1995), for example, reported that calcium supplementation of a diet habitually low in calcium intake reduced bone loss from the femoral neck in women with the BB VDR genotype. Greater rates of bone loss under conditions of low dietary calcium intakes would be consistent with a possible effect of the VDR genotype on vitamin D-dependent calcium absorption (see below). Moreover, this absorption defect might be masked in subjects with high calcium loads, via a vitamin D-independent pathway (Sheikh et al., 1988). A limited number of associational studies have examined whether a relationship between VDR genotype and bone was influenced by dietary calcium, and the results have been inconsistent (Garnero et al., 1996b; Kiel et al., 1997; Ferrari et al., 1998; Salamone et al., 1998). There have also been a number of studies which investigated the impact of VDR genotype on calcium absorption (Dawson-Hughes et al., 1995; Wishart et al., 1997; Ames et al., 1999). Dawson-Hughes et al. (1995), for example, compared fractional calcium absorption in healthy, late postmenopausal women with (bb) and without (BB) the Bsm 1 restriction site. Calcium absorption and plasma 1,25(OH)2D3 were measured in 60 women after two weeks on a high calcium (1500 mg/day) and two weeks on a low calcium (<300 mg/day) intake. 45Ca absorption was similar in the two groups on the high calcium intake but differed significantly in the groups on the low calcium intake (21% in the BB VDR group and 24% in the bb VDR group). In contrast, others have found no relationship between VDR polymorphisms and intestinal calcium absorption (Francis et al., 1997; Kinyamu et al., 1997). The mechanisms by which VDR genotype influences calcium absorption are still unclear. VDR genotype-vitamin D interaction There is compelling evidence for a protective role for vitamin D on bone health (see reviews by Institute of Medicine, 1997; and see Chapter 7). Studies have examined the differential responses of bone to vitamin D in subjects stratified by VDR genotype. For example, Graafmans et al. (1997) studied the effects of a two-year regimen of vitamin D supplementation (400 IU/d) on BMD in Caucasian (Dutch) women over 70 years old. They observed that the mean increase in BMD in the vitamin D group relative to placebo group was higher in subjects with the BB and Bb VDR genotype compared with those with the bb VDR genotype. The effect of vitamin D supplementation on bone mass accrual in young girls (aged 11.5±13.5 years) stratified by VDR genotype is currently under way as part of the EU Framework V OPTIFORD project.

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VDR genotype-dietary calciuric factors interactions Besides an effect of VDR genotype on the response of bone to calcium and vitamin D, there is also some evidence that there is an interaction between VDR genotype and caffeine, and high-sodium high-protein intake in determining bone loss. For example, Rapuri et al. (2002) showed that postmenopausal women with the tt genetic variant of VDR (defined by the Taq 1 restriction endonuclease enzyme) appeared to be at a greater risk for the deleterious effect of a high caffeine intake (>300 mg/d) on vertebral bone loss over three years, compared to women with the TT VDR genotype. Recent evidence from the European Commission 5th Framework OSTEODIET project would suggest an interaction between VDR genotype and the effect of a high sodium-high protein intake on the rate of bone resorption in postmenopausal women (Harrington et al. 2004). Methylenetetrahydrofolate reductase genotype-B vitamin interactions As mentioned previously, the common allelic polymorphism in the gene that encodes the methylenetetrahydrofolate reductase (MTHFR) enzyme has been variably associated with BMD in postmenopausal women (Miyao et al., 2000; Jorgensen et al., 2002; Abrahamsen et al., 2003). The polymorphism is located to nucleotide 677 in the MTHFR gene and is caused by a single base change (Cto-T), leading to an amino acid replacement of alanine with valine at position 222. This point mutation gives rise to a thermolabile variant of the MTHFR enzyme, which is less effective. Some of the discordant findings on its effect on bone may arise from a possible gene-nutrient interaction between one or more of the B-complex vitamins and the MTHFR genotype. The MTHFR enzyme, together with a number of the B complex vitamins, is required for clearing homocysteine from the circulation. A preliminary investigation of possible interactions between BMD, MTHFR genotype and B vitamin complex in peri- and early postmenopausal women in the Aberdeen Prospective Osteoporosis Screening Study suggest that folate, B12, and B6 had no effect on BMD in the three MTHFR genotype groups (personal communication from S. New). However, for women homozygous for the TT MTHFR genotype only (which is usually associated with elevated homocysteine levels), there was a positive relationship between energy-adjusted vitamin B2 intake and BMD. More studies are needed to understand this interaction, including the underlying mechanisms. Apolipoprotein E genotype-vitamin K interaction Apolipoprotein E (Apo E) phenotype may be linked to osteoporosis and fracture risk (see review by Cusack and Cashman, 2003) through its involvement in the metabolism and transport of vitamin K, an important cofactor for the carboxylation of osteocalcin (Vermeer et al., 1995). Several studies have reported an association between undercarboxylated osteocalcin, a status indicator for vitamin K, and loss of BMD and/or hip fracture (see reviews by Institute of Medicine, 2001 and Weber, 2001). Genetically determined subtypes of Apo E play a crucial role in the transport of chylomicrons and thus of vitamin

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K to the liver and other target tissues, including bone. Saupe et al. (1993), for example, reported that the serum level of vitamin K1 depended on the Apo E phenotype, namely E2>E3>E4. This distribution is in accordance with the relation between Apo E genotype and the rate of hepatic clearance of chylomicron remnants from circulation, with the Apo E4 allele having most rapid catabolism (Booth et al., 2000). This may have implications for supply of vitamin K to bone cells for metabolic activity. In the only study to date which has investigated the relationship between vitamin K, Apo E genotype and bone, Booth et al. (2000) failed to find evidence of an interaction of vitamin K intake and Apo E4 allele on BMD or fracture incidence in elderly men and women. However in that study, neither vitamin K intake nor Apo E genotype was associated with BMD or fracture, even though as mentioned earlier several studies have reported significant associations between vitamin K and bone outcomes and Apo E genotype and bone outcomes (see review by Cusack and Cashman, 2003). Vitamin K intake was estimated by a food frequency questionnaire and unfortunately, vitamin K status (such as undercarboxylated osteocalcin) data was unavailable. The influence of vitamin K supplementation on whole-body calcium retention and bone metabolism in postmenopausal women stratified by Apo E genotype is currently under way as part of the EU Framework V OPTIFORD project. Future studies will need to include measures of Apo E genotype, vitamin K1 intakes and status and BMD and possibly, bone quality measures to test the hypothesis that vitamin K1 may mediate the observed relationship between Apo E genotype and hip fracture. Possible oestrogen receptor genotype-phyto-oestrogen interactions While the mechanism by which polymorphisms in the oestrogen receptor  (OR) gene affects BMD (see review by Cusack and Cashman, 2003) is unclear, it may be that they confer some degree of oestrogen resistance. For example, Han et al. (1997) suggests that variants in the OR gene might account for the lack of response to HRT in some women despite good drug compliance and good health. If the OR genotype can lead to oestrogen resistance then there are also implications for women using dietary phyto-oestrogens as a natural alternative to HRT. Phyto-oestrogens are nonsteroidal compounds naturally occurring in foods of plant origin (especially soy foods) which are able to compete with the principal oestrogens of most mammals for binding ORs (see review by Cotter and Cashman, 2003). Such compounds have been shown to have a favourable effect on bone mass in postmenopausal women in several, but not all, studies (see review by Cotter and Cashman, 2003, and the issue will be dealt with in more detail in Chapter 9). Several studies have investigated the influence of OR genotype on responsiveness of bone to HRT in postmenopausal women (Han et al., 1997; Ongphiphadhanakul et al., 2000; Salmen et al., 2000). For example, Ongphiphadhanakul et al. (2000) reported that the OR gene polymorphism (as defined by the Pvu II endonuclease system) affects the vertebral BMD response to oestrogen in postmenopausal women, suggesting that OR genotype may help

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identify those women who will have more skeletal benefit from HRT. To date, no studies have investigated the influence of OR genotype on the responsiveness of bone to dietary phyto-oestrogen supplementation. Furthermore, phyto-oestrogens, which have been shown to have a relative molar binding affinity for OR between 100 and 1,000 times lower than 17 -oestradiol in vitro, have an even higher specificity for OR (Cotter and Cashman, 2003). OR is preferentially expressed in tissues such as bone, brain, vascular endothelia and bladder. However, to date, no studies have investigated the influence of OR genotype on the responsiveness of bone to phyto-oestrogen supplementation. As dietary phyto-oestrogens bind to both the OR and OR , polymorphisms in both receptor subtypes may influence the response of bone to phyto-oestrogen therapy. However, future research is needed to investigate the potential impact of genetic variation at the OR genes loci on the responsiveness of bone to phyto-oestrogen therapy. One such study, funded by the European Commission 5th Framework Programme funded research project (The Prevention of Osteoporosis by Nutritional Phytoestrogens (PHYTOS) (QLK1±2000±00431) see http://www.phytos.org/project.htm), is currently investigating the effect of OR genotypes on responsiveness of postmenopausal bone to dietary phyto-oestrogens.

4.6

Conclusions and future trends

The impact of nutrition on bone health has gained considerable research attention in recent years, with large cross-sectional studies and retrospective analysis providing new evidence for old hypotheses, such as the impact of protein and sodium intake on bone health, and have brought new arguments in favour of so far mostly hypothetical theories, such as the role of vegetables and fruits, and acid load on bone health or that of vitamin K on bone. It has also become apparent that vitamin D insufficiency is much more common than formerly believed. However, there is still an urgent need for more intervention trials in which the influence of these dietary factors are tested for true efficacy, i.e., does altering the dietary level of these factors impact on bone health, as assessed by BMC, BMD and bone turnover markers. Encouragingly, a number of the major research funding bodies, in particular the European Commission, as part of their Framework programmes, have recognised this need and invested considerably in human intervention trials in the area of diet and bone health (see below). The results of these intervention studies, together with evidence from cross-sectional studies, will allow us to identify nutritional factors that can modify bone health throughout life and subsequently will help in developing and implementing efficient and precocious nutritional strategies in the prevention of osteoporosis. In terms of future trends in the area of diet and bone health, it is likely that the nutrigenomic approach (i.e., assessing the impact of genotype on metabolic response of bone to diet) will become more commonplace within intervention studies, especially as new candidate genes for osteoporosis become apparent. It is also likely that the whole area of nutrition and male osteoporosis will gain

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more attention, as part of an overall increased research emphasis on the causes and treatment of osteoporosis in men.

4.7

Sources of further information and advice

The interested reader is referred to the following useful sources of new and relevant information on osteoporosis in general, but in particular on the impact of nutrition on bone health: European Commission (1998) Report on osteoporosis in the European Community: Action for prevention, Office for Official Publications for the European Commission, Luxembourg. Interim Report and Recommendations of the World Health Organization TaskForce for Osteoporosis. Osteoporosis International 1999; 10: 259±264. Important websites for osteoporosis/bone health societies: http://www.nof.org/ http://www.osteo.org/ http://www.osteofound.org/ http://www.asbmr.org/ EU Framework V funded `diet and bone health-related' research projects: http://osteodiet.ucc.ie http://www.optiford.org/ http://www.phytos.org/project.htm http://www.inra.fr/zenith/

4.8

References

ABRAHAMSEN B, MADSEN J S, TOFTENG C L, STILGREN L, BLADBJERG E M, KRISTENSEN S R,

and MOSEKILDE L (2003), `A common methylenetetrahydrofolate reductase (C677T) polymorphism is associated with low bone mineral density and increased fracture incidence after menopause: longitudinal data from the Danish osteoporosis prevention study', J Bone Miner Res 18(4), 723±9. AMES S K, ELLIS K J, GUNN S K, COPELAND K C and ABRAMS S A (1999), `Vitamin D receptor gene Fok I polymorphism predicts calcium absorption and bone mineral density in children', J Bone Miner Res, 14(5), 740±46. BRIXEN K

È RKKA È INEN M, ANDERSEN R, BROT C, CASHMAN K D, CHARZEWSKA J, FLYNN A, JAKOBSEN J, KA

and (2003), `The vitamin D status in two risk groups from four European countries', Proc Nutr Soc 62(4), 33A. ANDERSON J J and GARNER S C (2001), `Controversy over dietary phosphorus', J Am Coll Nutr, 20(4), 269±70. ANGUS R M, SAMBROOK P N, POCKOCK N A and EISMEN J A (1988), `Dietary intake and bone mineral density', Bone Min, 4(3), 265±77. BARGER-LUX M J and HEANEY R P (1993), `Effects of calcium restriction on metabolic KIELY M, LAMBERG-ALLARDT C, MOREIRAS O, MéLGAARD C, NATRI A M, O'BRIEN M OVESEN L

Diet and control of osteoporosis

107

characteristics of premenopausal women', J Clin Endocrinol Metab, 76(1), 103±7. (1995), `The skeleton as an ion exchange system: implications for the role of acidbase imbalance in the genesis of osteoporosis', J Bone Miner Res, 10(10), 1431±6. BINKLEY N C and SUTTIE J W (1995), `Vitamin K nutrition and osteoporosis', J Nutr, 125 (7), 1812±21. BIZIK B, DING W and CERKLEWSKI F (1996), `Evidence that bone resorption of young men is not increased by high dietary phosphorus obtained from milk and cheese', Nutr Res, 16, 11436. BARZEL, U S

BOOTH S L, TUCKER K L, CHEN H, HANNAN M T, GAGNON D R, CUPPLES L A, WILSON P W F,

and KIEL D P (2000), `Dietary vitamin K intakes are associated with hip fracture but not with bone mineral density in elderly men and women', Am J Clin Nutr 71(5), 1201±8. BRESLAU N A, MCGUIRE J L, ZERWEKH J E and PAK C Y C (1982), `The role of dietary sodium on renal excretion and intestinal absorption of calcium and on vitamin D metabolism', J Clin Endocrinol Metab, 55(2), 369±73. BRITISH NUTRITION FOUNDATION (1989), Calcium. London, British Nutrition Foundation. BROWNER W S, PRESSMAN A R, NEVITT M C and CUMMINGS S R (1996), `Mortality following fractures in older women. The study of Osteoporotic Fractures', Arch Intern Med,156(14), 1521±5. CALVO M S and PARK Y K (1996), `Changing phosphorus content of the U.S. diet: potential for adverse effects on bone', J Nutr, 126(4 Suppl), 1168S-80S. CALVO M S, KUMAR R and HEATH H (1988), `Elevated secretion and action of serum parathyroid hormone in young adults consuming high phosphorus, low calcium diets assembled from common foods', J Clin Endocrinol Metab, 66, 823±9. CALVO M S, KUMAR R and HEATH H (1990), `Persistently elevated parathyroid hormone secretion and action in young women after four weeks of ingesting high phosphorus, low calcium diets', J Clin Endocrinol Metab, 70, 1334±40. CASHMAN K D (2002a), `Calcium intake, calcium bioavailability and bone health', Br J Nutr, 87 Suppl 2, S169±77. CASHMAN K D (2002b), `Prebiotics and calium bioavailability'. In Tannock G W Probiotics and Prebiotics: Where are we going? Caister Academic Press: Norfolk, 149±74. CASHMAN K D (2003), `Prebiotics and calcium bioavailability', Curr Issues Intest Microbio, 4 (1), 21±32. CASHMAN K and FLYNN A (1998), `Trace Elements and Bone Metabolism', Bibl Nutr Dieta, 54, 150±64. CASHMAN K and FLYNN A (1999), `Optimal Nutrition ± Calcium, Magnesium and Phosphorus', Proc Nutr Soc, 58(2), 477±87. CASHMAN K D and FLYNN A (2003), `Sodium effects on bone and calcium metabolism'. In New S and Bonjour P-J, Nutritional Aspects of Bone Health, Cambridge: Royal Society of Chemistry, pp. 267±88. CASHMAN K D and GINTY F (2003), `Bone' in Caballero B, Trugo L and Finglas P, Encyclopedia of Food Sciences and Nutrition, Second Edition, London: Academic Press, 557±65. CHAPUY M C, ARLOT M E, DUBOEUF F, BRUN J, CROUZET B, ARNAUD S, DELMAS P D and MEUNIER P J (1992), `Vitamin D3 and calcium to prevent hip fractures in elderly women', N Engl J Med, 327(23), 1637±42. CHAPUY M C, ARLOT M E, DELMAS P D and MEUNIER P J (1994), `Effect of calcium and ORDOVAS J, SCHAEFER E J, DAWSON-HUGHES B

108

Functional foods, ageing and degenerative disease

cholecalciferol treatment for three years on hip fractures in elderly women', BMJ, 308(6936), 1081±2. CHEVALLEY T, RIZZOLI R, NYDEGGER V, SLOSMAN D, RAPIN C H, MICHEL J, VASEY H and BONJOUR J P (1994), `Effects of calcium supplements on femoral bone mineral density and vertebral fracture rate in vitamin D replete elderly patients', Osteoporos Int, 4(5), 245±52. COMPSTON J (1993), `Osteoporosis'. In Campbell G, Compston J and Crisp A, The Management of Common Metabolic Bone Disorders, Cambridge: Cambridge University Press, 29±62. CONSENSUS DEVELOPMENT CONFERENCE (1993), `Diagnosis, prophylaxis and treatment of osteoporosis', Am J Med, 94(6), 646±50. COOPER C, ATKINSON E J, JACOBSEN S J, O'FALLON W M and MELTON L J 3rd (1993), `A population-based study of survival following osteoporotic fractures', Am J Epidemiol, 137 (9), 1001±5. COTTER A and K D CASHMAN (2003), `Genistein, a soy-based dietary phytoestrogen, appears to prevent early postmenopausal bone loss as effectively as hormone replacement therapy', Nutr Rev, 61(10), 346±51. CUSACK S and CASHMAN K D (2003), `Impact of genetic variation on metabolic response of bone to diet', Proc Nutr Soc, 62(4), 901±12. DAWSON-HUGHES B (1991), `Calcium supplementation and bone loss: a review of controlled clinical trials', Am J Clin Nutr, 54(1 suppl), 274S-80S. DAWSON-HUGHES B (1996), `Calcium insufficiency and fracture risk', Osteoporos Int, 6(Suppl 3), S37±41. DAWSON-HUGHES B, HARRIS S S and FINNERAN S (1995), `Calcium absorption on high and low calcium intakes in relation to vitamin D receptor genotype', J Clin Endocrinol Metab, 80(12), 3657±61. DAWSON-HUGHES B., FOWLER S E, DALSKY G and GALLAGHER C (1996), `Sodium excretion influences calcium homeostasis in elderly men and women,' J Nutr, 126(9), 2107± 12. DAWSON-HUGHES B., HARRIS S S, KRALL E A and DALLAL G E (1997), `Effect of calcium and vitamin B supplementation on bone density in men and women 65 years of age or older', N Engl J Med., 337(10), 670±6. DEPARTMENT OF HEALTH (1991) `Calcium', In Dietary Reference Values for Food Energy and Nutrients for the United Kingdom. Report in Health and Social Subjects no. 41. pp. 150±7, London: H.M. Stationery Office. DEPARTMENT OF HEALTH (1998), Nutrition and Bone Health with Reference to Calcium and Vitamin D. Dietary Reference Values for Food Energy and Nutrients for the United Kingdom. Report in Health and Social Subjects no. 49. London: H.M. Stationery Office. DEVINE A, CRIDDLE R A, DICK I M, KERR D A and PRINCE R L (1995), `A longitudinal study of the effect of sodium and calcium intakes on regional bone density in postmenopausal women', Am J Clin Nutr, 62(4), 740±5. DIMAI H P, PORTA S, WIRNSBERGER G, LINDSCHINGER M, PAMPERL I, DOBNIG H, WILDERS-

and LAU K H. (1998), `Daily oral magnesium supplementation suppresses bone turnover in young adult males', J Clin Endocrinol Metab, 83(8), 2742±8. DOYLE L, FLYNN A and CASHMAN K (1999), `The effect of magnesium supplementation on biochemical markers of bone metabolism or blood pressure in healthy young adult females', Eur J Clin Nutr, 53(4) 255±61. TRUSCHNIG M

Diet and control of osteoporosis

109

ELFFORS I, ALLANDER E, KANIS J A, GULLBERG B, JOHNELL O, DEQUEKER J, DILSEN G, GENNARI

and LYRITIS G (1994), `The variable incidence of hip fracture in Southern Europe; The Medos study', Osteoporos Int, 4(5), 253±63. EUROPEAN COMMISSION (1998), Report on osteoporosis in the European Community: Action for prevention, Office for Official Publications for the European Commission, Luxembourg. EVANS C E L, CHUGHTAI A Y, BLUMSOHN A, GILES M and EASTELL R (1997), `The effect of dietary sodium on calcium metabolism in premenopausal and postmenopausal women', Eur J Clin Nutr, 51(6), 394±9. FERRARI S, RIZZOLI R, CHEVALLEY T, SLOSMAN D, EISMAN J A and BONJOUR J P (1995), `Vitamin D receptor gene polymorphisms and change in lumbar spine bone mineral density', Lancet, 345(8947), 423±4. FERRARI S, RIZZOLI R, MANEN D, SLOSMAN D and BONJOUR J P (1998), `Vitamin D receptor gene start codon polymorphisms (FokI) and bone mineral density: Interaction with age, dietary calcium, and 30 -end region polymorphisms', J Bone Miner Res, 13(6), 925±30. FESKANICH D, SINGH V, WILLETT W C and COLDITZ G A (2002), `Vitamin A intake and hip fractures among postmenopausal women', JAMA 287(1), 47±54. FESKANICH D, WEBER P, WILLETT W C, ROCKETT H, BOOTH S L and COLDITZ G A (1999), `Vitamin K intake and hip fractures in women: a prospective study' Am J Clin Nutr 69(1), 74±9. FRANCIS R M, HARRINGTON F, TURNER E, PAPIHA S S and DATTA H K (1997), `Vitamin D receptor gene polymorphism in men and its effect on bone density and calcium absorption', Clin Endocrinol (Oxf), 46(1), 83±6. FRASSETTO L A, TODD K M., MORRIS R C, JR and SEBASTIAN A (2000), `Worldwide incidence of hip fracture in elderly women: relation to consumption of animal and vegetable foods', J Gerontol 55A (10), M585±M592. FREUDENHEIM J L, JOHNSON N E and SMITH E L (1986), `Relationships between usual nutrient intake and bone-mineral content of women 35±65 years of age: Longitudinal and cross-sectional analysis', Am J Clin Nutr, 44(6), 863±76. C, LOPES VAZ A A

GARNERO P, HAUSHERR E, CHAPUY M C, MARCELLI C, GRANDJEAN H, MULLER C, CORMIER C,

and DELMAS P D (1996a), `Markers of bone resorption predict hip fracture in elderly women: the EPIDOS Prospective Study', J Bone Miner Res, 11(10), 1531±8. GARNERO P, BOREL O, SORNAY-RENDU E, ARLOT M E and DELMAS P D (1996b), `Vitamin D receptor gene polymorphisms are not related to bone turnover, rate of bone loss, and bone mass in postmenopausal women: The OFELY Study', J Bone Miner Res, 11(6), 827±34. GARNERO, P, SORNAY-RENDU, E, CLAUSTRAT, B and DELMAS, PD (2000), `Biochemical markers of bone turnover, endogenous hormones and the risk of fractures in postmenopausal women: the OFELY study', J Bone Miner Res, 15(8), 1526±36. GINTY F, FLYNN A and CASHMAN KD (1998), `The effect of short-term calcium supplementation on biochemical markers of bone metabolism in healthy young adults', Br J Nutr, 80(5), 437±43. GRAAFMANS W C, LIPS P, OOMS M E, VAN LEEUWEN J P, POLS H A and UITTERLINDEN A G (1997), `The effect of vitamin D supplementation on the bone mineral density of the femoral neck is associated with vitamin D receptor genotype' J Bone Miner Res 12(8), 203±5. GREENDALE G A, BARRETT-CONNOR E, EDELSTEIN S, INGLES S and HAILE R (1994), `Dietary BREART G, MEURIER P J

110

Functional foods, ageing and degenerative disease

sodium and bone mineral density: Results of a 16±year follow-up study', J Am Geriatr Soc, 42(10), 1050±5. GUEGUEN R, JOUANNY P, GUILLEMIN F, KNUTZ C, POUREL J and SISET G (1995), `Segregation analysis and variance components analysis on bone mineral density in healthy families', J Bone Miner Res, 10(12), 2017±22. HALL S L and GREENDALE G A (1998), `The relation of dietary vitamin C intake to bone mineral density: Results from the PEPI study', Calcif Tissue International, 63(3), 183±9. HAN K O, MOON I G, KANG Y S, CHUNG H Y, MIN H K and HAN I K (1997), `Non association of estrogen receptor genotypes with bone mineral density and estrogen responsiveness to hormone replacement therapy in Korean postmenopausal women', Journal of Clinical Endocrinology and Metabolism, 82(4), 991±5. HANSEN M A, OVERGAARD K, RIIS B J and CHRISTIANSEN C (1991), `Role of peak bone mass and bone loss in postmenopausal osteoporosis: 12 year study', BMJ, 303(6808), 961±4. HARRINGTON M and CASHMAN K D (2003), `High salt intake appears to increase bone resorption in postmenopausal women but high potassium intake ameliorates this adverse effect', Nutr Rev, 61(5) 179±83. HARRINGTON M, BENNETT T, JACOBSEN J, BROT C, OVENSEN L, FLYNN A and CASHMAN K D (2004), `The effect of a high-protein, high salt diet on calcium and bone metabolism in postmenopausal women and its interaction with vitamin D receptor genotype' British Journal of Nutrition, 91(1), 41±51. HEANEY R P and RECKER R R (1987), `Calcium supplements: anion effects', Bone Miner, 2(6), 4339. HOLICK M F (1994), `McCollum Award Lecture, 1994: Vitamin D: new horizons for the 21st century', Am J Clin Nutr, 60(4), 619±30. HOLICK M F (1995), `Environmental factors that influence the cutaneous production of vitamin D', Am J Clin Nutr, 61(3 Suppl), 638S-45S. INSTITUTE OF MEDICINE (1997), Food and Nutrition Board. Dietary Reference Intakes: Calcium, Magnesium, Phosphorus, Vitamin D, and Fluoride. Washington, DC, National Academy Press. INSTITUTE OF MEDICINE (2001), Dietary reference intakes for vitamin A, vitamin K, arsenic, boron, chromium, copper, iodine, iron, manganese, molybdenum, nickel, silicon, vanadium and zinc. Washington, DC: National Academy Press. JOHNELL O, GULLBERG B, ALLANDER E and KANIS J A (1992), `The apparent incidence of hip fracture in Europe: A study of national register sources', Osteoporos Int, 2(6), 298±302. JOHNSTON C C JR, MILLER J Z, SLEMENDA C W, REISTER T K, HUI S, CHRISTIAN J C and PEACOCK M (1992), `Calcium supplementation and increases in bone mineral density in children', N Engl J Med, 327(2), 82±7. JONES G, BEARD T, PARAMESWARAN V, GREENAWAY T and VON WITT R (1997), `A population-based study of the relationship between salt intake, bone resorption and bone mass', Eur J Clin Nutr, 5(18), 561±5. JONES G, RILEY M D and WHITING S (2001), `Association between urinary potassium, urinary sodium, current diet, and bone density in prepubertal children', Am J Clin Nutr, 73(4), 839±44. JORGENSEN H L, MADSEN J S, MADSEN B, SALEH M M, ABRAHAMSEN B, FENGER M and LAURITZEN J B (2002), `Association of a common allelic polymorphism (C677T) in the methylene tetrahydrofolate reductase gene with a reduced risk of osteoporotic

Diet and control of osteoporosis

111

fractures. A case control study in Danish postmenopausal women', Calcif Tissue International, 71(5), 386±92. KANDERS B, DEMPSTER D W and LINDSAY R (1988), `Interaction of calcium nutrition and physical activity on bone mass in young women', Journal of Bone Mineral Research, 3(2), 145±9. KERSTETTER J E, O'BRIEN K O and INSOGNA K L (2003), `Low protein intake: the impact on calcium and bone homeostasis in humans', J Nutr, 133(3), 855S-61S. KIEL D P, MYERS R H, CUPPLES L A, KONG X F, ZHU X H, ORDOVAS J, SCHAEFER E J, FELSON D T, RUSH D, WILSON P W F, EISMAN J A and HOLICK M F (1997), `The BsmI vitamin D receptor restriction fragment length polymorphism (bb) influences the effect of calcium intake on bone mineral density', J Bone Miner Res, 12(7), 1049±57. KINYAMU H K, GALLAGHER J C, KNEZETIC J A, DELUCA H F, PRAHL J M and LANSPA S J (1997), `Effect of vitamin D receptor genotypes on calcium absorption, duodenal vitamin D receptor concentration and serum 1,25 dihydroxyvitamin D levels in normal women', Calcified Tissue International, 60(6), 491±5. KRALL E A, PARRY P, LICHTER J B and DAWSON-HUGHES B (1995), `Vitamin D receptor alleles and rates of bone loss: influence of years since menopause and calcium intake'. J Bone Miner Res 10(6), 978±84. KRUGER M C and HORRBIN D F (1997), `Calcium metabolism, osteoporosis and essential fatty acids: a review', Prog Lipid Res, 36(2±3), 131±51. KRUGER M C, COETZER H, DE WINTER R, GERICKE G and PAPENDORP D H (1998), `Calcium, gamma-linolenic acid and eicosapentaenoic acid supplement in senile osteoporosis', Aging, 10(5), 385±94. LEITZ G, AVENELL A and ROBINS S P (1997), `Short-term effects of dietary sodium intake on bone metabolism in postmenopausal women measured using urinary deoxypyridinoline excretion', Br J Nutr, 78(1), 73±82. LEMANN JR J, PLEUSS J A, GRAY R W and HOFFMANN R G (1991), `Potassium administration reduces and potassium deprivation increases urinary calcium excretion in healthy adults', Kidney Int, 39(5), 973±83. LIN P, GINTY F, APPEL L J, AICKIN M, BOHANNON A, GARNERO P, BARCLAY D, and SVETKEY L P (2003), `The DASH diet and sodium reduction improve markers of bone turnover and calcium metabolism in adults', Journal of Nutrition, 133, 3130±6. MACKERRAS D and LUMLEY T (1997), `First- and second-year effects in trials of calcium supplementation on the loss of bone density in postmenopausal women', Bone, 21(6), 527±33. MASSEY L K (2003), `Dietary animal and plant protein and human bone health: a whole foods approach'. J Nutr, 133(3), 862S±5S. MASSEY L K and WHITING S J (1996), `Dietary salt, urinary calcium and bone loss', J Bone Min Res, 11(6), 731±6. MATKOVIC V, ILICH J Z, ANDON M B, HSIEH L C, TZAGOURNIS M A, LAGGER B J and GOEL P K (1995), `Urinary calcium, sodium, and bone mass of young females', Am J Clin Nutr, 62(2), 417±25. MELHUS H, MICHAEÈLSSON K, KINDMARK A, BERGSTROM R, HOLMBERG L, MALLMIN H, WOLK A

and LJUNGHALL S (1998), `Excessive dietary intake of vitamin A is associated with reduced bone mineral density and increased risk for hip fracture', Ann Intern Med, 129(10), 770±8. MELTON L J, CHRISCHILLES E A, COOPER C, LANE A W and RIGGS B L (1992), `How many women have osteoporosis?', Journal of Bone Mineral Research, 7(9), 1005±10. MICHAEÈLSSON K, LITHELL H, VESSBY B and MELHUS H (2003), `Serum retinol levels and the

112

Functional foods, ageing and degenerative disease risk of fracture', N Engl J Med, 348 287±94.

and (2000), `Association of methylenetetrahydrofolate reductase (MTHFR) polymorphism with bone mineral density in postmenopausal Japanese women', Calcif Tissue International, 66(3), 190±4. MORRIS R C, SEBASTIAN A, FORMAN A, TANAKA M and SCHMIDLIN O (1999), `Normotensive saltsensitivity: effects of race and dietary potassium', Hypertension, 33(1), 18±23. MORRISON N A, QI J C, TOKITA A, KELLY P J, CROFTS L, NGUYEN T V, SAMBROOK P N and EISMAN J A (1994), `Predicators of bone density from vitamin D receptor alleles', Nature, 367(6460), 284±7. MORTON D J, BARRETT-CONNOR E L and SCHNEIDER D L (1997), `Vitamin C supplement use and bone mineral density in older women: The Rancho Bernado Study', J Bone Miner Res, 12(Suppl), 283. NATIONAL INSTITUTES OF HEALTH (1994), Optimal Calcium Intake. NIH Consensus Statement 12:4. Bethesda, MD: NIH. NATIONAL INSTITUTES OF HEALTH (2000), Osteoporosis Prevention, Diagnosis, and Therapy. NIH Consensus Development Conference Statement. http://odp.od.nih.gov/consensus/cons/111/111_statement.htm NATIONAL RESEARCH COUNCIL (1989), Recommended Dietary Allowances: 10th edition. Report of the Subcommittee on the Tenth Edition of the RDA. Food and Nutrition Board and the Commission on Life Sciences. Washington, DC: National Academy Press. NEW S A, BOLTON-SMITH C, GRUBB D A and REID D M (1997), `Nutritional influences on bone mineral density: a cross-sectional study in premenopausal women', Am J Clin Nutr, 65(6), 1831±9. MIYAO M, MORITA H, HOSOI T, KURIHARA H, INOUE S, HOSHINO S, SHIRAKI M, YAZAKI Y OUCHI Y

NEW S A, ROBINS S P, CAMPBELL M K, MARTIB J C, GARTON M J, BOLTON-SMITH C, GRUBB D A,

and REID D M (2000), `Dietary influences on bone mass and bone metabolism: further evidence of a possible link between fruit and vegetable consumption and bone health', Am J Clin Nutr, 71 (1), 142±51. NORDIN B E C (1997), `Calcium in health and disease', Food, Nutrition and Agriculture, No. 20, 13±26. NORDIN B E C and POLLEY KJ (1987), `Metabolic consequences of the menopause: a crosssectional, longitudinal and intervention study on 557 normal postmenopausal women', Calcified Tissue International, 41 (Suppl. 1), S1±59. NORDIN B E C, NEED A G, MORRIS H A and HOROWITZ M (1993), `The nature and significance of the relationship between urinary sodium and urinary calcium in women', J Nutr, 123 (9), 1615±22. NORMAN A W (1990), `Intestinal calcium absorption: a vitamin D hormone mediated adaptive response', Am J Clin Nutr. 51(2), 290±300. LEE S J

ONGPHIPHADHANAKUL B, CHANPRASERTYOTHIN S, PAYATIKUL P, TUNG S S, PIASEU N,

CHAILURKIT L, CHANSIRIKARN S, PUAVILAI G and RAJATANAVIN R. (2000), `Oestrogen receptor-alpha gene polymorphism affects response in bone mineral density to oestrogen in post-menopausal women', Clinical Endocrinology, 52(5), 581±5. PORTALE A A, HALLORAN B P, MURPHY M M and MORRIS R C JR (1986), `Oral intake of phosphorus can determine the serum concentration of 1,25±dihydroxyvitamin D by determining its production rate in humans', J Clin Invest, 77(1), 7±12. PRENTICE A (1997), `Is nutrition important in osteoporosis?', Proceedings of the Nutrition Society, 56(1B), 357±67. PROMISLOW J H E, GOODMAN-GRUEN D, SLYMEN D J and BARRETT-CONNER E (2002), `Retinol

Diet and control of osteoporosis

113

intake and bone mineral density in the elderly: the Rancho Bernardo Study', J Bone Miner Res, 17(8), 1349±58. RAPURI P B, GALLAGHER J C, KINYAMU H K and RYSCHON K L (2001), `Caffeine intake increases the rate of bone loss in elderly women and interacts with vitamin D receptor genotypes', Am J Clin Nutr, 74(5), 694±700. REGINSTER J Y, STRAUSE L, DERIOSY R, LECART M P, SALTMAN P and FRANCHMONT P (1989), `Preliminary report of decreased serum magnesium in postmenopausal osteoporosis', Magnesium, 8(2), 106±9. REID D M and NEW S A (1997), `Nutritional influences on bone mass', Proceedings of the Nutrition Society, 56(3), 977±87. RIGGS B L (1988), `Osteoporosis'. In Smith L H and Wyngaarden J B, Cecil textbook of medicine. Philadelphia: WB Saunders Co., 1510±5. RUDE R K (1998), `Magnesium deficiency: a cause of heterogeneous disease in humans', J Bone Min Res, 13(4), 749±58. SALAMONE L M, GLYNN N W, BLACK D M, FERRELL R E, PALERMO L, EPSTEIN R S, KULLER L H

and CAULEY J A (1996), `Determinants of premenopausal bone mineral density: The interplay of genetic and lifestyle factors', J Bone Miner Res 11 (10), 1557±65.

SALMEN T, HEIKKINEN A M, MAHONEN A, KROGER H, KOMULAINEN M, SAARIKOSKI S, HONKANEN R and MAENPAA P H (2000), `Early postmenopausal bone loss is associated with Pvu II estrogen receptor gene polymorphism in Finnish women: effect of hormone replacement therapy', J Bone Miner Res, 15(2), 31521. SAUPE J, SHEARER M J and KOHLMEIER M (1993), `Phylloquinone transport and its influence on y-carboxylation residues of osteocalcin in patients on maintenance haemodialysis', Am J Clin Nutr, 58(2), 204±8. SAX L (2001), `The institute of medicine's ``dietary reference intake'' for phosphorus: a critical perspective', J Am Coll Nutr, 20(4), 271±8. SCHURCH M, RIZZOLI R, MERMILLOD B, VASEY H, MICHEL J and BONJOUR J (1996), `A prospective study on socioeconomic aspects of fracture of the proximal femur', J Bone Miner Res, 11(12), 1935±42. SELLMEYER D E, SCHLOETTER M and SEBASTIAN A (2002), `Potassium citrate prevents increased urine calcium excretion and bone resorption induced by a high sodium chloride diet', J Clin Endocrinol Metab, 87(5), 2008±12. SHEARER M J (2000), `Role of vitamin K and Gla proteins in the pathophysiology of osteoporosis and vascular calcification', Curr Opin Clin Nutr Metab Care, 3 (6), 433±8. SHIEKH M, RAMIREZ A, EMMETT M, SANTA ANA C and FORDTRAN J (1988), `Role of vitamin D dependent and vitamin D independent mechanisms in absorption of food calcium', J Clin Investig, 81(1), 126±32. SHORTT C and FLYNN A (1990), `Sodium-calcium inter-relationships with specific reference to osteoporosis', Nutr Res Rev, 3, 101±15. SILVERBERG S J, SHANE E, CLEMENS T L, DEMPSTER D W, SEGRE G V, LINDSAY R and BILEZIKIAN J P (1986), `The effect of oral phosphate administration on major indices of skeletal metabolism in normal subjects', J Bone Miner Res, 1(4), 383±8. SOKOLL L J and SADOWSKI J A (1996), `Comparison of biochemical indexes for assessing vitamin K status in a healthy adult population' Am J Clin Nut, 63(4), 566±73. STENDIG-LINDBERG G, TEPPER R and LEICHTER I (1993), `Trabecular bone density in a two year controlled trial of peroral magnesium in osteoporosis', Magnes Res, 6(2), 155±63. SZULC P, CHAPUY M C, MEUNIER P J and DELMAS P D (1993), `Serum undercarboxylated

114

Functional foods, ageing and degenerative disease

osteocalcin is a marker of the risk of hip fracture in elderly women', J Clin Invest, 91(4), 1769±74. SZULC P, ARLOT M, CHAPUY M C, DUBOEUF F, MUENIER P J and DELMAS P D (1994) `Serum undercarboxylated osteocalcin correlates with hip bone mineral density in elderly women` J Bone Miner Res, 9(10), 1591±5. TEEGARDEN D, PROULX W R, MARTIN B R, ZHAO J, MCCABE G P, LYLE R M, PEACOCK M,

and WEAVER C M (1995), `Peak bone mass in young women', Journal of Bone Mineral Research, 10(5), 711±15. TUCKER K L, HANNAN M T, CHEN H, CUPPLES L A, WILSON P W and KIEL D P (1999), `Potassium, magnesium, and fruit and vegetable intakes are associated with greater bone mineral density in elderly men and women', Am J Clin Nutr, 69(4), 727±36. SLEMENDA C, JOHNSTON C C JR

È LIMA È KI M J, KA È RKKA È INEN M, LAMBERG-ALLARDT C, LAITINEN K, ALHAVA E, HEIKKINEN J, VA

and SEPPANEN R (1994), `Exercise, smoking and calcium intake during adolescence and early adulthood as determinants of peak bone mass', BMJ, 309(6949), 230±5. VAN DOKKUM W, CLOUGHLEY F A, HULSHOF K F and OOSTERVEEN L A (1983), `Effect of variations in fat and linoleic acid intake on the calcium, magnesium and iron balance of young men' Ann Nutr Metab, 27(5), 361±9. VERGNAUD P, GARNERO P, MEUNIER P J, BREART G, KAMIHAGI K and DELMAS P D (1997), `Undercarboxylated osteocalcin measured with a specific immunoassay predicts hip fracture in elderly women: the EPIDOS study', J Clin Endocrinol Metab, 82(3), 719±24. VERMEER C, JIE K-S G and KNAPEN M H J (1995), `Role of vitamin K in bone metabolism', Ann Rev Nutr, 15, 1±22. VERMEER C, GIJSBERS B L M G, CRACIUN A M, GROENEN-VAN DOOREN M M C L and KNAPEN M H J (1996), `Effects of vitamin K on bone mass and bone metabolism', J Nutr, 126(4 Suppl.), 1187S±91S. WATTS N B (1999), `Clinical utility of biochemical markers of bone remodeling', Clin Chem, 45(8), 1359±68. WEAVER C M (2000), `The growing years and prevention of osteoporosis in later life', Proc Nutr Soc, 59(2), 303±6. WEBB, A R, KLINE, L W and HOLICK, M F (1988), `Influence of season and latitude on the cutaneous synthesis of vitamin D3: exposure to winter sunlight in Boston and Edmonton will not promote vitamin D3 synthesis in human skin', J Clin Endocrinol Metab, 67(2), 373±8. WEBER P (2001), `Vitamin K and bone health', Nutr, 17(10), 880±7. WELTEN D, KEMPER H C G, POST G B and VAN STAVEREN W (1995), `A meta-analysis of the effect of calcium and bone mass in young and middle aged females and males', J Nutr, 125(11), 2802±13. WISHART J M, HOROWITZ M, NEED A G, SCOPACASA F, MORRIS H A, CLIFTON P M and NORDIN C (1997), `Relations between calcium intake, calcitriol, polymorphisms of the vitamin D receptor gene, and calcium absorption in premenopausal women', Am J Clin Nutr, 65, 798±802. WOOD R J and FLEET J C (1998), `The genetics of osteoporosis: Vitamin D receptor polymorphisms', Ann Rev Nutr, 18, 233±58. WORLD HEALTH ORGANISATION (1994), Assessment of Fracture Risk and its Application to Screening for Postmenopausal Osteoporosis. Technical Report Series 843. Geneva, World Health Organisation. IMPIVAARA O, MAKELA P, PALMGREN J

5 Phytoestrogens and the control of osteoporosis S. Lorenzetti and F. Branca, Instituto Nazionale di Ricerca per gli Alimenti e la Nutrizione (INRAN), Italy

5.1

Introduction

Phytoestrogens are non-steroidal, diphenolic compounds of plant origin structurally similar to estradiol. Several classes of phytoestrogens are known and, despite their different structures, they have been shown to have both estrogenic and anti-estrogenic activities, depending on the concentrations of endogenous estrogens and on the tissue expression profile of estrogen receptors. However, the most widely studied compounds are isoflavones that have indeed stimulated the initial interest of scientists who observed a lower incidence of menopausal symptoms and of several chronic diseases in Asian women and postulated a possible role of soy isoflavones in preventing cardiovascular disease and osteoporosis in Western countries. In this chapter we are going to review epidemiological and human studies, as well as in vitro and animal studies, on the effect of phytoestrogens in comparison with other compounds currently used to prevent and to treat osteoporosis and other bone diseases characterised by an unbalanced bone resorption. Phytoestrogens are part of a larger group of non-steroidal compounds. The best known phytoestrogens are flavonoids (including isoflavones, flavonols, flavones, catechins, anthocyanidins), lignans, coumestans and resorcylic acid lactones. Flavonoids include thousands of molecules that are present mainly in the outer part of legumes and fruits and in leafy vegetables, whereas lignans and coumestans are also present in cereals and oilseeds as well as in fruits and vegetables. The estimation of phytoestrogen intake in human populations is based on a few molecules such as the two isoflavones, genistein and daidzein;

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the three flavonols, quercetin, myricetin, and kaempferol; and the two flavones, luteolin and apigenin. Clearly, this approach means the total intake of phytoestrogens is underestimated. Up to now, studies based on analytical methods of selected phytoestrogens have reported that isoflavone intake in Western countries (USA and Europe) reaches only about 1 mg/day (de Kleijn et al., 2001; van Erp-Baart et al., 2003), whereas flavonol intake (only quercetin and kaempferol) ranges between 1 and 81 mg/day (de Vries et al., 1997). In contrast, the isoflavone content of a typical Asian diet is about 50 mg/day (Chen et al., 1999; Lorenzetti and Branca, 2003), but intake can be up to 100 mg/day in the Hong-Kong Chinese population (Ho et al., 2000). If a more comprehensive estimate of phytoestrogen intake was made, values could probably be close to 1 g/day, as Kunhau (1976) calculated for the USA.

5.2

Osteoporosis: prevention and treatment

The internationally agreed definition of osteoporosis is `a systemic skeletal disease characterised by low bone mass and microarchitectural deterioration of bone tissue, with a consequent increase in bone fragility and susceptibility to fracture' (AJM, 1993). From an operational point of view, the World Health Organization (WHO Technical Report Series 843, 1994) indicated that the diagnosis of osteoporosis should be based on the measurement of bone mineral density (BMD), obtained by dual energy X-ray absorptiometry, compared to the values observed in healthy young women. A correlation has in fact been established between BMD and the prospective risk of fracture. Due to such a gradient of risk, the WHO panel of experts suggested that an individual with a Tscore of BMD measured at the hip below ÿ2.5 have osteoporosis. Osteoporosis is considered severe or established when one or more prior fragility fractures are present. When the BMD value has a T-score in between ÿ2.5 and ÿ1 an individual has osteopenia. Analysing the WHO definition for osteoporosis it is therefore apparent that the current method to diagnose osteoporosis does not take into account other aspects of bone quality which depend on bone microarchitecture and remodelling rates. Osteoporosis affects mainly white postmenopausal women reaching 50 years of age. 5.2.1 Prevention of osteoporosis Osteoporosis prevention can be achieved by maximising bone accretion during adolescence, so as to ensure the achievement of an optimal bone mass, and by reducing post-menopausal bone loss. For both purposes, regular weight-bearing physical activity (e.g. walking, jumping) as well as good nutrition is required. Nutrients with a demonstrable effect on bone health include calcium, that in postmenopausal women is required in the amount of 1,200 mg per day and vitamin D (5ÿ10 g/day). Other minerals (potassium, zinc, copper, manganese, boron) and vitamins K, C and B are also required, although there is no clear

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evidence as to their precise role and optimal intake. Post-menopausal women are also advised to reduce sodium intake, to increase consumption of fruits and vegetables, to maintain a healthy body weight, to avoid smoking and to limit alcohol intake (WHO, 2003). Fruits and vegetables are important as they are a good source of potassium, vitamin C and K and bioactive compounds with antioxidant and estrogenic action. 5.2.2 Treatment of osteoporosis Pharmacological treatment might reduce the risk of fractures in women with osteoporosis. Therapies accepted include hormone replacement therapy (HRT), calcitonin (CT), bisphosphonates (BPs) such as alendronate and risedronate, selective estrogen-receptor modulators (SERMs) such as raloxifene, and selective tissue estrogen activity regulators (STEARs) such as tibolone. Hormone replacement therapy (HRT) The role of estrogen deficiency in bone remodelling imbalance that occurs in post-menopausal women is well established. This is why hormone replacement therapy (HRT) has been so far considered the first option in post-menopausal women. HRT prevents bone loss in a dose-dependent manner and reduces the risk of fractures (Setchell and Lydeking-Olsen, 2003). The long-term administration of HRT has been challenged due to the increased risk of breast and endometrial cancers, despite estrogen formulations being associated to progestins (WHI, 2002; Lacey et al., 2002). Estrogen regulation of bone metabolism occurs at multiple levels. An indirect action, via the modulation of several hormones and cytokines known to influence bone density and calcium homeostasis, and a direct action, through estrogen receptors (ERs) (Riggs, 2000). Estrogens modulate both osteoclast formation and activity in terms of the number of formed cells, rate of apoptosis and ability to resorb bone (Riggs, 2000). Estrogen treatment inhibits bone loss and bone turnover mainly in early post-menopausal women and increases both spine and hip bone mineral density (Hosking et al., 1998). In late post-menopausal women the preventive role of estrogens is less pronounced and a recent five-year WHI mega-trial has also cast doubt on the efficiency of long-term HRT treatments in healthy postmenopausal women because the slight improvement in the reduction of osteoporotic (hip) fractures were concomitant with an unexpected higher risk of cardiovascular disease (WHI, 2002). Calcitonin Calcitonin (CT) is a 32 amino acid-long peptide synthesised by the C cells of the thyroid gland and originally discovered as a hypocalcemic factor. CT acts via its receptors (CTRs), mainly localised in bone and kidney (Warshawsky et al., 1980). CT has been shown to inhibit bone loss by acting directly on the osteoclast-mediated bone resorption via the regulation of cAMP-signalling pathway (Chambers and Magnus, 1982; Nicholson et al., 1986). Endogenous CT

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deficiency has been reported to favour post-menopausal osteoporosis (Taggart et al., 1982). Hence, salmon CT has been used to treat osteoporosis (Overgaard et al., 1992). Currently, even if widely used in the treatment of hypercalcemia as well as in osteoporosis and Paget's disease due to its ability to block rapidly osteoclast activity, CT has been proved to have more of a main role in acute, short-term treatments than in longer ones (Zaidi et al., 2002). Furthermore, the effective bioavailability of CT raises questions about its usefulness in bone disease treatments. The main limitation remains the CT-induced downregulation of CTRs transcription (Inoue et al., 1999) and hence the CT resistance (Rodan and Martin, 2000) or ligand-induced desensitisation (Schneider et al., 1993), a phenomenon well documented both in vitro and in vivo (Zaidi et al., 2002), and recently shown to take place also in postmenopausal women under CT treatment (Beaudreuil et al., 2000). Bisphosphonates Bisphosphonates (BPs) are a class of compounds, analogues of pyrophosphate, suggested to have have a strong effect on the skeleton because of their ability to interfere with calcification, mainly by inhibiting both formation and dissolution of calcium phosphate crystals (Fleisch et al., 2002). BPs are characterised by 2 carbon-phosphate bonds (P-C-P) but each BP has its own biological properties due the peculiar identity of the lateral chains. The ability of BPs to inhibit bone resorption has been demonstrated in normal animals as well as in experimental animal models of bone diseases including osteoporosis, tumour-induced bone resorption, tumour-induced hypercalcemia, and arthritis (Fleisch et al., 2002). Many BPs have been investigated in human bone diseases and their activities on bone resorption vary greatly. At present, only some of them are commercially available (alendronate, clodronate, etidronate, ibandronate, pamidronate, risedronate, tiludronate and zoledronate) and recognised as potent inhibitors of osteoclast-mediated bone resorption (Reid et al., 2002; Watts et al., 2003). BPs act preferentially on bone tissue since they target it with high affinity (Sato and Grasser, 1990). At low pharmacological doses, it has been shown that alendronate, the most powerful BP, binds preferentially the resorption surface under the osteoclast and not the newly formed bone (Sato et al., 1991), whereas at higher amounts, etidronate, a less powerful BP, binds equally the resorption and formation surfaces (Azuma et al., 1995). Overall, the most active BPs are the nitrogen-containing side chain (N-BPs) and it appears that the non-N-BPs have also a different mechanism of action. N-BPs might in fact act through the inhibition of a key enzyme in the mevalonate pathway, or cholesterol synthesis pathway, the farnesyl diphosphate (FPP) synthase (Rodan and Reszka, 2002). Interestingly, such a pathway is involved not only in the synthesis of cholesterol but also in protein prenylation since the mevalonate pathway produces also the lipid moieties (farnesyl-, geranyl- and geranylgeranyldiphosphates) of intracellular proteins, such as Ras and the Rab protein family, essential for vesicular transport. Recently, alendronate has been shown to impair intracellular vesicular transport and to cause the accumulation within the

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osteoclast of tartrate-resistant acid phosphatase, a key effector of bone resorption (Alakangas et al., 2002). N-BPs might also decrease bone resorption by inhibiting the activity of matrix metalloproteinase (MMPs), namely matrix degradation and osteoclast migration/podosome disassembly, as indicated by the ability of different BPs (including also some non-N-BPs) to inhibit the proteolytic activity of many MMPs (Teronen et al., 1999; Goto et al., 2002) and to impair migration and invasiveness of bone tumorigenic cells (Teronen et al., 1999). Finally, N-BPs such as zoledronate and incadronate act by inhibiting angiogenesis (Wood et al., 2002; Okamoto et al., 2002). The non-N-BPs appear to act differently from the N-BPs. They are metabolised within the cell to form toxic analogues of adenosine triphosphate (ATP) and hence could impair many intracellular metabolic processes. Clodronate, for instance, has been shown to induce apoptosis both in osteoclast and macrophage cells once it is transformed in a nonhydrolyzable ATP analogue (Frith et al., 1997). Although BPs are generally the most potent drugs actually known to inhibit bone resorption ± and as such the most widely used in the treatment of osteoporosis ± the main complaint reported in their therapeutical use is about upper gastrointestinal disturbances (Fleisch, 2003). BPs have been shown to reduce the risk of hip fracture in large randomised trials. Risedronate (5 mg/day) has been shown to significantly reduce the risk of vertebral fractures in established osteoporosis in one year in randomised, controlled clinical trials (Harris et al., 1999; Fogelman et al., 2000; Reginster et al., 2000; Reid et al., 2000; McClung et al., 2001). Alendronate (5ÿ10 mg/day) has been also shown to increase BMD in both early post-menopausal women and in those with established osteoporosis in at least 11 different clinical trials and to reduce significantly the vertebral fracture rate over 2±3 years of treatment (Cranney et al., 2002). Selective estrogen-receptor modulators (SERMs) Besides the known estrogens' beneficial effects in early post-menopausal women (see above in 5.2.2) as well as in peri-menopausal women (maintenance of the thickness and elasticity of skin, of the vagina and perineal connective tissue and the prevention of hot flushes), some side effects suggest limiting estrogen use to a short-time period. Vaginal bleeding, breast tenderness, a general fear and anxiety of developing cancer (mainly breast) and the rare but serious possibility of a thromboembolic event, limit the long-term use of estrogens. Since the SERMs, originally referred to as antiestrogens, have been shown to act as full or partial agonist/antagonist of estrogens through the estrogen receptors (ERs) high affinity binding (see section 5.3) (Love et al., 1992), they have raised great interest for long-term employment by virtue of their tissue-selective role. Indeed, tamoxifen (the first identified SERM) has been shown to prevent bone loss (Love et al., 1992) without having a negative effect on breast cancer but having a positive effect on uterine cancers due to its uterotrophic effect. The following generation of SERMs include also raloxifene, a benzothiophene molecule identified about 20 years ago, whose promising role

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in preventing E2 deficiency-induced bone loss was shown in post-menopausal women, in a clinical trial showing about 40% reduction in the relative risk of vertebral fractures without negative effects in both breast and uterine cancers (Delmas et al., 1997; Ettinger et al., 1999; Cummings et al., 1999). The main concern about raloxifene remains its inability to improve non-vertebral and hip fracture risk (Ettinger, 2003). Selective tissue estrogen action regulators Tibolone is a synthetic estrogen that has estrogenic, androgenic and progestogenic action, according to the level that its different metabolites can reach in different tissues (Kloosterboer, 2001). For such properties, it has been defined a selective tissue estrogen action regulator (STEAR). Several randomised clinical trials (RCT) have demonstrated that tibolone decreases bone turnover and significantly improves BMD, especially trabecular BMD. At the same time, this molecule can have positive clinical effects on vagina and brain, may have beneficial, androgenic effects on sexual function and avoids stimulation of the endometrium and breast tissue. However, no data about the effects of tibolone on fracture risk are yet available. (Modelska and Cummings, 2002).

5.3 Mechanisms of action of phytoestrogens in bone metabolism Two different ERs, members of the nuclear receptors transcription factors, have been identified and characterised as ER- and ER- (Kuiper et al., 1996; Mosselman et al., 1996; Tremblay et al., 1997). They differ in their ligandbinding properties and transactivation activities and both of them have been shown to possess different isoforms (Kuiper et al., 1997; De Lisle et al., 2001; Lewandowski et al., 2002). Different tissues express a different proportion of the two ERs, although in reproductive tissues it appears to exert an almost complete dominance of either one of the two known receptors (Batra et al., 2003). In bone, ERs are present and co-expressed in both cell types, even if it is still a matter of discussion whether they are present at all stages of osteoblastogenesis and osteoclastogenesis (Batra et al., 2003). The role of either ER is described by the outcome of several studies on bone cells and in vitro, in mice whose ERs were activated by gene deletion (knock-out mice). ER expression is specifically required for bone remodelling in the male skeleton, whereas both ER- and - appear to be involved in the female one (Vidal et al., 2000; Lindberg et al., 2001a; Sims et al., 2002). Only ER- seems to modulate the inverted ratio (OPG/RANKL ratio) of the osteoclast differentiation factor RANKL and of its direct inhibitor, the decoy receptor OPG, as well as to regulate the osteoclast differentiation marker TRAP5b and the serum levels of IL-6 (Lindberg et al., 2001b). The bone anabolic response to mechanical loading requires functional ER- (Lee et al., 2003). ER- expression is greatly increased during bone mineralisation in an osteoblast cell line (Arts et al., 1997). ER-

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might be responsible for an overall reduction of the ER- regulated gene transcription in bone and such a function seems to occur in the presence of the ER- whereas in its absence, ER- can partially replace it (Lindberg et al., 2003). Up to now this inhibitory role is the only function solely attributed to the ER- (Couse and Korach, 1999). The relative importance of ER- and ER- in bone metabolism is crucial to understand the function of phytoestrogens since the selectivity difference in ERs binding might give a difference in the strength of action, more than in the specific role of each compound. Phytoestrogens bind estrogen receptors (ERs), triggering their so-called classical, or genomic, action, although with a different receptor binding affinity. Genistein is one of the strongest ligands for both ER- and ER- (even if affinity is much lower compared to E2) and possesses a higher affinity (about 25-fold more) for ER- than for ER- (Kuiper et al., 1997; Kuiper et al., 1998). Binding of compounds with estrogen-like activities to ERs triggers a change in the transcriptional status of ER-regulated genes, that is obviously not the same in all tissues due to the differential tissue distribution of the receptors and to their possibility to form both types of homodimers and even to heterodimerise, as has been shown both in vivo and in vitro (Ogawa et al., 1998). Moreover, an additional level of complexity is obviously due to the contemporary action of dozens of interacting proteins (co-repressors and coactivators) acting in a transcriptional complex in their respective cellular context (Pettersson and Gustafsson, 2001). Genistein has been shown to possess a wide array of ER-mediated biological actions (Barnes et al., 2000a; Polkowski and Mazurek, 2000; Dixon and Ferreira, 2002). Genistein shares structural features with natural estrogens and with the SERM tamoxifen. Genistein binds to estrogen receptors and sex hormone binding proteins. Hence, it can exert both estrogenic and antiestrogenic activity by either cooperating or competing for receptor binding by estradiol. Moreover, a regulative role of genistein on estrogen and testosterone availability to target cells might be obtained by competition for the human sex steroid binding proteins by estrogens and androgens (Dixon and Ferreira, 2002). Besides the ER-mediated role, the action of genistein and other phytoestrogens might be mediated through non-ER mechanisms, inhibition of different classes of proteins such as protein tyrosine kinases, DNA topoisomerases, and ribosomal S6 kinase (Akiyama et al., 1987; Kaufmann 1989; Linassier et al., 1990). Non-ER mediated genistein roles might be due to modulation of transcriptional processes (Barnes et al., 2000b) or based on the unique property of genistein to bind proteins in their NTP-binding pocket (Lorenzetti and Branca, 2003). The discovery of a ligand-independent activation of ER- upon cross-talk with tyrosine kinase receptors (RTKs)-mediated signalling (Hall et al., 2001) broadened the research on ERs toward alternative modes of action that led also to the recognition of a direct involvement of several signal transduction pathways in the fate regulation of bone cell precursors (Hotokezaka et al., 2002), and to the working hypothesis that at least some of the estrogen bone protective effects might be due to nongenomic actions (Kousteni et al., 2001).

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5.4

Phytoestrogen action on bone cells

Several in vitro studies have shown that phytoestrogens might affect both bone cell types and, by doing so, modulate both bone formation and bone resorption. Genistein and daidzein have been shown to have an anabolic effect in the osteoblastic cell line, MC3T3-E1 (Gao and Yamaguchi, 1999a), via a general increase in protein synthesis (Yamaguchi and Sugimoto, 2000; Sugimoto and Yamaguchi 2000a, b). Moreover, in primary cell cultures obtained from rat metaphyseal tissue (Yamaguchi and Gao, 1999), genistein has been shown to increase alkaline phosphatase (ALP) activity as well as DNA and calcium content of the bone tissue. A significant elevation of protein and DNA content as well as of ALP activity have been shown to occur in an ER-depending manner in the MC3T3-E1 cell line for both genistein and daidzein (Sugimoto and Yamaguchi 2000a, b). A direct stimulatory effect on the same osteoblastic cell line has been shown also for resveratrol, a stilbene found mainly in grape skins and in red wine. The trans form of resveratrol has been demonstrated to promote osteoblastogenesis by a dose-dependent increase in DNA synthesis and ALP activity. Such effects are thought to be ER-mediated since they are blocked by tamoxifen (Mizutani et al., 1998). Resveratrol has been shown to support an increase of the prolyl hydroxylase activity, thus stimulating collagen synthesis, to inhibit prostaglandin E2 production, thus inhibiting osteoclast differentiation, and to induce bone mineralisation (Morita et al., 1992). Resveratrol has been shown to prevent OVX-induced decreases in femoral bone strength (Mizutani et al., 2000), and to antagonise the deleterious dioxin effects on bone formation in stromal bone marrow cells (Singh et al., 2000). Dioxin is one of the products of cigarette smoking and the number of smoked cigarettes per day is related to the decrease in bone mineral density in a dose-dependent manner (Hollenbach et al., 1993; Franceschi et al., 1996) and is associated with increased fracture risk (Hollenbach et al., 1993; Grisso et al., 1997). Furthermore, dioxin, as with many environmental hydrocarbons, is an aryl hydrocarbon receptor (AhR) ligand and resveratrol has been shown to compete (Ciolino et al., 1998; Casper et al., 1999) for the AhR ligand, behaving like a partial or full antagonist (Singh et al., 2000). In osteoclast-like cells, different phytoestrogens have been shown to inhibit osteoclast differentiation. The suppression of osteoclastogenesis by genistein has been shown to occur via an overall inhibition of protein kinases associated to a general activation of protein tyrosine phosphatases (Gao and Yamaguchi, 2000), but also through the inactivation of the cAMP signalling pathway (Gao and Yamaguchi, 1999b), a common positive signalling for the osteoclastogenic stimuli of different hormones and cytokines affecting bone metabolism. Furthermore, it has been demonstrated that genistein affects osteoclast activity by inducing apoptosis. The reduction of the apoptosis rate in osteoclasts is one of the known mechanisms by which some cytokines, growth factors and hormones positively regulate physiological osteoclast activity (Riggs, 2000). Significantly, estrogens and bisphosphonates partially act via the induction of

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osteoclast apoptosis (Hughes et al., 1995; Hughes et al., 1996). In rat primary cultures of osteoclasts, genistein has been shown to increase apoptotic events via the induction of an intracellular calcium signalling pathway, in a manner similar to calcitonin (Gao and Yamaguchi, 1999c). Flavonols, another subfamily of flavonoids, commonly found in tea, onions and other vegetables, have also been shown to affect bone resorption via the induction of apoptosis in osteoclast cells. Quercetin and kaempferol, two of the most widely distributed flavonols in the human diet, have been shown to induce apoptosis and to potently reduce bone resorption in purified primary cultures of rabbit osteoclasts (Wattel et al., 2003). In such a system, kaempferol has been shown to be more efficient than quercetin (being active at a physiological concentration of about 0.1ÿ1 M), an efficiency that the authors correlated with the higher ER-binding capability of kaempferol. Other mechanisms known to be affected by estrogens and bisphosphonates (Riggs, 2000; Rodan and Reszka, 2002) are the recruitment and the differentiation of new osteoclasts and the following maturation steps, processes that are regulated among several other factors by the matrix metalloproteinases (MMPs) and by the vitronectin receptor. In a murine model of osteoclastogenesis, the monocyte/macrophage cell line RAW264.7 (Srivastava et al., 2001), Lorenzetti et al., (2003) have recently shown that both genistein and daidzein might reduce osteoclast differentiation (about 50% of reduction with each isoflavone) to an extent similar or even better than 17 -estradiol (about 40% of reduction). A role for soy isoflavones in modulating MMPs was also suggested in primary cultures of isolated human osteoclasts (Lorenzetti et al., 2001), where the activity of both gelatinases appear to be down-regulated by purified genistein and daidzein. Other phytoestrogens, namely green tea catechins, are already known to inhibit both MMP-2 and MMP-9 (but also MMP-12 and MMP-14) activity in different cell-culture systems (Demeule et al., 2000; Garbisa et al., 2001; Annabi et al., 2002; Dell'Aica et al., 2002; Oku et al., 2003), thus impairing the ability of such cells to cross the basal lamina and delaying tumour invasion, inflammation, neovascularisation and cell migration. Notably, the action of catechins on MMPs appears to be mediated also by an enhancement of the levels of protein expression and binding activity of the MMPs endogenous inhibitors (Cheng et al., 2003; Maeda et al., 2003). Finally, a key molecule in osteoclast differentiation is the protein osteoprotegerin (Simonet et al., 1997), defined as the decoy receptor of the osteoclast differentiation factor, RANKL. OPG has been shown to block the expression of RANKL and compete for the same receptor (c-fms) on the osteoclast cells and by doing so to decrease the activation of the signal transduction pathways leading to the osteoclast differentiation and, hence, prevent bone resorption (Teitelbaum, 2000). The balance between bone formation and resorption is maintained modulating the proper ratio of the expression levels of RANKL and OPG, and almost all the bone regulators alter the normal ratio in one way or the other. Similar to E2, genistein has been recently demonstrated to increase OPG

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expression via osteoblast stimulation (human trabecular osteoblasts) and so to reduce osteoclastogenesis in a paracrine manner (Viereck et al., 2002). Such an effect has been shown to be ER-mediated and obtained by an up-regulation of the OPG gene transcription levels.

5.5 Investigating phytoestrogen action on bone: animal and human studies 5.5.1 Animal studies Bone loss and its prevention by drugs and nutritional factors have been widely studied in ovariectomised (OVX) rodent models (FDA, 1994; Thorndike and Turner, 1998). The effects of soy isoflavones, pure or in soy extracts, in such animal models have been recently reviewed (Coxam, 2003; Offord, 2003; Setchell and Lydeking-Olsen, 2003): overall, most studies point to an osteoprotective role of isoflavones independently of the contemporary presence of soy proteins. Despite this, animal studies are not conclusive due the contradictory results depending on time and length of exposure, dose and method of administration and even age of the rodents (Coxam, 2003; Offord, 2003; Setchell and Lydeking-Olsen, 2003). Among the most convincing studies to suggest a beneficial effect of genistein to prevent OVX-induced bone loss, one has been particularly significant since it has been performed using different concentrations (0.5, 1.6, and 5 mg per day) of genistein, and above all since the resulting dose response pattern (Anderson et al., 1998) has been proved to be biphasic. Indeed, the low dose has been more effective than the high dose and the low dose effect was almost as effective as estrogens in the retention of cancellous bone tissue and on the maintainance of the architecture and morphology of the endosteal surfaces. Following the first report on the beneficial effect of soy proteins in preventing OVX-induced bone loss in rats, many other studies have confirmed how soy isoflavones decreased OVX-induced femur bone loss (reviewed in Coxam, 2003; Offord, 2003; Setchell and Lydeking-Olsen, 2003) and in one case also the ability of each soy isoflavones (at 50 mg per kg body weight per day) to reduce also the urinary excretion of pyridoline and deoxypyridinoline has been proved (Uesugi et al., 2001). The optimal isoflavone safe dose for a beneficial effect on bone without uterotrophic effects has been estimated to be 40 mg per kg of body weight (Picherit et al., 2001). Furthermore, at least in one case (Picherit et al., 2000) daidzein has been shown to be more effective than genistein in preventing bone loss. Daidzein prevented total femoral BMD and vertebral trabecular bone losses as efficiently as estradiol, whereas genistein did not. On the contrary, few studies investigated the effect of other phytoestrogens, even if environmental estrogens, prenylflavonoids and lignans have also been shown to be effective at different levels on bone metabolism (Setchell and Lydeking-Olsen, 2003). The ovariectomised old cynomolgus monkey represents a recognised model

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of human osteoporosis (Legrand, 2003) and is considered more reliable than the rat model in the assessment of the efficacy and potential toxicity of agents intended to prevent or treat osteoporosis in humans. The most recent investigation on primates has been performed on OVX cynomolgus macaques (Register et al., 2003) and the main result of such a longitudinal study pointed out that a long-term (greater than one year) consumption of a soy protein-based diet containing minimal soy isoflavones (1.4 mg/day) had little or no effect on bone loss after ovariectomy. No adverse effects on the reproductive system have been reported and a slight improvement in cardiovascular risk factors has been shown. As a whole, even if animal studies on soy isoflavones appear to significantly prevent bone loss due to estrogen deficiency, attention should be paid to species-specific differences in responsiveness to phytoestrogens. 5.5.2 Human studies A beneficial effect of high soy consumption, and hence of high isoflavone intake, on bone health in post-menopausal women has been first suggested by population studies indicating a lower rate of hip fractures (50±60% less) in Asian women than in Western women (Ross et al., 1991; Lauderdale et al., 1997). However, factors other then diet might contribute to such a difference. A higher amount of physical activity and better body balancing have been reported in Asian women, who may then have a reduced incidence of falls (Davis et al., 1999). Genetic factors also affect the shape and geometry of the skeleton (Nakamura et al., 1994; Melton, 2000). Supporting data for an association between isoflavone intake and bone mineral density (BMD) have been reported in several observational studies as recently reviewed (Anthony et al., 2002; Setchell and Lydeking-Olsen, 2003). Crosssectional studies on Asian women confirmed the view that a life-long high intake of soy foods (containing 15±50 mg IF per day) positively affect lumbar spine BMD in both pre- and post-menopausal women (Horiuchi et al., 2000; Wangen et al., 2000, Ho et al., 2001; Somekawa et al., 2001). Consistently, some of such cross-sectional studies have been correlated also to a decrease in bone turnover since markers of bone formation and resorption (osteocalcin and urinary pyridinoline and deoxypyridinoline, respectively) have been proved to show a lower level of resorption (Horiuchi et al., 2000; Wagen et al., 2000). Intervention studies have recently been reviewed by Branca (2003). The design of such studies has not always been adequate, particularly as far as duration is concerned. Bone is in fact a body compartment that is subjected to adaptation (bone remodelling transient) and at least 12±24 months are necessary to demonstrate the effect of a drug or environmental factor on BMD. By analysing the published literature (some studies are available only in abstract form), we have identified seven studies carried out for a short period (mostly three months) and seven studies carried out for at least six months. All studies have used an amount of isoflavones of 50±100 mg/day. Short-term studies have

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used both BMD and bone biomarkers as outcome measures and have not shown consistent results, some showing an increase in BMD (Alekel et al., 2000) and some other showing no effect (Dalais et al., 1998; Gallagher et al., 2000). The first convincing intervention study (Potter et al., 1998) has been performed over a six-month period using two different doses of isoflavones (45 and 90 mg per day in a soy protein rich diet) and only the higher one has proved effective in increasing BMD at the lumbar spine. In a study by Morabito et al. (2002), 90 post-menopausal women were randomly assigned to 54 mg/day genistein, to HRT or to placebo and followed for 12 months. In the genistein group and HRT group they observed an increase in BMD at femoral neck, Ward's triangle and lumbar spine and reduced PYD and DPD excretion at six and 12 months. Interestingly, they also observed increased serum bone alkaline phosphatase and serum osteocalcin at six and 12 months in the genistein-treated group. Two long-term studies (two years' duration) have been performed in post-menopausal women and again results have been controversial since only in one case was a bone-sparing effect observed (Lydeking-Olsen et al., 2002; Vitolins et al., 2002). A possible explanation for the conflicting results is that the effects of soy isoflavones might depend on equol formation (Setchell et al., 2002). Equol is a metabolite of daidzein that is produced in humans in only about 40% of the adult population. Equol formation is absolutely dependent on intestinal microflora (Setchell et al., 2002) and equol has been proved to possess a higher estrogenic activity and antioxidant properties than soy isoflavones. The `equol status' hypothesis suggests that the beneficial effects of soy proteins might be present only in `equol producers' (Setchell et al., 2002). This hypothesis is supported by the already mentioned two-year long-term intervention study (Lydeking-Olsen et al., 2002), in which the increase in lumbar spine BMD observed in all recruited postmenopausal women was even greater among the `equol producers' (45% of the recruited women) than in `nonequol producers' (2.4% vs. 0.6%). The role of phytoestrogens in osteoporosis prevention might be clarified by the outcome of two ongoing trials, ending respectively in December 2004 and December 2005. A 12-month double blind RCT intervention study on 300 postmenopausal women is being carried out with 100 mg/day soy isoflavones in three European countries (PHYTOS project), while a two-year follow-up, randomised, double-blind, placebo-controlled study is going on in the United States (the OPUS project).

5.6

Conclusions

As described earlier in this chapter, the current therapies to prevent postmenopausal bone loss include ERT, calcitonin, bisphosphonates and SERMs. Reported adverse effects of such therapies mainly include upper gastrointestinal distress for BPs, a slight increase of breast and endometrial cancer risk for ERT, only an increase in endometrial cancer risk for the SERM tamoxifen, and still an

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increase in hot flashes for the SERM raloxifene. Furthermore, those drugs have a very high cost. The search for drug substitutes pointed toward alternative therapies, particularly those obtained directly from plants and therefore perceived as `natural' and free of any deleterious side effects. In the past few years a great deal of work has been done to clarify the role of phytoestrogens in bone health and confidence about their possible use in osteoporosis prevention has increased. In 2000, the NIH Consensus Conference stated that `There is a great deal of public interest in natural estrogens, particularly plant-derived phytoestrogens. These compounds have weak estrogen-like effects, and although some animal studies are promising, no effects on fracture reduction in humans have been shown' (NIH, 2000). In 2003 the Report of the UK Food Standard Agency concluded that `Clinical data on the effects of phytoestrogens on bone density are limited but results of short-term human studies suggest small protective effects in the lumbar spine. The data for protective effects at other sites are equivocal' (Committee on Toxicity of Chemicals in Food, Consumer Products and the Environment, 2003). However, the role of phytoestrogens in osteoporosis has not yet been established. While a 50 mg/day dose seems appropriate for the prevention of cardiovascular disease, a higher dose (50ÿ100 mg/day) seems necessary for osteoporosis prevention in post-menopausal women. At present, there is no rationale for considering phytoestrogens as a possible option in the treatment of osteoporosis, despite the indication that they may have an effect on bone formation. Isoflavones are present at generally low concentrations in most foods used in Europe and the Western world. Switching to the use of foods typical of South East Asian diets is an option that is not going to be feasible for the majority of the population. In order to increase their intake phytochemical preparations or fortified foods should be used. However, phytoestrogens are a wide definition that include many compounds normally present in fruits and vegetables. We need to explore the whole range and possibly to evaluate jointly their effects on bone health.

5.7

Sources of further information and advice

Among the literature already mentioned in the text we suggest the comprehensive scientific reports in the following reviews or books. Principle of Bone Biology, 2nd edition, Bilezikian JP, Raisz LG, and Rodan GA, eds, Academic Press, San Diego (CA-USA). Science, 2000, vol. 289, no. 5484, pp. 1497±1514; reviews on bone remodelling and repair. British Journal of Nutrition, 2003, vol. 89, suppl. 1, `Effects of Phytoestrogens on Bone Health: the VENUS concerted action'. A general overview on osteoporosis and bone diseases for a wider community could be obtained from the web pages of:

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Functional foods, ageing and degenerative disease The National Institute of Health (NIH), http://www.osteo.org. The International Osteoporosis Foundation (IOF), http:// www.osteofound.org/, containing also the link to the European Commission `Summary reports on osteoporosis in the European Community', http:// www.connect.ie/effo/sreports.htm. The National Osteoporosis Organization (NOF), a USA non profit organization, http://www.nof.org/. The Foundation for Osteoporosis Research and Eradication (FORE), a nonprofit research center, http://www.fore.org/. The National Osteoporosis Society, a UK national charity, http:// www.nos.org.uk/.

For updated data sets on the phyto-oestrogens content in foods and diets, we suggest checking the following websites: http://www.venus-ca.org; http:// www.nal.usda.gov/fnic/foodcomp/Data/isoflav/isoflav.html (USDA-Iowa State University database on the isoflavones content of foods) and http:// www.ifr.bbsrc.ac.uk/phytochemicals/Links.htm (Institute of Food Research Database on the levels of bioactive compounds in plant foods). For recent advances on the effects of phyto-oestrogens on hormonaldependent diseases as well as on human supplementation trials, it might be useful to refer to http://www.phytos.org (EU-funded project on the prevention of osteoporosis by nutritional phyto-oestrogens); http://www.phytoprevent.org (EU-funded project on the role of phyto-oestrogens in the prevention of breast and prostate cancer); http://www.nutrition.tum.de/isoheart.htm (EU-funded project on cardiovascular health of postmenopausal women). Other information about osteoporosis and bone diseases as well as on therapies can be found on the above-mentioned web pages as well as on the World Health Organization web pages, in the drug information section, www.who.int/druginformation/; on the Food and Drug Administration website, in the `women's health' area of special interest, http://www.cfsan.fda.gov/~dms/ wh-osteo.html; and at the European Institute for Women's Health (EIWH) website, http://www.eurohealth.ie/. On GMOs debate and biosafety research, a review of results performed under the European Commission supervision (`EC-sponsored Research on Safety of Genetically Modified Organisms', edited by C. Kessler and I. Economidis) is available also online at the EU website http://europa.eu.int/comm/research/ quality-of-life/gmo/; moreover, an update on current research in food safety, nutrition and food-related disease might be found in Committee on Toxicity of Chemicals in Food, Consumer Products and the Environment (2003). Phytoestrogens and Health. London and on the websites of the World Health Organization, http://www.who.int/fsf/GMfood/index.htm, and of the Food Standards Agency, http://www.foodstandards.gov.uk/.

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129

References

(1993). Consensus development conference report. Diagnosis, prophylaxis and treatment of osteoporosis', Am J Med 94(6): 646±650. AKIYAMA T, ISHIDA J, NAKAGAWA S, OGAWARA H, WATANABE S, ITOH N, SHIBUYA M and FUKAMI Y (1987) Genistein, a specific inhibitor of tyrosine-specific protein kinases. J Biol Chem 262(12): 5592±5595. ALAKANGAS A, SELANDER K, MULARI M, HALLEEN J, LEHENKARI P, MONKKONEN J, SALO J and VAANANEN K (2002). Alendronate disturbs vesicular trafficking in osteoclasts. Calcif Tissue Int 70(1): 40±47. ALEKEL DL, GERMAIN AS, PETERSON CT, HANSON KB, STEWART JW and TODA T (2000). Isoflavone-rich soy protein isolate attenuates bone loss in the lumbar spine of perimenopausal women. Am J Clin Nutr 72(3): 844±852. ANDERSON JJ, AMBROSE WW and GARNER SC (1998). Biphasic effects of genistein on bone tissue in the ovariectomized, lactating rat model. Proc Soc Exp Biol Med 217(3): 345±350. ANNABI B, LACHAMBRE MP, BOUSQUET-GAGNON N, PAGE M, GINGRAS D and BELIVEAU R (2002). Green tea polyphenol (-)-epigallocatechin 3-gallate inhibits MMP-2 secretion and MT1-MMP-driven migration in glioblastoma cells. Biochim Biophys Acta. 1542(1±3): 209±220. ANTHONY MS, ANDERSON JJ and ALEKEL DL (2002). Association between soy and/or soy isoflavones and bone: evidence from epidemiologic studies. In: Gilani, GS and Anderson, JJ eds, Phytoestrogens and health. Champaign, IL: AOCS Press, 331± 340. ARTS J, KRUIPER GG, JANSSEN JM, GUSTAFSSON JA, LOWIK CW, POLS HA and VAN LEEUWEN JP (1997). Differential expression of estrogen receptors alpha and beta mRNA during differentiation of human osteoblast SV-HFO cells. Endocrinology 138(11): 5067± 5070. AZUMA Y, SATO H, OUE Y, OKABE K, OHTA T, TSUCHIMOTO M and KIYOKI M (1995). Alendronate distributed on bone surfaces inhibits osteoclastic bone resorption in vitro and in experimental hypercalcemia models. Bone 16(2): 235±245. BARNES S, BOERSMA B, PATEL R, KIRK M, DARLEY-USMAR VM, KIM H and XU J (2000a). Isoflavonoids and chronic disease: mechanisms of action. Biofactors 12(1±4): 209± 215. BARNES S, KIM H, DARLEY-USMAR V, PATEL R, XU J, BOERSMA B and LUO M (2000b). Beyond ERalpha and ERbeta: estrogen receptor binding is only part of the isoflavone story. J Nutr 130(3): 656S±657S. BATRA GS, HAINEY L, FREEMONT AJ, ANDREW G, SAUNDERS PTK, HOYLAND JA and BRAIDMAN IP (2003). Evidence for cell-specific changes with age in expression of oestrogen receptor (ER)  and in bone fractures from men and women. J Pathol 200(1): 65±73. BEAUDREUIL J, TABOULET J, ORCEL P, GRAULET AM, DENNE MA, BAUDOIN C, JULLIENNE A and DE VERNEJOUL MC (2000). Calcitonin receptor mRNA in mononuclear leucocytes from postmenopausal women: decrease during osteoporosis and link to bone markers with specific isoform involvement. Bone 27(1): 161±168. BRANCA F (2003). Dietary phytoestrogens and bone health. Proc Nutr Soc 62(4): 877±887. CASPER RF, QUESNE M, ROGERS IM, SHIROTA T, JOLIVET A, MILGROM E and SAVOURET JF (1999). Resveratrol has antagonist activity on the aryl hydrocarbon receptor: implications for prevention of dioxin toxicity. Mol Pharmacol. 56(4): 784±790. AJM

130

Functional foods, ageing and degenerative disease and MAGNUS CJ (1982). Calcitonin alters behaviour of isolated osteoclasts. J Pathol. 136(1): 27±39. Z, ZHENG W, CUSTER LJ, DAI Q, SHU XO, JIN F and FRANKE AA (1999). Usual dietary consumption of soy foods and its correlation with the excretion rate of isoflavonoids in overnight urine samples among Chinese women in Shanghai. Nutr Cancer 33: 82±87.

CHAMBERS TJ CHEN

CHENG XW, KUZUYA M, KANDA S, MAEDA K, SASAKI T, WANG QL, TAMAYA-MORI N, SHIBATA T

and IGUCHI A (2003). Epigallocatechin-3-gallate binding to MMP-2 inhibits gelatinolytic activity without influencing the attachment to extracellular matrix proteins but enhances MMP-2 binding to TIMP-2. Arch Biochem Biophys. 415(1): 126±132. CIOLINO HP, DASCHNER PJ and YEH GC (1998). Resveratrol inhibits transcription of CYP1A1 in vitro by preventing activation of the aryl hydrocarbon receptor. Cancer Res. 58(24): 5707±5712. COMMITTEE ON TOXICITY OF CHEMICALS IN FOOD, CONSUMER PRODUCTS AND THE

(2003). Phytoestrogens and Health. London. and KORACK KS (1999). Estrogen Receptor Null Mice: What Have We Learned and Where Will They Lead Us? Endocrine Rev 20(3): 358±417. COXAM V (2003). Prevention of osteopaenia by phytoestrogens: animal studies. Br J Nutr 89 (Suppl. 1): S75±S85. ENVIRONMENT

COUSE JF

CRANNEY A, WELLS G, WILLAN A, GRIFFITH L, ZYTARUK N, ROBINSON V, BLACK D, ADACHI J, SHEA B, TUGWELL P and GUYATT G (2002). Meta-analyses of therapies for postmenopausal osteoporosis. II. Meta-analysis of alendronate for the treatment of postmenopausal women. Endocr Rev. 23(4): 508±516. CUMMINGS SR, ECKERT S, KRUEGER KA, GRADY D, POWLES TJ, CAULEY JA, NORTON L, NICKELSEN T, BJARNASON NH, MORROW M, LIPPMAN ME, BLACK D, GLUSMAN JE, COSTA

and JORDAN VC (1999). The effect of raloxifene on risk of breast cancer in postmenopausal women: results from the MORE randomized trial. Multiple Outcomes of Raloxifene Evaluation. JAMA 281(23): 2189±2197. DALAIS FS, RICE GE, WAHLQUIST ML, GREHAN M, MURKIES AL, MEDLEY G, AYTON R and STRAUSS BJ (1998). Effects of dietary phytoestrogens in postmenopausal women. Climacteric 1(2): 124±129. DAVIS JW, NEVITT MC, WASNICH RD and ROSS PD (1999). A cross-cultural comparison of neuromuscular performance, functional status, and falls between Japanese and white women. J Gerontol A Biol Sci Med Sci. 54(6): M288±M292. DELISLE RK, YU SJ, NAIR AC and WELSH WJ (2001). Homology modeling of the estrogen receptor subtype beta (ER-beta) and calculation of ligand binding affinities. J Mol Graph Model. 20(2): 155±167. DELL'AICA I, DONA M, SARTOR L, PEZZATO E and GARBISA S (2002). (-)Epigallocatechin-3gallate directly inhibits MT1-MMP activity, leading to accumulation of nonactivated MMP-2 at the cell surface. Lab Invest. 82(12): 1685±93. DELMAS PD, BJARNASON NH, MITLAK BH, RAVOUX AC, SHAH AS, HUSTER WJ, DRAPER M and CHRISTIANSEN C (1997). Effects of raloxifene on bone mineral density, serum cholesterol concentrations, and uterine endometrium in postmenopausal women. N Engl J Med 337(23): 1641±1647. DEMEULE M, BROSSARD M, PAGE M, GINGRAS D and BELIVEAU R (2000). Matrix metalloproteinase inhibition by green tea catechins. Biochim Biophys Acta. 1478(1): 51±60. A

DE KLEIJN MJ, VAN DER SCHOUW YT, WILSON PW, ADLERCREUTZ H, MAZUR W, GROBBEE DE

Phytoestrogens and the prevention of osteoporosis

131

and JACQUES PF (2001). Intake of dietary phytoestrogens is low in postmenopausal women in the United States: the Framingham Study (1±4). J Nutr 131: 1826±1832. DE VRIES JH, JANSSEN PL, HOLLMAN PC, VAN STAVEREN WA and KATAN MB (1997). Consumption of quercetin and kaempferol in free-living subjects eating a variety of diets. Cancer Lett. 1997 Mar 19;114(1±2): 141±144. DIXON RA and FERREIRA D (2002). Genistein. Phytochemistry 60(3): 205±211. ETTINGER B, BLACK DM, MITLAK BH, KNICKERBOCKER RK, NICKELSEN T, GENANT HK, CHRISTIANSEN C, DELMAS PD, ZANCHETTA JR, STAKKESTAD J, GLUER CC, KRUEGER K, COHEN FJ, ECKERT S, ENSRUD KE, AVIOLI LV, LIPS P and CUMMINGS SR (1999). Reduction of vertebral fracture risk in postmenopausal women with osteoporosis treated with raloxifene: results from a 3-year randomized clinical trial. Multiple Outcomes of Raloxifene Evaluation (MORE) Investigators. JAMA 282(7): 637± 645. ETTINGER MP (2003). Ageing bone and osteoporosis: strategies for preventing fractures in the elderly. Arch Intern Med. 163(18): 2237±2246. FLEISCH H (2003). Bisphosphonates in osteoporosis. Eur Spine J. Online. FLEISCH H, RESZKA A, RODAN G and ROGERS M (2002). Bisphosphonates ± mechanisms of action. In: Principles of Bone Biology, 2nd edition, vol. 2, ch. 78, pp. 1361±1385. Bilezikian JP, Raisz LG, and Rodan GA, eds, Academic Press, San Diego (CAUSA). FOGELMAN I, RIBOT C, SMITH R, ETHGEN D, SOD E and REGINSTER J-Y (2000). Risedronate reverses bone loss in postmenopausal women with low bone mass: results from a multinational, double-blind, placebo-controlled trial. J Clin Endocrinol Metab. 85(5): 1895±1900. FOOD AND DRUG ADMINISTRATION (1994). Guidelines for preclinical and clinical evaluation of agents used in the prevention or treatment of postmenopausal osteoporosis. FRANCESCHI S, SCHINELLA D, BIDOLI E, DAL MASO L, LA VECCHIA C, PARAZZINI F and ZECCHIN R (1996). The influence of body size, smoking, and diet on bone density in pre- and postmenopausal women. Epidemiology. 7(4): 411±414. FRITH JC, MONKKONEN J, BLACKBURN GM, RUSSELL RG and ROGERS MJ (1997). Clodronate and liposome-encapsulated clodronate are metabolized to a toxic ATP analog, adenosine 5'-(beta, gamma-dichloromethylene) triphosphate, by mammalian cells in vitro. J Bone Miner Res 12(9): 1358±67. GALLAGHER JC, RAFFERTY K, HAYNATZKA V and WILSON M (2000). The effect of soy protein on bone metabolism. J Nutr 130(3): 667S. GAO YH and YAMAGUCHI M (1999a). Anabolic effect of daidzein on cortical bone in tissue culture: comparison with genistein effect. Mol Cell Biochem. 194(1±2): 93±97. GAO YH and YAMAGUCHI M (1999b). Inhibitory effect of genistein on osteoclast-like cell formation in mouse marrow cultures. Biochem Pharmacol. 58(5): 767±772. GAO YH and YAMAGUCHI M (1999c). Suppressive effect of genistein on rat bone osteoclasts: apoptosis is induced through Ca2+ signaling. Biol Pharm Bull. 22(8): 805±809. GAO YH and YAMAGUCHI M (2000). Suppressive effect of genistein on rat bone osteoclasts: involvement of protein kinase inhibition and protein tyrosine phosphatase activation. Int J Mol Med. 5(3): 261±267. GARBISA S, SARTOR L, BIGGIN S, SALVATO B, BENELLI R and ALBINI A (2001). Tumor gelatinases and invasion inhibited by the green tea flavanol epigallocatechin-3gallate. Cancer. 91(4): 822±32.

132

Functional foods, ageing and degenerative disease

and TANAKA T (2002). A selective inhibitor of matrix metalloproteinases inhibits the migration of isolated osteoclast by increasing the life span of podosomes. J Bone Miner Res 20(2): 98±105. GRISSO JA, KELSEY JL, O'BRIEN LA, MILES CG, SIDNEY S, MAISLIN G, LAPANN K, MORITZ D and PETERS B (1997). Risk factors for hip fracture in men. Hip Fracture Study Group. Am J Epidemiol. 145(9): 786±793. HALL JM, COUSE JF and KORACH KS (2001). The Multifaceted Mechanisms of Estradiol and Estrogen Receptor Signaling. J Biol Chem. 276(40): 36869±36872. GOTO T, MAEDA H

HARRIS ST, WATTS NB, GENANT HK, MCKEEVER CD, HANGARTNER T, KELLER M, CHESNUT III

and MILLER PD (1999). Effects of risedronate treatment on vertebral and nonvertebral fractures in women with postmenopausal osteoporosis. A randomized controlled trial. JAMA 282: 1344± 1352. HO SC, WOO JL, LEUNG SS, SHAM AL, LAM TH and JANUS ED (2000). Intake of soy products is associated with better plasma lipid profiles in the Hong Kong Chinese population. J Nutr 130: 2590±2593. HO SC, CHAN SG, YI Q, WONG E and LEUNG PC (2001). Soy intake and the maintenance of peak bone mass in Hong Kong Chinese women. J Bone Miner Res. 16(7): 1363± 1369. HOLLENBACH KA, BARRETT-CONNOR E, EDELSTEIN SL and HOLBROOK T (1993). Cigarette smoking and bone mineral density in older men and women. Am J Public Health. 83(9): 1265±1270. HORIUCHI T, ONOUCHI T, TAKAHASHI M, ITO H and ORIMO H (2000). Effect of soy protein on bone metabolism in postmenopausal Japanese women. Osteoporos Int. 11(8): 721± 724. CH, BROWN J, ERIKSEN EF, HOSEYNI MS, AXELROD DW

HOSKING D, CHILVERS CED, CHRISTIANSEN C, RAVN P, WASNICH R, ROSS P, MCCLUNG M,

and YATES AJ (1998). Prevention of Bone Loss with Alendronate in Postmenopausal Women under 60 Years of Age. N Engl J Med. 338: 485±492. HOTOKEZAKA H, SAKAI E, KANAOKA K, SAITO K, MATSUO K, KITAURA H, YOSHIDA N and NAKAYAMA K (2002). U0126 and PD98059, specific inhibitors of MEK, accelerate differentiation of RAW264.7 cells into osteoclast-like cells. J Biol Chem. 277(49): 47366±47372. HUGHES DE, WRIGHT KR, UY HL, SASAKI A, YONEDA T, ROODMAN GD, MUNDY GR and BOYCE BF (1995). Bisphosphonates promote apoptosis in murine osteoclasts in vitro and in vivo. J Bone Miner Res. 10(10): 1478±1487. HUGHES DE, DAI A, TIFFEE JC, LI HH, MUNDY GR and BOYCE BF (1996). Estrogen promotes apoptosis of murine osteoclasts mediated by TGF-beta. Nat Med. 2(10): 1132± 1136. INOUE D, SHIH C, GALSON DL, GOLDRING SR, HORNE WC and BARON R (1999). Calcitonindependent down-regulation of the mouse C1a calcitonin receptor in cells of the osteoclast lineage involves a transcriptional mechanism. Endocrinology 140(3): 1060±1068. KAUFMANN WK (1989). Human topoisomerase II function, tyrosine phosphorylation and cell cycle checkpoints Proc Soc Exp Biol Med. 217(3): 327±334. KLOOSTERBOER HJ. Tibolone: a steroid with a tissue-specific mode of action. J Steroid Biochem Mol Biol. 2001; 76: 231±8. BALSKE A, THOMPSON D, DALEY M

KOUSTENI S, BELLIDO T, PLOTKIN LI, O'BRIEN CA, BODENNER DL, HAN L, HAN K, DIGREGORIO GB, KATZENELLENBOGEN JA, KATZENELLENBOGEN BS, ROBERSON PK, WEINSTEIN RS,

Phytoestrogens and the prevention of osteoporosis

133

and MANOLAGAS SC (2001). Nongenotropic, sex-nonspecific signaling through the estrogen or androgen receptors: dissociation from transcriptional activity. Cell. 104(5): 719±730. KUIPER GG, ENMARK E, PELTO-HUIKKO M, NILSSON S and GUSTAFSSON JA (1996). Cloning of a novel receptor expressed in rat prostate and ovary. Proc Natl Acad Sci USA 93(12): 5925±5930. KUIPER GG, CARLSSON B, GRANDIEN K, ENMARK E, HAGGBLAD J, NILSSON S and GUSTAFSSON JA (1997). Comparison of the ligand binding specificity and transcript tissue distribution of estrogen receptor  and . Endocrinol. 138(3): 863±870. JILKA RL

KUIPER GG, LEMMEN JG, CARLSSON B, CORTON JC, SAFE SH, VAN DER SAAG PT, VAN DER BURG

and GUSTAFSSON JA (1998). Interaction of estrogenic chemicals and phytoestrogens with estrogen receptor beta, Endocrinology, 139(10): 4252±4263. KUNHAU J (1976). The flavonoids, a class of semi-essential food components: their role in human nutrition. World Rev Nutr Diet 24: 117. LACEY JV JR, MINK PJ, LUBIN JH, SHERMAN ME, TROISI R, HARTGE P, SCHATZKIN A and SCHAIRER C (2002). Menopausal hormone replacement therapy and risk of ovarian cancer. JAMA 288(3): 334±341. LAUDERDALE DS, JACOBSEN SJ, FURNER SE, LEVY PS, BRODY JA and GOLDBERG J (1997). Hip fracture incidence among elderly Asian-American populations. Am J Epidemiol. 146(6): 502±509. LEE K, JESSOP H, SUSWILLO R, ZAMAN G and CANYON L (2003). Bone adaptation requires oestrogen receptor alpha. Nature 424(6947): 389. LEGRAND JJ, FISCH C, GUILLAUMAT PO, PAVARD JM, ATTIA M, DE JOUFFREY S and CLAUDE JR (2003). Use of biochemical markers to monitor changes in bone turnover in cynomolgus monkeys. Biomarkers. 8(1): 63±77. LEWANDOWSKI S, KALITA K and KACZMAREK L (2002). Estrogen receptor beta. Potential functional significance of a variety of mRNA isoforms. FEBS Lett. 524(1±3): 1±5. LINASSIER C, PIERRE M, LE PECQ JB and PIERRE J (1990). Mechanisms of action in NIH-3T3 cells of genistein, an inhibitor of EGF receptor tyrosine kinase activity. Biochem Pharmacol. 39(1): 187±193. LINDBERG MK, ALATALO SL, HALLEEN JM, MOHAN S, GUSTAFSSON JA and OHLSSON C (2001a). Estrogen receptor specificity in the regulation of the skeleton in female mice. J Endocrinol 171(2): 229±236. B

LINDBERG MK, ERLANDSSON M, ALATALO SL, WINDAHL S, ANDERSSON G, HALLEEN JM,

and OHLSSON C (2001b). Estrogen receptor alpha, but not estrogen receptor beta, is involved in the regulation of the OPG/RANKL (osteoprotegerin/receptor activator of NF-kappa B ligand) ratio and serum interleukin-6 in male mice. J Endocrinol 171(3): 425±433. LINDBERG MK, MOVERARE S, SKRTIC S, GAO H, DAHLMAN-WRIGHT K, GUSTAFSSON JA and OHLSSON C (2003). Estrogen receptor (ER)-beta reduces ERalpha-regulated gene transcription, supporting a `ying yang' relationship between ERalpha and ERbeta in mice. Mol Endocrinol 17(2): 203±208. LORENZETTI S and BRANCA F (2003). Assessing the intake of phytoestrogens: isoflavones. In: Johnson, I and Williamson, G, eds, Phytochemical Functional Food, Cambridge, Woodhead Publishing Ltd., pp. 189±221. Á A, GERMANI D, CIANFARANI S and BRANCA F (2001). Phytoestrogens LORENZETTI S, PATERNO and IGF-I in vitro regulation of bone resorption by osteoclasts. Ann Nutr Metab 45(5): 229. LORENZETTI S, GERMANI D, FARINI D, CORSI A, BIANCO P, CIANFARANI S and BRANCA F (2003). CARLSTEN H, GUSTAFSSON JA

134

Functional foods, ageing and degenerative disease

A murine model of RANKL-induced osteoclastogenesis to study the role of phytoestrogens on osteoclast differentiation. Ann Nutr Metab 47(6): 484. LOVE RR, MAZESS RB, BARDEN HS, EPSTEIN S, NEWCOMB PA, JORDAN VC, CARBONE PP and DEMETS DL (1992). Effects of tamoxifen on bone mineral density in postmenopausal women with breast cancer. N Engl J Med 326(13): 852±856. LYDEKING-OLSEN E, JENSEN JB, SETCHELL KD, DAMHUS M and JENSEN TH (2002). Isoflavonerich soy milk prevents bone loss in the lumbar spine of postmenopausal women. A 2-yr study. J Nutr 132(3): 581S. MCCLUNG MR, GEUSENS P, MILLER PD, ZIPPEL H, BENSEN WG, ROUX C, ADAMI S, FOGELMAN I, DIAMOND T, EASTELL R, MEUNIER PJ and REGINSTER J-Y (2001). Effect of risedronate on the risk of hip fracture in elderly women. New Engl J Med. 344(5): 333±340. MAEDA, K, KUZUYA M, CHENG XW, ASAI T, KANDA S, TAMAYA-NORI N, SASAKI T, SHIBITA T and IGUCHI A (2003). Green tea catechins inhibit the cultured smooth muscle cell invasion through the basement barrier. Atherosclerosis 166(1): 23±30. MELTON LJ 3rd (2000). Who has osteoporosis? A conflict between clinical and public health perspectives. J Bone Miner Res. 15(12): 2309±2314. MIZUTANI K, IKEDA K, KAWAI Y and YAMORI Y (1998). Resveratrol stimulates the proliferation and differentiation of osteoblastic MC3T3-E1 cells. Biochem Biophys Res Commun. 253(3): 859±863. MIZUTANI K, IKEDA K, KAWAI Y and Yamori Y (2000). Resveratrol attenuates ovariectomyinduced hypertension and bone loss in stroke-prone spontaneously hypertensive rats. J Nutr Sci Vitaminol. 46(2): 78±83. MODELSKA K and CUMMINGS S. Tibolone for Postmenopausal Women: Systematic Review of Randomized Trials. J Clin Endocrinol Metab 2002; 87(1): 16±23.

MORABITO N, CRISAFULLI A, VERGARA C, GAUDIO A, LASCO A, FRISINA N, D'ANNA R, CORRADO

F, PIZZOLEO MA, CINCOTTA M, ALTAVILLA D, IENTILE R and SQUADRITO F (2002). Effects of genistein and hormone-replacement therapy on bone loss in early postmenopausal women: a randomized double-blind placebo-controlled study. J Bone Miner Res. 17(10): 1904±1912. MORITA I, SAKAGUCHI K, KURACHI T and MUROTA S (1992). Ipriflavone inhibits murine osteoclast formation in vitro. Calcif Tissue Int. 51 (Suppl 1): S7±S10. MOSSELMAN S, POLMAN J and DIJKEMA R (1996). ER beta: identification and characterization of a novel human estrogen receptor. FEBS Lett 392(1): 49±53. NAKAMURA T, TURNER CH, YOSHIKAWA T, SLEMENDA CW, PEACOCK M, BURR DB, MIZUNO Y,

and JOHNSTON CC JR (1994). Do variations in hip geometry explain differences in hip fracture risk between Japanese and white Americans? J Bone Miner Res. 9(7): 1071±6. NICHOLSON GC, MOSELEY JM, SEXTON PM, MENDELSOHN FA and MARTIN TJ (1986). Abundant calcitonin receptors in isolated rat osteoclasts. Biochemical and autoradiographic characterization. J Clin Invest 78(2): 355±360. NIH (2000). National Health Institute Consensus Conference: Osteoporosis Prevention, Diagnosis, and Therapy. NIH Consens Statement 17(1): 1±36. OFFORD E (2003). Phytoestrogens and bone health. In: Johnson, I and Williamson, G, eds, Phytochemical Functional Food, Cambridge, Woodhead Publishing Ltd., pp. 88± 106. OGAWA S, INOUE S, WATANABE T, HIROI H, ORIMO A, HOSOI T, OUCHI Y and MURAMATSU M (1998). The complete primary structure of human estrogen receptor beta (hER beta) and its heterodimerization with ER alpha in vivo and in vitro. Biochem Biophys Res Commun. 243(1): 122±126. ORIMO H, OUCHI Y

Phytoestrogens and the prevention of osteoporosis

135

and (2002). Incadronate disodium inhibits advanced glycation end products-induced angiogenesis in vitro. Biochem Biophys Res Commun 297(2): 419±424. OKU N, MATSUKAWA M, YAMAKAWA S, ASAI T, YAHARA S, HASHIMOTO F and AKIZAWA T (2003). Inhibitory effect of green tea polyphenols on membrane-type 1 matrix metalloproteinase, MT1-MMP. Biol Pharm Bull. 26(9): 1235±1238. OVERGAARD K, HANSEN MA, JENSEN SB and CHRISTIANSEN C (1992). Effect of salmon calcitonin given intranasally on bone mass and fracture rates in established osteoporosis: a dose-response study. Br Med J 305: 556±561. PETTERSSON K and GUSTAFSSON JA (2001). Role of estrogen receptor beta in estrogen action. Annu Rev Physiol. 63: 165±192. OKAMOTO T, YAMAGISHI S, INAGAKI Y, AMANO S, TAKEUCHI M, KIKUCHI S, OHNO S YOSHIMURA A

PICHERIT C, COXAM V, BENNETAU-PELISSERO C, KATI-COULIBALY S, DAVICCO MJ, LEBECQUE P

and BARLET JP (2000). Daidzein is more efficient than genistein in preventing ovariectomy-induced bone loss in rats. J Nutr. 130(7): 1675±1681.

PICHERIT C, CHANTERANNE B, BENNETAU-PELISSERO C, DAVICCO MJ, LEBECQUE P, BARLET JP

and COXAM V (2001). Dose-dependent bone-sparing effects of dietary isoflavones in the ovariectomised rat. Br J Nutr. 85(3): 307±316. POLKOWSKI K and MAZUREK AP (2000). Biological properties of genistein. A review of in vitro and in vivo data. Acta Pol Pharm 57(2): 135±155. POTTER SM, BAUM JA, TENG H, STILLMAN RJ, SHAY NF and ERDMAN JW JR (1998). Soy protein and isoflavones: their effects on blood lipids and bone density in postmenopausal women. Am J Clin Nutr 68 (suppl. 6): 1375S±1379S. REGINSTER J, MINNE HW, SORENSEN OH, HOOPER M, ROUX C, BRANDI ML, LUND B, ETHGEN D,

and EASTELL R (2000). Randomized trial of the effects of risedronate on vertebral fractures in women with established postmenopausal osteoporosis. Osteoporos Int 11(1): 83±91. REGISTER TC, JAYO MJ and ANTHONY MS (2003). Soy phytoestrogens do not prevent bone loss in postmenopausal monkeys. J Clin Endocrinol Metab. 88(9): 4362±4370. REID DM, HUGHES RA, LAAN RF, SACCO-GIBSON NA, WENDEROTH DH, ADAMI S, EUSEBIO RA and DEVOGELAER JP (2000). Efficacy and safety of daily risedronate in the treatment of corticosteroid-induced osteoporosis in men and women: a randomized trial. J Bone Miner Res. 15(6): 1006±1013. PACK S, ROUMAGNAC I

REID IR, BROWN JP, BURCKHARDT P, HOROWITZ Z, RICHARDSON P, TRECHSEL U, WIDMER A, DEVOGELAER JP, KAUFMAN JM, JAEGER P, BODY JJ, BRANDI ML, BROELL J, DI MICCO R, GENAZZANI AR, FELSENBERG D, HAPP J, HOOPER MJ, ITTNER J, LEB G, MALLMIN H,

and (2002). Intravenous zoledronic acid in postmenopausal women with low bone mineral density. N Engl J Med. 346(9): 653±661. RIGGS BL (2000). The mechanisms of estrogen regulation of bone resorption. J Clin Invest 106(10): 1203±1204. RODAN GA and RESZKA AA (2002). Bisphosphonate mechanism of action. Curr Mol Med 2(6): 571±577. RODAN GA and MARTIN TJ (2000). Therapeutic approaches to bone diseases. Science 289(5484): 1508±1514. ROSS PD, NORIMATSU H, DAVIS JW, YANO K, WASNICH RD, FUJIWARA S, HOSODA Y and MELTON LJ 3RD (1991). A comparison of hip fracture incidence among native Japanese, Japanese Americans, and American Caucasians. Am J Epidemiol. 133(8): 801±809. SATO M and GRASSER W (1990). Effects of bisphosphonates on isolated rat osteoclasts as MURRAY T, ORTOLANI S, RUBINACCI A, SAAF M, SAMSIOE G, VERBRUGGEN L MEUNIER PJ

136

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examined by reflected light microscopy. J Bone Miner Res 5(1): 31±40. and RODAN GA (1991). Bisphosphonate action. Alendronate localization in rat bone and effects on osteoclast ultrastructure. J Clin Invest 88(6): 2095±2105. SCHNEIDER HG, RAUE F, KOPPOLD A, RUF W and ZIEGLER R (1993). Homologous desensitization of calcitonin receptors and calcitonin-dependent adenylate cyclase in T47D cells. Acta Endocrinol. 128(4): 373±378. SETCHELL KD, BROWN NM and LYDEKING-OLSEN E (2002). The clinical importance of the metabolite equol-a clue to the effectiveness of soy and its isoflavones. J Nutr 132(12): 3577±3584. SETCHELL KDR and LYDEKING-OLSEN E (2003). Dietary phytoestrogens and their effect on bone: evidence from in vitro and in vivo, human observational, and dietary intervention studies. Am J Clin Nutr 78 (Suppl. 3): 593S±609S. SATO M, GRASSER W, ENDO N, AKINS R, SIMMONS H, THOMPSON DD, GOLUB E

SIMONET WS, LACEY DL, DUNSTAN CR, KELLEY M, CHANG MS, LUTHY R, NGUYEN HQ, WOODEN S, BENNETT L, BOONE T, SHIMAMOTO G, DEROSE M, ELLIOTT R, COLOMBERO A, TAN HL, TRAIL G, SULLIVAN J, DAVY E, BUCAY N, RENSHAW-GEGG L, HUGHES TM, HILL D,

PATTISON W, CAMPBELL P, SANDER S, VAN G, TARPLEY J, DERBY P, LEE R and BOYLE WJ (1997). Osteoprotegerin: a novel secreted protein involved in the regulation of bone density. Cell. 89(2): 309±319. SIMS NA, DUPONT S, KRUST A, CLEMENT-LACROIX P, MINET D, RESCHE-RIGON M, GAILLARD-

and BARON R (2002). Deletion of estrogen receptors reveals a regulatory role for estrogen receptors-beta in bone remodeling in females but not in males. Bone 30(1): 18±25. SINGH SU, CASPER RF, FRITZ PC, SUKHU B, GANSS B, GIRARD B JR, SAVOURET JF and TENENBAUM HC (2000). Inhibition of dioxin effects on bone formation in vitro by a newly described aryl hydrocarbon receptor antagonist, resveratrol. J Endocrinol. 167(1): 183±195. SOMEKAWA Y, CHIGUCHI M, ISHIBASHI T and ASO T (2001). Soy intake related to menopausal symptoms, serum lipids, and bone mineral density in postmenopausal Japanese women. Obstet Gynecol. 97(1): 109±115. SRIVASTAVA S, TORALDO G, WEITZMANN MN, CENCI S, ROSS FP and PACIFICI R (2001). Estrogen decreases osteoclast formation by down-regulating receptor activator of NF-kappa B ligand (RANKL)-induced JNK activation. J Biol Chem. 276(12): 8836±8840. SUGIMOTO E and YAMAGUCHI M (2000a). Anabolic effect of genistein in osteoblastic MC3T3-E1 cells. Int J Mol Med. 5(5): 515±520. SUGIMOTO E and YAMAGUCHI M (2000b). Stimulatory effect of Daidzein in osteoblastic MC3T3-E1 cells. Biochem Pharmacol. 59(5): 471±475. TAGGART HM, CHESNUT CH 3RD, IVEY JL, BAYLINK DJ, SISOM K, HUBER MB and ROOS BA (1982). Deficient calcitonin response to calcium stimulation in postmenopausal osteoporosis? Lancet 1(8270): 475±8. TEITELBAUM SL (2000). Bone resorption by osteoclasts. Science 289(5484): 1504±1508. KELLY M

TERONEN O, HEIKKILA P, KONTTINEN YT, LAITINEN M, SALO T, HANEMAAIJER R, TERONEN A,

and SORSA T (1999). MMP inhibition and downregulation by bisphosphonates. Ann N Y Acad Sci 878: 453±465. THORNDIKE EA and TURNER AS (1998). In search of an animal model for postmenopausal diseases. Front Biosci. 3: c17±26. TREMBLAY GB, TREMBLAY A, COPELAND NG, GILBERT DJ, JENKINS NA, LABRIE F and GIGUERE V (1997). Cloning, chromosomal localization, and functional analysis of the murine estrogen receptor beta. Mol Endocrinol 11(3): 353±365. MAISI P

Phytoestrogens and the prevention of osteoporosis

137

and ISPIDA H (2001). Comparative study on reduction of bone loss and lipid metabolism abnormality in ovariectomized rats by soy isoflavones, daidzin, genistin, and glycitin. Biol Pharm Bull 24(4): 368±372.

UESUGI T, TODA T, TSUJI K

VAN ERP-BAART AMJ, BRANTS HAM, KIELY M, MULLIGAN A, TURRINI A, SERMONETA C,

and VALSTA LM (2002). Isoflavone intake in different European countries: the VENUS approach. Br J Nutr, 89 (suppl. 1): S25±S30.

KILKINNEN A

VIDAL O, LINDBERG MK, HOLLBERG K, BAYLINK DJ, ANDERSSON G, LUBAHN DB, MOHAN S, GUSTAFSSON JA and OHLSSON C (2000). Estrogen receptor specificity in the regulation of skeletal growth and maturation in male mice. PNAS 97(10): 5474± 5479. VIERECK V, GRUNDKER C, BLASCHKE S, SIGGELKOW H, EMONS G and HOFBAUER LC (2002). Phytoestrogen genistein stimulates the production of osteoprotegerin by human trabecular osteoblasts. J Cell Biochem. 84(4): 725±735. VITOLINS M, ANTHONY M, LENSCHIK L, BLAND DR and BURKE GL (2002). Does soy protein and its isoflavones prevent bone loss in peri- and post-menopausal women? Results of a two year randomized clinical trial. J Nutr 132(3): 582S. WANGEN KE, DUNCAN AM, MERZ-DEMLOW BE, XU X, MARCUS R, PHIPPS WR and KURZER MS (2000). Effects of soy isoflavones on markers of bone turnover in premenopausal and postmenopausal women. J Clin Endocrinol Metab. 85: 3043±3048. WARSHAWSKY H, GOLTZMAN D, ROULEAU MF and BERGERON JJ (1980). Direct in vivo demonstration by radioautography of specific binding sites for calcitonin in skeletal and renal tissues of the rat. J Cell Biol. 85(3): 682±694. WATTEL A, KAMEL S, MENTAVERRI R, LORGET F, PROUILLET C, PETIT JP, FARDELONNE P and BRAZIER M (2003). Potent inhibitory effect of naturally occurring flavonoids quercetin and kaempferol on in vitro osteoclastic bone resorption. Biochem Pharmacol 65(1): 35±42. WATTS NB, JOSSE RG, HAMDY RC, HUGHES RA, MANHART MD, BARTON I, CALLIGEROS D and FELSENBERG D (2003). Risedronate prevents new vertebral fractures in postmenopausal women at high risk. J Clin Endocrinol Metab. 88(2): 542±549. WOOD J, BONJEAN K, RUETZ S, BELLAHCENE A, DEVY L, FOIDART JM, CASTRONOVO V and GREEN JR (2002) Novel antiangiogenic effects of the bisphosphonate compound zoledronic acid. J Pharmacol Exp Ther 302(3): 1055±61. WHI, WRITING GROUP FOR THE WOMEN'S HEALTH INITIATIVE INVESTIGATORS (2002). Risks and benefits of estrogen plus progestin in healthy postmenopausal women: principal results from the Women's Health Initiative randomizied control trial. JAMA 288: 321±333. WHO (1994) Assessment of fracture risk and its application to screening for postmenopausal osteoporosis. WHO Technical Report Series 843. WHO, Geneva. WHO (2003). Diet, nutrition and the prevention of chronic diseases. WHO Technical Report Series 916. WHO, Geneva. YAMAGUCHI M and GAO YH (1999). Anabolic effect of genistein and genistin on bone metabolism in the femoral-metaphyseal tissues of elderly rats: the genistein effect is enhanced by zinc. Mol Cell Biochem. 178(1-2): 377±382. YAMAGUCHI M and SUGIMOTO E (2000). Stimulatory effect of genistein and daidzein on protein synthesis in osteoblastic MC3T3-E1 cells: activation of aminoacyl-tRNA synthetase. Mol Cell Biochem. 214(1±2): 97±102. YAMAGUCHI M, GAO YH and MA ZJ (2000). Synergistic effect of genistein and zinc on bone components in the femoral-metaphyseal tissues of female rats. J Bone Miner Metab 18(2):77-83.

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Functional foods, ageing and degenerative disease and BURCKHARDT P (2002). Molecular and clinical pharmacology of calcitonin. In: Principles of Bone Biology, 2nd edition, vol. 2, ch. 82, pp. 1423±1440. Bilezikian JP, Raisz LG, and Rodan GA, ed., Academic Press, San Diego (CA-USA).

ZAIDI M, INZERILLO AM, TROEN B, MOONGA BS, ABE E

6 Vitamin D fortification and bone health L. Ovesen, Institute of Food Safety and Nutrition, Denmark

6.1

Introduction

Food fortification has for decades been used to improve the nutritional quality of the food supply. The rare occurrence today of rickets in children, the classical vitamin D deficiency disease, so widespread in many industrialised countries at the turn of the century, has in part been ascribed to fortification programmes. Osteoporosis is a disorder of the bones, which increases susceptibility to fractures. Osteoporosis increases in incidence exponentially with age and causes high morbidity and mortality, and heavy burdens on health care expenditure. Nutritional factors, primarily a deficient supply of vitamin D, have been implicated in the pathogenesis of osteoporosis. An increased intake of vitamin D through fortification of foods should therefore benefit bone health. Food fortification may be a relevant strategy to increase vitamin D intake, however, to be safe and effective, fortification must reach people in need, and at the same time not contribute to excessive intakes. The success of a fortification programme depends on several factors, among which control of fortification levels, monitoring of intakes and evaluation of clinical efficacy are critical. The present chapter opens with a review of the present knowledge of the metabolism and functions of vitamin D, including a short discussion of new roles for vitamin D and the problems of defining hypovitaminosis. The chapter then goes on to give a brief introduction to osteoporosis and lists the published randomised controlled studies of vitamin D supplementation in the prevention of fractures. To be effective, fortification has to supply enough vitamin D to cover the recommended dietary allowances in the target group without exposing other people at risk of an overdose. Consequently, recommended dietary

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allowances and the safety of vitamin D are discussed. The final theme concerns the efficacy, possible side effects and practical aspects of food fortification with vitamin D.

6.2

Vitamin D: sources, metabolism, function and deficiency

Vitamin D ± or calciferol ± is a generic term that refers to cholecalciferol (vitamin D3) and ergocalciferol (vitamin D2). Vitamin D is a derivative of sterol with a chemical structure similar to other steroid hormones. The two forms of vitamin D differ only by the side chain to the sterol skeleton. The major vitamin D supply for humans comes from cholecalciferol. Ergocalciferol is formed by ultraviolet (UV) radiation from its precursor ergosterol in yeast and fungi, and does not contribute to dietary intake, unless supplements or fortification with vitamin D2 are used. The active metabolite of vitamin D, 1,25±dihydroxyvitamin D (1,25(OH)2D), maintains serum calcium and phosphorus concentrations within the normal range to support a wide variety of organ functions, including neural transmission, muscle contraction, cardiac function and blood coagulation, as well as optimising bone health. Vitamin D fulfils this role by enhancing the efficiency of the small intestine and the kidney to absorb calcium and phosphorus and by mobilising calcium and phosphorus from the bone. 6.2.1 Sources of vitamin D Cholecalciferol is produced endogenously when the skin is exposed to sunlight and can be obtained exogenously from the natural contents in foods, from food fortification and supplements. Vitamin D is found naturally in only few foods, mainly in fish and in lesser amounts in eggs (yolk), meat and milk products. Animal foods contributing to dietary vitamin D also contain 25±hydroxyvitamin D (25(OH)D). This vitamin D metabolite has a higher potency than native vitamin D. The precise potency of dietary 25(OH)D is not known, but it varies between 1.5 and 5 times that of vitamin D (Ovesen et al., 2003). Vitamin D is produced from the precursor 7-dehydrocholesterol in the skin when exposed to sunlight (Pilai and Bikle, 1991). In the skin 7-dehydrocholesterol is photochemically converted into precholecalciferol. Precholecalciferol is thermodynamically unstable and is isomerised into the more stable cholecalciferol, which slowly moves into the bloodstream. Endogenous vitamin D production depends on the length of time spent outside, the atmospheric conditions, clothing and sunscreen, season of the year, and most importantly the latitude. At subtropical latitudes cutaneous production continues throughout the winter, however in subjects living at high latitudes little or no vitamin D is produced in the skin during the winter months (Holick, 1995).

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6.2.2 Metabolism Vitamin D from food is absorbed mainly into the lymph (Thompson et al., 1966). In man and in many animals, adipose and muscular tissue is the principal storage site of vitamin D (Mawer et al., 1972). There is no evidence of a reduced intestinal absorption of vitamin D in the elderly (Clemens et al., 1986). Vitamin D from food and from the skin has no biological activity, and requires activation by two successive hydroxylation steps in, respectively, the liver and the kidney (Parfitt et al., 1982). The 25±hydroxylation in the liver is very fast and almost unregulated. Thus, 25(OH)D is the most abundant form of the vitamin in the circulation (Lund and DeLuca, 1966), which circulates in the blood with a biological half-life of one month (Clements et al., 1992). Further metabolism in the kidney of 25(OH)D to the physiologically active metabolite 1,25(OH)2D is strictly regulated by parathyroid hormone (PTH) (Shepard and DeLuca, 1980). Free 1,25(OH)2D is in equilibrium with the bound form, and it is only the free fraction, i.e., only 0.5% of the total amount of 1,25(OH)2D, which is hormonally active. The blood concentration of 1,25(OH)2D is maintained within a narrow range (normal range: 40±140 pmol/l), independent of normal variations of vitamin D supply and in circulating 25(OH)D (Vieth et al., 1990). The half-life of 1,25(OH)2D is 4±6 hours. In the blood 1,25(OH)2D is transported bound to a vitamin D binding protein (DBP). DPB seems to have a higher affinity for 25(OH)D than for 1,25(OH)2D and native vitamin D (Haddad and Walgate, 1976). DPB circulates in the plasma at much higher molar concentrations than the normal blood contents of its ligands. In the liver 25(OH)D and 1,25(OH)2D undergo degradation to several biologically inactive metabolites that are excreted in the bile. Under physiological conditions bile contains only trivial amounts of active metabolites (and native vitamin D); consequently enterohepatic circulation does not seem to be involved in the maintenance of vitamin D status (Clements et al., 1984). 6.2.3 Function of vitamin D The active vitamin D metabolite, 1,25(OH)2D functions as a steroid hormone through binding to its specific intranuclear receptor (VDR) causing changes in gene transcription (Brown et al., 1999). Several genetic polymorphisms of the VDR have been identified. The exact role of these in the expression of vitamin D function has not been clarified. Non-genomic functions have also been found for the active vitamin D metabolite. In the intestine 1,25(OH)2D binds to the VDR, and stimulates synthesis of several proteins, which participate in the transport of calcium from the intestinal lumen into the bloodstream (Reichel et al., 1989). Calcium absorption from the intestine is dependent on the amount of calcium in the diet and on physiological requirements. When dietary calcium is low almost all calcium is absorbed. 1,25(OH)2D also promotes the absorption of phosphorous and magnesium. The action of 1,25(OH)2D on bone is not well understood. It stimulates bone resorption by increasing the formation of osteoclasts (Suda et al., 1992),

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probably in part through stimulation of osteblast formation (Suda et al., 1995). The effect of 1,25(OH)2D on bone mineralisation appears to be indirect by stimulating the calcium and phosphorous supply, mainly by absorption from the intestine (Slovik et al., 1981). In the kidneys 1,25(OH)2D to a limited extent increases the reabsorption of calcium and phosphorous. The inhibition of PTH secretion by 1,25(OH)2D is yet another calcium regulatory function (Slatopolsky et al., 1992). Although 1,25(OH)2D is normally recognised as the active vitamin D metabolite, its precursor 25(OH)D, is absorbed more efficiently than vitamin D from the diet and has metabolic functions of its own in regulating cell growth and calcium metabolism (Barger-Lux et al., 1995; Heaney et al., 1997). Based on comparisons of molar calcium absorption potencies and blood concentrations of 25(OH)D and 1,25(OH)2D, it can be calculated that as much as 80% of circulating vitamin D activity would have to be due to 25(OH)D (Colodro et al., 1978). 6.2.4 New roles for vitamin D In the last couple of decades it has become apparent that vitamin D has other (noncalcaemic) functions in tissues. In fact, VDRs have been found in most body tissues suggesting a more fundamental role for vitamin D in human biology (Walters, 1992). Vitamin D has a role in modulating immune defence mechanisms. The receptor is found in significant concentrations in the Tlymphocyte, and animal studies have shown that vitamin D can suppress experimental autoimmune disorders (DeLuca and Cantorna, 2001). Epidemiological studies have suggested that providing supplemental vitamin D to infants may prevent the development of type 1 diabetes (Harris, 2002). However, human studies are presently too few to evaluate the potential of vitamin D in the prevention and treatment of diabetes and other autoimmune diseases. Vitamin D reduces proliferation, increases differentiation and induces apoptosis in different cell lines suggesting that the vitamin may be protective against cancers (Guyton et al., 2001). Some prospective epidemiological studies have found an inverse association between vitamin D intake and serum 25(OH)D concentration, and cancer risk, primarily colon (Garland et al., 1985; Martinez et al., 1996; Tangrea et al., 1997) and breast cancer (Shin et al., 2002). Well-designed clinical trials need to be conducted to determine if an increased intake of vitamin D is protective against some cancers. In the skin, vitamin D causes differentiation of keratinocytes and other skin cells. This has led to the use of topical 1,25(OH)2D and analogs in the treatment of psoriasis with some success (Mason et al., 2002). 6.2.5 Vitamin D status For assessment of vitamin D status the concentration of 25(OH)D in serum is considered as an accurate, integrative measure reflecting an individual's dietary

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intake and cutaneous production (Parfitt, 1998). Degree of long-term solar exposure and time spent outdoors are probably better predictors of serum 25(OH)D than is dietary intake (Thomas et al., 1998). The threshold concentration of 25(OH)D, which delimits insufficiency from sufficiency is controversial with proposed cut-off values ranging from 20 nmol/l to over 100 nmol/l (Chapuy et al., 1997; Dawson-Hughes et al., 1997a; Gloth et al., 1995; Holick, 1998; Ooms et al., 1995a; Thomas et al., 1998). Alternatively, a gradual scale has been proposed in which hypovitaminosis D is defined as a 25(OH)D concentration <100 nmol/l, vitamin D insufficiency as a 25(OH)D concentration <50 nmol/l, and vitamin D deficiency as a 25(OH)D concentration <25 nmol/l (McKenna and Freaney, 1998). Many studies have suggested that there is a value of 25(OH)D above which there is little further decrease in PTH (Chapuy et al., 1997). Consequently, there has been interest in the use of PTH as a functional indicator of vitamin D status but at the moment, there is no clear definition of an absolute value of 25(OH)D above which a subject is vitamin D replete (McKenna and Freaney, 1998). The concentration of 1,25(OH)2D, the biologically active form of vitamin D, will usually be normal or even slightly elevated in vitamin D deficiency, and therefore provides no information with respect to nutritional status (Hollis, 1996). Blood concentration of native vitamin D reflects recent intake of vitamin D and/or exposure to sunlight, and therefore may vary greatly over a short time in an individual. 6.2.6 Vitamin D deficiency The classical vitamin D deficiency diseases, rickets in children and osteomalacia in adults, are caused by severe lack of vitamin D. Rickets and osteomalacia are characterised by inhibited mineralisation of newly formed bone leading to uncalcified osteoid tissue (Hutchison and Bell, 1992). Vitamin D deficiency is accompanied by decreased serum 25(OH)D (usually <10 nmol/l), hypocalcaemia, hypophosphataemia and increased serum alkaline phosphatase, and secondary hyperparathyroidism. Vitamin D deficiency (osteomalacia) is associated with muscle weakness, especially of the proximal muscle groups, and falls (Mowe et al., 1999), however prospective studies on the effect of vitamin D supplementation on muscle strength and risk of falls have given conflicting results (Janssen et al., 2002). Low concentrations of 25(OH)D are associated with congestive heart failure (Zittermann et al., 2003) and higher blood pressure (Kristal-Boneh et al., 1997), and increasing 25OHD concentration through UV irradiation (Krause et al., 1998) or vitamin D supplementation (Pfeifer et al., 2001) may decrease blood pressure. Although vitamin D deficiency may have potentially important consequences along these pathways, the effects on health status in the elderly are uncertain. More recently, the concept of vitamin D insufficiency (inadequacy, marginal deficiency) has emerged. Vitamin D insufficiency may contribute to

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osteoporosis in the elderly by decreasing calcium absorption, with subsequent secondary hyperparathyroidism and bone loss. As people age the concentration of 25(OH)D decreases mainly because of a declining efficiency of skin to produce vitamin D (MacLaughlin and Holick, 1985). Also, elderly people may spend less time outdoors, especially if institutionalised, and their food intake, including fish intake decreases, adding to the risk of vitamin D insufficiency and consequently osteoporosis (Lips et al., 1987).

6.3

Vitamin D fortification and osteoporosis

Osteoporosis is defined as a systemic skeletal disease characterised by low bone mass and microarchitectural deterioration of bone tissue, with a consequent increase in bone fragility and susceptibility predisposing to an increased risk of a fracture (NIH Consensus Development Panel on Osteoporosis Prevention, Diagnosis and Therapy, 2001). Osteoporosis is without symptoms until fractures occur. Many data point towards a relationship between low vitamin D status and increased risk of fractures. Seasonal variations have been shown in the incidence of hip fractures (Jacobson et al., 1991), incidences being higher during winterspring than during summer-autumn months, similar to the seasonal fluctuations in 25(OH)D concentrations (Bouillon et al., 1987). Further, postmenopausal women have greater losses of bone density in winter months (Dawson-Hughes et al., 1991). Patients with recent osteoporotic fractures frequently, but not always, present lower 25(OH)D and higher PTH concentrations (Baker et al., 1979; Compston et al., 1989; Cooper et al., 1989; Falch et al., 1992; Lips et al., 1987; Weatherall, 2000). Concurrent osteomalacia may increase fracture risk. Biochemical (LeBoff et al., 1999; Rapin et al., 1982) or histological signs (Aaron et al., 1974; Chalmers et al., 1969; HaÈrmaÈ et al., 1987; Hordon and Peacock, 1990) of osteomalacia, have been reported in 10±30% of patients with hip fractures. Even a very sunny climate does not exclude the occurrence of osteomalacia (Eid, 1978; GannageÂ-Yared et al., 1998). Bone loss is an inevitable accompaniment to normal ageing starting at midlife in women as well as men, however, women experience a more rapid bone loss following menopause due to the reduction in oestrogen production. Family history is a significant predictor for osteoporosis; the risk is doubled in the daughter, if her mother has osteoporosis (Seeman et al., 1989). There are a variety of other factors, apart from family history and low vitamin D supply, which may hasten bone loss, including ethnicity, low body weight, low lifetime dietary calcium intake, caffeine intake, cigarette smoking, and inactivity (Cummings et al., 1995). Bone mass, at least up to the age of 75 years, is a function of peak bone mass achieved during childhood and adolescence. Optimisation of peak bone mass gives perhaps the best protection against the development of osteoporosis later

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in life (Heaney et al., 2000). Twin studies indicate that between 60 and 80% of peak bone mass is genetically determined (Kelly et al., 1993). 6.3.1 Significance of osteoporosis The clinical significance of osteoporosis lies in the increased risk of a fragility fracture. Common fractures include vertebral compression fractures, and fractures of forearm and the proximal femur (hip fractures). Osteoporotic fracture risk increases exponentially with age. Forearm fractures commonly occur at the perimenopause, vertebral fractures in the mid-sixties and the average for hip fractures is 80 years. Osteoporosis is particularly common in North America, Europe and Oceania where more than one-third of women and one-sixth of men will sustain one or more osteoporotic fractures in their lifetime. Osteoporotic fractures constitute a major public health problem. Fractures markedly increase mortality, decrease the quality of life, and represent a major source of health costs (Kanis et al., 2002). The most serious consequences arise in patients with hip fracture, which requires hospital admission for an average of 20±30 days and is associated with a 20% increase in mortality (European Commission, 1998). The osteoporotic fracture burden is expected to worsen, e.g., the number of hip fractures is expected to quadruple over the next 30 years, exceeding six million cases per year by 2050 (Cooper et al., 1992). 6.3.2 Does vitamin D reduce the risk of fractures? Vitamin D supplementation in elderly people rapidly improves vitamin D status, i.e., increases the concentration of 25(OH)D, decreases the concentration of PTH, and increases intestinal calcium absorption, suggesting a protective role against fracture (Chapuy et al., 1987; Lips et al., 1988). Observational studies on vitamin D deficiency as a predictor of bone loss (Hannan et al., 2000) or osteoporotic fractures (Cummings et al., 1998) are scarce, and have given conflicting results. In the Nurses' Health Study, which followed 72,337 postmenopausal women for 18 years, a significant inverse association between vitamin D intake from foods plus supplements and risk of hip fractures was demonstrated (Feskanich et al., 2003). Women consuming 12.5 g vitamin D per day had a 37% lower risk of hip fracture compared to women consuming <3.5 g/day. Because lengthy and expensive clinical trials are necessary to determine the effects of vitamin D on fracture risk, most studies have used surrogate endpoints, primarily changes in bone mineral density (BMD). In such trials Vitamin D supplementation (often in combination with calcium) has repeatedly been shown to increase BMD in several regions of the skeleton (Chapuy et al., 2002; DawsonHughes et al., 1995; Ooms et al., 1995b; Peacock et al., 2000). Although the role of BMD to predict fracture risk is well established (Marshall et al., 1996), the relationship between a change in BMD from a given intervention and a change in

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fracture risk is less secure. Consequently, the effectiveness of an intervention can ultimately be gauged only through fracture outcomes. An insufficient number of randomised, controlled trials have been conducted to clarify the effectiveness on fracture rates of community prevention programmes employing vitamin D supplementation, with and without calcium co-supplementation (Gillespie et al., 2003). Reduction of risk of vertebral fractures has been found in a meta-analysis by including studies that have used hydroxylated derivatives (1±alpha hydroxylated forms) of vitamin D (Papadimitropoulos et al., 2002). However, hydroxylated derivatives may have an effect of their own on bone, and are more expensive and less safe than vitamin D, which makes them unsuitable for primary intervention strategies such as fortification. Consequently, only, randomised controlled studies that have employed standard vitamin D will be shortly mentioned below. Chapuy et al. (1992; 1994) compared the administration of a placebo with oral vitamin D3 (20 g/day) with calcium (1.2 g/day) on the frequency of hip fractures in 2,790 women with a mean age of 84 years living in nursing homes. At three years, significantly fewer subjects in the active treatment group had sustained a hip fracture or any non-vertebral fracture. Oral vitamin D3 (10 g/day) (no calcium supplementation) given for a maximum of 3.5 years compared to placebo did not decrease the incidence of hip or other peripheral fractures in 2,578 independently living elderly Dutch men and women (Lips et al., 1996). It is possible that the lack of efficacy relates to the low dose of vitamin D used and the high habitual intake of calcium in Holland. Vitamin D3 (17.5 g/day) and calcium (0.5 g/day) supplementation reduced the incidence of osteoporotic non-vertebral fractures after three years in 389 healthy, independently living men and women in the United States with mostly normal serum 25(OH)D concentrations (Dawson-Hughes et al., 1997b). By far the most fractures occurred in the women. In a study from Norway performed on a population of 1,144 frail nursing home residents with a mean age of 84.7 years, intervention with 10 g of vitamin D3 did not prevent hip fracture or all non-vertebral fractures after two years of therapy (Meyer et al., 2002). Vitamin D was given as cod liver oil and the placebo as cod liver oil where vitamin D had been removed. Also, in this study lack of efficacy may be related to the low vitamin D dosage ± although about 40% of the population already took vitamin D supplements before entering the study ± and the fact that cod liver oil is rich in vitamin A, which has been suggested to promote bone loss. A study from the United Kingdom randomised 2,686 non-institutionalised men and women (most of them physicians) aged 65±85 to receive oral vitamin D3, 2,500 g every four months corresponding to an average of 20 g/day, or a matching placebo, sent to the participants by mail (Trivedi et al., 2003). After five years those allocated to supplementation with vitamin D had a significant 22% lower rate for fracture at any site and a 33% lower rate at any major osteoporotic site.

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Published randomised intervention studies have therefore shown discrepant results. Three studies have demonstrated an effect on fracture rates with an average dose of around 20 g per day; one of these studies used intermittent high-dose vitamin D supplementation and the other two studies cosupplemented with calcium. There is therefore presently insufficient scientific evidence, based on intervention studies, that fortification of food with vitamin D in doses relevant for fortification will have a beneficial effect on fracture risk in primary prevention programmes.

6.4

Dietary intake of vitamin D

Dietary intake surveys from various parts of Europe show that a substantial part of the elderly population has a vitamin D intake below the recommended dietary intakes (see below). Generally, vitamin D intake in the elderly is around 2±3 g/ day or less in Europe except in Iceland and Norway where fish intake is high and where the use of cod liver oil is common, especially among the elderly (Table 6.1). Direct comparison between countries is, however, difficult due to the different methods used for food intake estimation. Further, food tables differ in the way vitamin D content is expressed. Some food tables use content values derived from imprecise bioassay techniques, others use analysis data from more modern chemical methods of vitamin D determination, which do not include 25(OH)D (Deharveng et al., 1999). The imprecision is further augmented by the extreme paucity of systematic analytical data, and the uncertainty of the biological activity of 25(OH)D (Ovesen et al., 2003). A number of independent studies from around the world have shown that the elderly often have low serum concentrations of 25(OH)D coupled with elevations of PTH (Lips, 2001). Concentrations of 25(OH)D are somewhat higher in independent healthy subjects compared to patients in hospitals and residents of nursing homes, indicating the increased reliance on vitamin D in food in the institutionalised elderly. Serum concentrations of 25(OH)D varies between countries but is higher in the United States and Canada and in Scandinavia compared to the rest of Europe, a difference that at least partly can be explained by the higher intakes of vitamin D from fortification and supplements in the North American continent and in Scandinavia (McKenna, 1992). However, as indicated by international comparative studies the assays for 25(OH)D lack sufficient standardisation to allow comparison between countries, due to large inter- and intra-assay variations between laboratories (Lips et al., 1999). The European Euronut SENECA Study was done in 19 centres in 11 countries using a central laboratory facility (van der Wielen et al., 1995). Lowest mean winter serum 25(OH)D concentrations (21 nmol/l) were found in study centres in Greece and Spain and the highest in Switzerland and Norway (46 nmol/l). Low 25(OH)D concentrations could be explained by reduced sunlight exposure (time spent outside, clothing in sunshine) and low physical health

Table 6.1 Mean dietary vitamin D intake in representative samples of independent elderly from several countries (intake from supplements are not included unless indicated) Country

Survey

Method

Intake (g/day)

Denmark

The Danish Dietary Survey, 1995 Seven±day record (Danish Food Agency, 1996)

Males 45±54 55±64 65±74 75±80

Finland

The 1997 Dietary Survey of Finnish adults (National Public Health Institute, 1998)

24±hour recall

Males 45±54 y (n ˆ 346): 5.79 55±64 y (n ˆ 380): 6.33

France

INCA 1999 (Volatier, 2000)

Seven±day record

Males and females 45±64 y (n ˆ 389): 3.0 >65 y (n ˆ 245): 2.5

Germany

ErnaÈhrungsbericht 2000 (Deutsche Gesellschaft fuÈr ErnaÈhrung)

Three±day record

Males 65±74 y (n ˆ 361): 3.65 75±84 y (n ˆ 126): 3.47 >65 y (n ˆ 23): 4.08

y y y y

(n (n (n (n

ˆ ˆ ˆ ˆ

155): 3.6 128): 4.0 103): 3.3 44): 3.2

Females 45±54 y 55±64 y 65±74 y 75±80 y

(n (n (n (n

ˆ ˆ ˆ ˆ

147): 3.7 140): 3.9 122): 4.1 64): 3.7

Females 45±54 y (n ˆ 383): 3.87 55±64 y (n ˆ 398): 4.75

Females 65±74 y (n ˆ 503): 3.05 75±84 y (n ˆ 285): 2.95 >65 y (n ˆ 73): 2.55

Seven±day record. Intake from supplements included

Males 51±64 y (n ˆ 173): 4.4

Females 51±64 y (n ˆ 162): 5.8

Netherlands The Third Dutch National Food Consumption Survey 1997/1998 (Hulshof et al., 1998)

Two±day record

Males 50±65 y (n ˆ 454): 4.9 >65 y (n ˆ 185): 4.8

Females 50±65 y (n ˆ 512): 3.3 >65 y (n ˆ 236): 3.6

Norway

Norkost 1997 (Johansen and Solvoll, 1999)

Food frequency questionnaire

Males 50±59 y (n ˆ 196): 6.3 60±69 y (n ˆ 131): 5.6 70±79 y (n ˆ 106): 6.0

Females 50±59 y (n ˆ 196): 4.5 60±69 y (n ˆ 137): 4.0 70±79 y (n ˆ 109): 4.0

Sweden

Riksmaten 1997±98 (Becker and Pearson, 2002)

Seven±day record

Males 45±54 y (n ˆ 118): 6.6 55±64 y (n ˆ 68): 6.6 >65 y (n ˆ 65): 7.1

Females 45±54 y (n ˆ 153): 5.8 55±64 y (n ˆ 81): 6.1 >65 y (n ˆ 58): 4.9

United Kingdom

National Diet and Nutrition Survey. People aged 65 years and over (Finch et al., 1998)

Four±day record

Males 65±74 y (n ˆ 271): 4.25 75±84 y (n ˆ 265): 3.81 >85 y (n ˆ 96): 3.18

Females 65±74 y (n ˆ 256): 2.96 75±84 y (n ˆ 217): 3.03 >85 y (n ˆ 170): 2.31

Ireland

North±South Ireland Food Consumption Survey (O'Brien et al., 2001)

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status. The low concentrations of 25(OH)D in the south might also be explained by limited fortification of foods with vitamin D in this part of Europe. An inverse correlation between latitude and 25(OH)D has been demonstrated within countries (Chapuy et al., 1997). 6.4.1 Dietary recommendations There is a wide variation in dietary recommendations for vitamin D (Table 6.2). The difficulty in setting daily recommendations for vitamin D arises from the dual nature of its supply, and since various amounts of vitamin D originate from endogenous production, a recommendation cannot be determined accurately (Prentice, 2002). People, including the elderly, exposed to adequate amounts of sunlight probably need little vitamin D in the diet. Dependence of dietary vitamin D occurs when there is restricted skin exposure to sunlight (housebound or institutionalised, protective clothing, limited mobility) and reduced capacity for endogenous synthesis (dark skin, habitual use of sunscreen). In most countries the recommended daily intake ranges from 5 to 10 g, often at the higher intake levels in the elderly (and in infants) with less opportunity for skin production of vitamin D. Some committees recommend a higher intake for the elderly due to their high risk of vitamin D deficiency. It should be pointed out that there is controversy about how much input of vitamin D is required each day to meet or sustain `normal' concentrations of 25(OH)D in the blood, and that some groups advocate a much higher intake of vitamin D (around 100 g/ day) in populations without substantial body stores of vitamin D (Heaney et al., 2003; Vieth et al., 2001). 6.4.2 Safe level of intake High doses of vitamin D may lead to hypercalcaemia and hypercalciuria, increasing the likelihood of deposition of calcium in soft tissues, diffuse demineralisation of bones and irreversible renal and cardiovascular toxicity. The tolerance to vitamin D seems to be quite high because the conversion into 1,25(OH)2D is under tight feed-back control. A rare problem is an increased conversion of 25(OH)D to 1,25(OH)2D, which may occur in patients with primary hyperparathyroidism and granulomatous diseases such as sarcoidosis, tuberculosis or malignant lymphoma (Fuss et al., 1992). There are only a few data from which to establish vitamin D safety and toxicity limits in healthy subjects, however, based on available data, different scientific bodies have laid down upper intake levels of vitamin D. Upper intake level is the maximum level of total (from all sources) chronic daily intake of a nutrient judged to be unlikely to pose a risk of adverse health effects to humans. The American Food and Nutrition Board (Standing Committee on the Scientific Evaluation of Dietary Reference Intakes, 1997) guidelines specify 50 g/day as the highest vitamin D intake that healthy adults can consume (the noadverse-effect level; NOAEL) and 95 g/day as the lowest observed adverse-

Table 6.2

Examples of current vitamin D recommendations* for people over 50 years

Country

Responsible body

Recommendation*

Comments

Denmark, Norway, Sweden

Nordic Council of Ministers (1996)

Recommended dietary intake: <60 y: 5 g 61 y: 10 g

Subjects over 65 y are recommended 10 g as supplement (only in Denmark)

United Kingdom

Committee on Medical Aspects of Food Policy (COMA) (1991)

Reference nutrient intake: >65 y: 10 g

Ten g for those confined indoors, irrespective of age

Germany, Austria, Switzerland

Deutsche Gesellschaft fuÈr ErnaÈhrung, Population reference intake: È sterreichische Gesellschaft fuÈr O 51±65 y: 5 g ErnaÈhrung, Schweizerische 66 y: 10 g Vereinigung fuÈr ErnaÈhrung (2000)

Netherlands

Gezondheidsraad (2000)

Adequate intake: 51±60 y: 5 g 61±70 y: 7.5 g 71 y: 12.5 g

Higher recommendations with limited exposure to sunlight and dark skin colour

Belgium

MinisteÁre des Affairs Sociale, de la Sante Publique et de l'Environment (2000)

Recommended dietary intake: 61 y: 10 g

France

Agence FrancËaise de SecuriteÂ, Sanitaire des Aliments (AFFSA) (2001)

Recommended dietary intake Adults: 5 g Old: 10 g

United States, Canada

Institute of Medicine (Standing Committee on the Scientific Evaluation of Dietary Reference Intakes, 1997)

Adequate intake: 51±70 y: 10 g 71 y: 15 g

Australia

National Health and Medical Research Council (NHMRC) (1991)

Recommended dietary intake: 0 g

No recommendations for adults under 60 y, only a satisfactory intake (2.5±10 g). Ten g is recommended for postmenopausal women

Ten g for those who are housebound, if not exposed for 1±2 hours per week to direct sunlight in summer

* Different names are given to the recommendation, e.g., recommended dietary allowance (RDA), recommended dietary intake (RDI), recommended nutrient intake (RNI) or population reference intake (PRI). The recommended intake is defined as the intake of an essential nutrient considered being adequate to meet known nutritional needs of practically all healthy persons. The recommended intake is normally calculated as the average requirement +2 SD. An adequate intake is set instead of an RDA if sufficient scientific evidence is not judged to be available to calculate an estimated average requirement. The average intake is based on observed or experimentally determined estimates of average nutrient intake by a group of healthy people.

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effect level (LOAEL). The EC Scientific Committee on Food (2002) considers a dose of 100 g vitamin D/day and a serum concentration of 200 nmol 25(OH)D/l as the NOAEL, and by including an uncertainty factor of two to account for interindividual variation, the Scientific Committee comes to an upper intake level of 50 g vitamin D/day for adults. Other national and international expert groups also have dealt with upper safe levels of vitamin D (Bernier, 1995; Committee on Medical Aspects of Food Policy, 1991; Nordic Council of Ministers, 1996). Vitamin D2 may have a greater potential for harm since DPB may have a weaker affinity for vitamin D2 than for D3 metabolites, increasing the proportions of free vitamin D2 metabolites (Nilsson et al., 1972).

6.5

Strategies to improve vitamin D supply

There are other strategies than nutritional addition of vitamin D to foods (fortification), which can be used to improve vitamin D supply at the population level. Such strategies include education; dietary supplementation and increased exposure to UV light. Often more than one strategy must be undertaken simultaneously to secure the supply of a given nutrient to the target population. This is the case with vitamin D, where securing the more elderly an adequate supply requires consideration of all four strategies. 6.5.1 Education The basic aim of nutrition education is to get consumers to eat a diet that promotes health and decreases the risk of nutrition-related diseases. Recommendations for a healthy diet vary somewhat between countries, however most nutritionists would agree that a healthy diet may contain about 250±300 g lean and fatty fish, 3±4 eggs, and 700 g lean meat and 3,500 ml lean milk per week (Table 6.3). With no fortified foods available, the vitamin D content of a diet in accordance with recommendations could bring the intake up to about 5 g per day. In addition, it is considered difficult to introduce foods with a high natural vitamin D content into the daily diet of the elderly. Thus, a recommended intake of vitamin D of more than 5 g/day for the elderly cannot be realised unless the vitamin is supplied by other means. 6.5.2 Supplementation Vitamin D supplementation can effectively increase vitamin D status in the elderly (Byrne et al., 1995) and offers some advantages compared to fortification. The recommendation can be aimed specifically at the persons who need an increased supply, thereby reducing the risk that others get an inexpedient high intake. The disadvantage with supplementation is that to be effective it requires a positive action by the persons who need the supplement

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Functional foods, ageing and degenerative disease

Table 6.3 Typical vitamin D content in a recommended diet (values are taken from the Danish Food Tables (National Food Agency, 1996)) Per week 150 g trout* 50 g smoked mackerel 50 g smoked halibut 50 g canned sardine 3 (180 g) eggs 700 g lean meat 3,500 ml low fat milk Total

19.5 g 2.9 g 2.5 g 6.0 g 2.5 g 1.4 g 2.9 g 37.7 g per week = 5.4 g per day

*Vitamin D content in fatty fish shows large variations

(or by health care personnel taking care of the elderly), and for that to happen educational programmes are necessary. No data exist on the efficacy of a vitamin D supplementation programme, however, experience of other supplementation programmes suggests that compliance will be low, despite intensive public health messages promoting supplement use (Chan et al., 2001; Health Education Authority, 1998). Vitamin D can be given as a continuous low-dose replacement in doses of 10±20 g/day. An alternative to continuous low-dose replacement is single highdose therapy (2,500 to 5,000 g) repeated on a biannual or yearly basis (Davies et al., 1985; Weisman et al., 1986). Intermittent high-dose supplementation may be useful in elderly subjects in whom compliance with daily supplementation regimes is poor. 6.5.3 Exposure to UV light Regular exposure to sunlight is an alternative form of prophylaxis that involves no risk of vitamin D intoxication. As little as 30 minutes per day spent outdoors during summertime increases serum concentration of 25(OH)D in the elderly (Reid et al., 1986). Skin synthesis of vitamin D may also be enhanced by exposure to artificial UV light (Dunnigan et al., 1986 Snell et al., 1978). A study has shown that ultraviolet irradiation with half the minimal erythemal dose on the lower back three times per week for 12 weeks is as effective as 10 g vitamin D orally in increasing 25(OH)D in old people (Chel et al., 1998). However, the increased exposure to ultraviolet light may increase the risk of skin malignancy, skin burns and eye disease (kerato-conjunctivitis and cataract). Advising older people to spend more time outdoors should be encouraged, but is not always practical for the infirm who are most at risk.

Vitamin D fortification and bone health

6.6

155

Food fortification: reducing deficiency diseases

Food fortification (or enrichment) is commonly defined as the addition of one or more essential nutrients to a food, whether or not it is normally contained in the food, for the purpose of preventing or correcting a demonstrated nutrient deficiency in the population or specific population groups (Food and Agriculture Organization, 1996). The goal for any food fortification is to increase the nutrient intake for the target population to as close as possible to the recommended intake, while at the same time maintaining safe levels of intake for all persons. Two other terms for the addition of nutrients to foods are used: restoration (the replacement compensates for losses during production, e.g., vitamin C to juices and nectars, B vitamins to flour); and substitution (addition to a substitute product to the levels in the food, which it is designed to resemble; e.g., vitamin A to margarine). The practice of adding essential nutrients to foods was first introduced in the 1920s to reduce deficiency disorders, which were prevalent at that time in the United States and Europe. Food fortification has most likely played an important role in the decline of deficiency diseases, e.g., niacin fortification of flour and bread in the elimination of pellagra (Park et al., 2000), iodine fortification of salt in the decline of goitre (Wu et al., 2003), and vitamin D fortification of margarine and milk in the disappearance of rickets (Council on Foods and Nutrition, 1955). More recently folic acid fortification of cereal products has proved effective in the decline of neural tube defects (Honein et al., 2001; Ray et al., 2002). Over the last couple of decades there has been a steep increase in fortification programmes in the developing countries, and considerable progress has been made in reducing particularly vitamin A and iodine deficiencies in some of these countries (Darnton-Hill and Nalubola, 2002). From a public health perspective, food fortification should serve the nutritional needs of the population. Within this framework, fortification policies should be judged on whether the health and function of the population is improved or harmed. The risk and benefit of food fortification is a function of the distribution of the nutritional requirements and susceptibility to toxicity, neither of which are well characterised for most nutrients, including vitamin D. At least for some nutrients (e.g., addition of folic acid to cereal products) fortification has proved to be a cost-effective method of increasing micronutrient intakes in populations. Fortification also has the distinct advantage of requiring less change in consumer behaviours than the other nutrient interventions. However, fortification implies much more than just deciding to add a nutrient to a food and putting the fortified food on the market. Without convincing consumers and consumer organisations, food industry and trade organisations, and policy makers and health professionals of the need and benefits of fortification, its sustainability will always be at stake. The Codex Alimentarius Commission (FAO/WHO, 1994) has outlined `general principles for the addition of essential nutrients to foods' for the

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Functional foods, ageing and degenerative disease

`rational addition of essential nutrients to foods'. According to these principles any fortification programme should meet the following conditions: 1.

2. 3. 4. 5.

There should be a demonstrated need for increasing the intake of the nutrient (vitamin D) in one or more target groups. This may be in the form of actual clinical or subclinical evidence of deficiency, estimates indicating low levels of intake of the nutrient or possible deficiency likely to develop because of changes taking place in food habits. The food selected as a vehicle for the nutrient should be consumed by the population at risk. The intake of the food selected as a vehicle for the essential nutrient should be stable and uniform, and the lower and upper levels of intake should be known. The amount of nutrient added should be sufficient to correct or prevent deficiency when the food is consumed in normal amounts by the population at risk. The amount of nutrient added should not result in excessive intakes by individuals with a high intake of a fortified food.

Consequently an ongoing process and impact monitoring (including monitoring of side effects), and a periodic evaluation of the efficacy of the fortification is necessary. Unfortunately, vitamin D fortification programmes to combat bone disease in the elderly have not been adequately monitored for their impact on vitamin D status, efficacy and side effects.

6.7

Issues in vitamin D fortification of food

While food fortification continues to be a widely used mechanism to increase vitamin D intake in many industrialised countries, prevailing attitudes and legislation towards it differ, and there is no general consensus regarding the extent to which vitamin D fortification should be practised (Nordic Council of Ministers, 1995). Differences in legislation particularly pertain to whether general permission is given for the addition, or whether the authorities require an individual authorisation or a notification process in connection with the addition. In some countries vitamin D addition is completely free, while the most restrictive countries permit addition only after authorisation based on a scientifically documented public health need. The most common foods used for fortification purposes are margarine, vegetable oils and milk, however several other products are on the market with added vitamin D, e.g., dairy products (other than milk), breakfast cereals, bread and juices. The amounts of vitamin D maximally allowed to be added to foods differ widely between countries, e.g., in margarine from around 2 g/100 g to more than 10 g/100 g, however in most countries the level is around 7±8 g/100 g. In Australia there is mandatory fortification of low fat spreads and table margarine, and voluntary fortification of modified skim milks, and powdered

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milk and other dairy products. In New Zealand voluntary fortification of margarine and dairy foods is permitted. In the United States it is voluntary to add vitamin D to milk products and margarine based on a general permission granted on food standards, while there are no regulations controlling addition to unstandardised foods. In the United States vitamin D is also added to most breakfast cereals and a few fruit juices. Legislation in Canada requires vitamin D to be added to margarine and all milk products. In Canada control is exerted via a positive list of foods to which vitamin D must be added. On the European continent fortification with vitamin D also varies between countries (a directive is presently under way, harmonising the regulations for voluntary addition of vitamins and minerals to foods in the European Union). In the Scandinavian countries the addition of vitamins to food is quite strictly controlled, and preferably justified by a public health need. Denmark allows voluntary addition of vitamin D to margarine, Norway addition to margarine, butter and oils, and Sweden allows addition to oils and margarine, while Finland has voluntary addition of vitamin D to milk and margarine. In the United Kingdom, the addition of vitamin D to margarine is mandatory, however, voluntary addition is not restricted in terms of types of food, nutrients or levels of nutrients, provided the addition is not injurious to health, whereas for example Belgium adopts a middle approach whereby additions of vitamins and minerals are allowed for all foods, but notification is needed. 6.7.1 Basic characteristics of fortificant and food vehicle The vitamin D forms used in fortification are D3 and D2. They are formed by irradiation of the appropriate sterol followed by purification procedures. Commercially available forms include fat-soluble crystals for use in high fat content foods, and encapsulated, stabilised versions of the fortificant, suitable for use in dry products to be reconstituted with water. Vitamin D is not inactivated by pasteurisation or sterilisation (Hartman and Dryden, 1974; Upreti et al., 2002). Conflicting results have been published regarding the stability of vitamin D to oxidation, light, pH and heat (Cremin and Power, 1985; Kreutler, 1980; Kutsky, 1981). It is relatively stable in fat solutions, and under proper storage conditions, vitamin D has been demonstrated to be stable throughout product shelf life (Renken and Warthesen, 1993; Upreti et al., 2002). In contrast, vitamin D is unstable in aqueous sugar solutions, in which vitamin D, particularly vitamin D2, breaks down within days (Trang et al., 1998). Powders of vitamin D2 have long been known to degrade faster than dry vitamin D3 when exposed to high temperatures and humidity, and under storage conditions where the vitamin comes in contact with air and light (Grady and Thacker, 1980). Synthetic vitamin D2 used to be the form added to food. During the past two to three decades vitamin D3 has increasingly been used to fortify milk, margarine and other foods worldwide. The requirements for a potential food vehicle for fortification are well established (Table 6.4). The selection of an appropriate vehicle is a critical step

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Functional foods, ageing and degenerative disease

Table 6.4 Requirements for a food vehicle for fortification (Food and Agriculture Organization, 1996) Commonly consumed by the target organisation Constant consumption pattern with a low risk of excess consumption Good stability during storage Relatively low cost Centrally processed with a minimal stratification of the fortificant No interactions between the fortificant and the carrier food Contained in most meals, with the availability unrelated to socio-economic status Linked to energy intake

for fortification to be successful. In many cases identification of suitable vehicles is made difficult by the absence of reliable information on the dietary habits of the target population. 6.7.2 Bioavailability When a food has added vitamin D, then attention must also be paid to the bioavailability of the vitamin. Components of the food matrix may affect the functionality of the fortificant, and selection of the vehicle in fortification programmes must be such as to avoid reduced bioavailability of nutrients due to the presence of anti-nutritional compounds. Early studies have indicated that the vehicle used in which vitamin D is administered could influence bioavailability. For instance, clinical reports (measurements of growth rates in children) indicated an approximately threefold increase in the potency of vitamin D when cod liver oil was given emulsified in milk compared to vitamin D given in pure cod liver oil (Stearns et al., 1936). The overall picture is that vitamin D added to milk and margarine is considered to be highly bioavailable, however bioavailability studies for other fortified foods in a normal eating pattern are needed. It has been assumed that vitamin D2 and D3 have equal potencies, however, there is some evidence that vitamin D3 is more effective in raising serum 25(OH)D concentrations (Trang et al., 1998). The importance of the chemical form of vitamin D, i.e., a lower biological efficiency of vitamin D2 compared to vitamin D3, should be noted. 6.7.3 Effectiveness of fortification on vitamin D status There is probably little doubt that a certain amount of a fortified food consumed regularly and for an extended period improves vitamin D status (Subar and Bowering, 1988). This was demonstrated in young adults in whom the seasonal decline of 25(OH)D concentration was reduced by 50% with the daily consumption of 350 ml of fortified milk (vitamin D content: 12 g/l) over the winter (McKenna et al., 1995). However, in elderly people the effect of inclusion of fortified foods on vitamin D status has been modest at best. A cross-

Vitamin D fortification and bone health

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sectional epidemiological study of elderly people conducted in the United Kingdom found that 25(OH)D concentrations were somewhat higher in those consuming fortified margarine daily compared with those using it less frequently (36.0 vs. 30.0 nmol/l) (Scragg et al., 1995). A study from the United States using data from the third National Health and Nutrition Examination Survey (NHANES III) in women of reproductive age demonstrated that the prevalence of vitamin D insufficiency (<37.5 nmol/l) was reduced in women using fortified foods, however the difference in 25(OH)D concentrations between non-users of fortified foods (milk and cereals) and those consuming three or more servings per week was modest (6±7 nmol/l) (NesbyO'Dell et al., 2002). A small rise in serum concentration of 25(OH)D, insufficient to correct deficiency, was also demonstrated in a dependent (Keane et al., 1992) and a community-based (Keane et al., 1998) elderly population with low baseline 25(OH)D concentrations who were encouraged to drink 400±500 ml of vitamin D fortified milk (10 g/l). In frail elderly subjects daily consumption for 17 weeks of fortified dairy products, which brought the intake of vitamin D up to recommended levels (from 3.2 to 11.6 g/day). Significant increases in 25(OH)D were observed (de Jong et al., 1999). In contrast, no increases were found in a study in long-stay male and female residents of geriatric wards who received fortified foods (margarine, butter or milk) as part of their daily diet for a period of six months to a year (Dunnigan et al., 1986). There is only little scientific evidence therefore that consumption of fortified products, with the fortification levels practised today, markedly improves the vitamin D status of the most important target group, the frail elderly. 6.7.4 The ideal food for fortification For fortification to be an effective way to increase intake and improve status of vitamin D in a population a suitable food or a combination of foods must be identified, i.e., foods that are consumed by almost everyone in the target group, and preferably with a Gaussian intake distribution and a narrow dispersion of intake. There are, however, only few foods ± if any ± with these intake characteristics. Model calculations of vitamin D intakes in a target population (women, 65± 74 years) based on known intake distributions of butter, margarine, cheese and milk with the addition of defined amounts of vitamin D, underline some of the problems with fortification (Table 6.5). The calculations are made for total vitamin D intakes of around 5 g/day and 10 g/day, respectively, as the goal for the mean of the population. It can be seen that fortification of milk, cheese, and butter and margarine alone is far from optimal due to the skewed distribution of the intake of these foods in the elderly population. The combined fortification of all milk (with 1.1 g/100 g) and butter plus margarine (with 9.5 g/100 g) would probably be the best option, and would leave few people with an intake above 40 g/day, whilst more than half of the target group would have

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Table 6.5 Total vitamin D intake (g/day) with different vitamin fortification approaches. Data are based on individual intakes of the chosen foods and vitamin D in the Danish adult population from the Danish Dietary Survey, 1995 (Danish Food Agency, 1996) Men and women, 15±80 years (n ˆ 1,837) Food (fortification

Mean

10 percentile

Median

90 percentile

Milk (0.5 g/100g) Milk (2.2 g/100g) Butter/margarine (4.5 g/100g) Butter/margarine (18.5 g/100g) Cheese (6 g/100g) Cheese (26 g/100g) Milk (0.3 g/100g) and butter/margarine (2 g/100g) Milk (1.1 g/100g) and butter/margarine (9.5 g/100g)

4.6 9.3 4.8 10.0 5.3 12.1 4.7

2.0 3.0 2.3 4.6 2.7 5.1 2.4

4.1 8.1 4.2 9.2 4.8 11.4 4.2

7.8 17.0 7.7 16.3 8.3 20.0 7.5

10.0

4.9

9.3

15.7

Food (fortification

Mean

10 percentile

Median

90 percentile

Milk (0.5 g/100g) Milk (2.2 g/100g) Butter/margarine (4.5 g/100g) Butter/margarine (18.5 g/100g) Cheese (6 g/100g) Cheese (26 g/100g) Milk (0.3 g/100g) and butter/margarine (2 g/100g) Milk (1.1 g/100g) and butter/margarine (9.5 g/100g)

4.8 8.6 5.6 11.5 5.6 11.9 5.2

2.3 3.1 3.1 6.0 2.7 4.6 2.8

4.2 8.0 4.8 11.4 5.1 11.9 4.3

8.1 14.9 8.6 18.6 9.8 19.4 8.5

10.1

5.8

9.4

15.9

Women, 65±74 years (n ˆ 103)

an intake of 10 g/day. However, this would still leave a large proportion of the elderly with a low vitamin D intake (those elderly who consume insignificant amounts of milk, butter and margarine). It should also be noted that only 10% of the elderly would have an intake of 15 g of vitamin D per day, the adequate intake for the elderly over 65 years, proposed by the Food and Nutrition Board. The suggested fortifications will leave only a few with intakes above the upper limit of intake (however, the consumption of 2 l/day of fortified milk (2.2 g/100 g) ± not an unusual amount in adolescent boys ± will increase vitamin D intake to 44 g/day). Since there does not seem to be an ideal food for vitamin D fortification, another approach to secure everyone in the target group a certain amount of

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vitamin D is to expand the number of foods to which the vitamin is added. There has been some concern that a free addition to several foods may lead to an excessive intake of vitamin D (Meltzer et al., 2002). Model calculations have shown that even with fortification of 25% of all potentially fortifiable foods, vitamin D can be added (at nutritionally relevant levels defined as 0.16  recommended daily allowance per 100 kcal portion) safely to foods, i.e., the addition does not exceed upper safe levels in high consumers (Flynn et al., 2003). However, such calculations are notoriously imprecise mainly because intakes from dietary intake studies, particularly at the upper (and lower) intake levels of nutrients, are imprecise. A factor to take into account is that the real level being added to food often is two- or threefold the declared amount, because the producer is assuming nutrient losses during storage (Choumenkovitch et al., 2002; Quinlivan and Gregory, 2003). Even if nutrient addition to foods does not lead to a toxic intake, an imbalance of the nutrient intakes might have negative effects, an issue that is still poorly understood. An example of this could be the finding that chronic excess intake of vitamin A is associated with decreased bone mineral density and increased hip fracture risk (Feskanich et al., 2002). The high content of vitamin A in fortified milk and margarine might thus nullify the beneficial effect of vitamin D on bone metabolism. The intake of vitamin A in fortified milk has been suggested to be the explanation for the high incidence of osteoporosis in northern European countries despite a high calcium intake in these countries (Whiting and Lemke, 1999). 6.7.5 Over- and under-fortification Large variations in vitamin D fortification levels in milk have repeatedly been demonstrated (Chen et al., 1993; Holick et al., 1992; Murphy et al., 2001; Tanner et al., 1988), to a large degree due to inconsistencies in the procedures used for fortification (Hicks et al., 1996; Murphy et al., 2001). Outbreaks of idiopathic infantile hypercalcaemia in the United Kingdom in the post-World War 2 era led to the withdrawal of vitamin D fortification from all foods in the country because of concern that they were due to hypervitaminosis D caused by technical problems with over-fortification (Anon., 1956). Although manufacturing control is better nowadays, reports of hypervitaminosis D caused by over-fortification of food have been published (intake of >100 g of vitamin D from the fortified food) with important health consequences for the consumer (Blank et al., 1995; Jacobus et al., 1992). Conversely, a low level of vitamin D in the finished product has been found in other studies (Chen et al., 1993; Holick et al., 1992), and could render it nutritionally ineffective. In the United States the frequent finding of low levels of vitamin D, particularly in milk of lower fat content, has been ascribed to the addition of vitamin D to the raw milk (Tanner et al., 1988). Because vitamin D is added before fat separation, the vitamin is removed when milk fat is removed.

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Further research is needed to determine how addition of vitamin D at different sites affects final vitamin concentrations in fortified milk products. 6.7.6 Monitoring, control and evaluation Monitoring, control and evaluation are critical aspects of a programme of vitamin D fortification, but are unfortunately largely ignored. Monitoring and control include measures to ensure that the food is fortified at adequate levels and is properly labelled when it reaches the consumer. The addition of any nutrient to a food for the purpose of fortification must be monitored and controlled within the framework of an effective quality assurance programme, which includes issuance of laws and regulations, inspection and analyses and quality assurance, by the food industry based on Hazard Analysis and Critical Control Points (HACCP). A good monitoring and control system needs specific mechanisms for prompt corrective actions to be taken when problems are identified. Evaluation includes identifying patterns of consumer behaviour in terms of consumption of fortified foods; determining the supply of vitamin D and the contribution of the fortified food to this intake; and the impact on the public health problem being addressed, e.g., fracture risk reduction. Monitoring and evaluation is also helpful, and may be necessary, to address concerns of safety of food fortification. Demonstrating public health benefits and safety through monitoring and evaluation increases public confidence, and is thus crucial for the sustainability of the fortification programme. 6.7.7 Claims A particular issue of interest is that of nutrition claims and health claims to be made for foods to which nutrients are added. However, care should be taken that the use of claims does not result in practices that could mislead the consumer. There is always the risk that the information on food labels overemphasises or distorts the role of a single food or component in enhancing good health. 6.7.8 What characterises a successful fortification programme? Fortification of foods with vitamin D, together with other strategies mentioned can probably be a useful tool in increasing vitamin D intake and improving a low vitamin D status in the population (i.e., increase concentrations of 25(OH)D and decrease concentrations of PTH), that might decrease bone loss and hopefully reduce the risk of fractures in the elderly. However, to be successful a vitamin D fortification programme needs at least the following basic issues to be properly taken care of: · a strong political commitment and the ability to enforce regulations in a facilitative manner

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· early private-sector involvement and willingness to comply with regulations · public sector-backing including endorsement by professional medical organisations · a sound scientific basis in the design of the fortification programme, including ± documentation of the severity and effects of the deficiency in the relevant population groups ± good data on food consumption on the individual level ± measures to monitor and evaluate the implementation of the fortification and ± timely and effective corrective actions where needed.

6.8

Future trends

There are several gaps in our knowledge concerning the role of vitamin D in the occurrence of bone loss and fractures in the elderly. Science will most likely give us some answers to the many questions crucial for the argument to justify the introduction of fortification programmes as a tool to improve vitamin D status in the population. One very important question that has not, as of now, been answered scientifically is: to what extent ± if any ± does vitamin D insufficiency contribute to osteoporosis and fracture? Also, the criteria for the assessment of vitamin D status in individuals must and will most likely be refined, and reliable, accurate, simple and rapid technology for the assessment of vitamin D status must be developed. The establishment of the lowest effective dose of vitamin D is decisive to ensure that a vitamin D fortification programme has the potential to fulfil its basic requirements to be maximally effective with minimum harm. It is equally important to have up-to-date and reliable food and nutrient intake data on the individual level. This and the data basis for the vitamin D contents in food will in the coming years be refined by the increased use of modern analytical procedures (HPLC). Standardisation of fortification procedures will improve, guided by research to determine how the addition of vitamin D at different sites affects final vitamin concentrations in fortified food products. Technical problems such as possible interactions with the food carrier matrix or with other nutrients may not be an issue with respect to the currently used foods for vitamin D fortification. However, there is reason to believe that an increasing variety of foods will be fortified making such problems relevant. Finally, national legislation concerning fortified foods will be harmonised, e.g., within the EU. The movement of fortified foods between countries with populations with different eating habits will require the establishment of an effective control, monitoring and evaluation programme.

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6.9

Sources of further information

Currently a major research programme with relevance for vitamin D fortification is being undertaken (Towards an Optimal Strategy to Vitamin D Fortification, OPTIFORD, under the EU Framework 5 Quality of Life Work Program). The purpose of the OPTIFORD project is to examine whether fortification of food with vitamin D is a feasible strategy to remedy the insufficient vitamin D status of large population groups in Europe, and to determine at what level the fortification should be pitched (for further information, see http://optiford.org). Other research projects, which may be pertinent to seek information from, are the Venus Project (http://www.venus-ca.org) and the Osteodiet Project (http:// osteodiet.ucc.ie). The Report on Osteoporosis in the European Community ± Action for Prevention published by the European Commission (1998) provides a common basis for action by presenting recommendations and detailed information on all relevant areas: epidemiology and demographics, nutrition, physical exercise, diagnosis, therapy and facilitating communication on osteoporosis. The report can be downloaded from http://www.osteofound.org/publications/pdf/ eu_report_98. Another recent Health and Technology report (Kanis et al., 2002) focusing on the cost-effectiveness of the different interventions in the management of established osteoporosis can be downloaded from http://www.ncchta.org. Readers seeking information about the regulatory principles and practices for fortification of foods in different countries are referred to the report by the Nordic Council of Ministers (1995). Information about the technology and quality control of fortification programmes can be read in the FAO report: Food fortification. Technology and quality control (1996) or downloaded from http:// www.fao.org/docrep/W2840E/w2840e00.htm.

6.10

References

AARON JE, GALLAGHER JC, ANDERSON J, STASIAK L, LONGTON, EB, NORDIN BEC, NICHOLSON M

(1974), `Frequency of osteomalacia and osteoporosis in fractures of the proximal femur', Lancet, 1 229±233. AGENCE FRANCËAISE DE SECURITEÂ, SANITAIRE DES ALIMENTS (AFFSA) (2001), Apports Nutritionnels ConseilleÂs. ANONYMOUS (1956) `Hypercalcaemia in infants and vitamin D', BMJ, 2, 149. BAKER MR, MCDONNELL H, PEACOCK M, NORDIN BEC (1979), `Plasma 25±hydroxy vitamin D concentrations in patients with fractures of the femoral neck', BMJ, 1, 589. BARGER-LUX MJ, HEANEY RP, LANSPA SJ, HEALY JC, DELUCA HF (1995), `An investigation of sources of variation in calcium absorption efficiency', J Clin Endocrinol Metab, 80, 406±411. BECKER W, PEARSON M (2002), Kostvanor och na È ringsintag i Sverige, Livsmedelsverket.  curite dans les consummations BERNIER JJ (1995), Rapport sur les limites de se alimentaires des vitamines et des mineraux, Conseil SupeÂrieur d'HygieÁne Publique

Vitamin D fortification and bone health

165

de France.

(1995), `An outbreak of hypervitaminosis D associated with overfortification of milk from home-delivery dairy', Am J Public Health, 85, 656±659. BOUILLON R, AUXWERX J, LISSENS W, PELEMANS W (1987), `Vitamin D status in the elderly: seasonal substrate deficiency causes 1,25±dihydroxycholecalciferol deficiency', Am J Clin Nutr, 45, 755±763. BROWN AJ, DUSSO A, SLATOPOLSKY S (1999), `Vitamin D', Am J Physiol, 277, F157±F175. BYRNE PM, FREANEY R, MCKENNA MJ (1995), `Vitamin D supplementation in the elderly: review of safety and effectiveness of different regimes', Calcif Tissue Int, 56, 518± 520. CHALMERS J, BARCLAY A, DAVISON AM, MACLEOD DAD, WILLIAMS DA (1969), `Quantitative measurements of oseoid in health and disease', Clin Orthopaed Rel Res, 63, 196± 209. CHAN A, PICKERING J, HAAN E, NETTING M, BURFORD A, JOHNSON A, KEANE RJ (2001), ` ``Folate before pregnancy'': the impact on women and health professionals of a population-based health promotion campaign in South Australia', Med J Aust, 74, 631±636. CHAPUY MC, CHAPUY P, MEUNIER PJ (1987), `Effect of calcium and vitamin D supplements on calcium metabolism in the elderly', Am J Clin Nutr, 46, 324±328. BLANK S, SCANLON KS, SINKS TH, LETT S, FALK H

CHAPUY MC, ARLOT ME, DUBOEUFF F, BRUN J, CROUZET B, ARNAUD S, DELAMS PD, MEUNIER PJ

(1992), `Vitamin D3 and calcium to prevent hip fractures in elderly women', N Engl J Med, 327, 1637±1642. CHAPUY MC, ARLOT ME, DELMAS PD, MEUNIER PJ (1994), `Effect of calcium and cholecalciferol treatment for three years on hip fractures in elderly women', BMJ, 308, 1081±1082. CHAPUY MC, PREZIOSI P, MAAMER M, ARNAUD S, GALAN, P, HERCBERG S, MEUNIER PJ (1997), `Prevalence of vitamin D insufficiency in an adult normal population', Osteoporosis Int, 7, 439±443. CHAPUY MC, PAMPHILE R, PARIS E, KEMPF C, SCHLICHTING M, ARNAUD S, GARNERO P, MEUNIER

(2002), `Combined calcium and vitamin D3 supplementation in elderly women: confirmation of reversal of secondary hyperparathyroidism and hip fracture risk: the Decalyos II study', Osteoporos Int, 13, 257±264.

PJ

CHEL VG, OOMS ME, POPP-SNIJDERS C, PAVEL S, SCHOTHORST AA, MEULEMANS CCE, LIPS P

(1998), `Ultraviolet irradiation corrects vitamin D deficiency and suppresses secondary hyperparathyroidism in the elderly', J Bone Miner Res, 13, 1238±1242. CHEN TC, SHAO Q, HEATH H, HOLICK MF (1993), `An update on the vitamin D content of fortified milk from the United States and Canada', N Engl J Med, 329, 1507. CHOUMENKOVITCH SF, SELHUB J, WILSON PWF, RADER JI, ROSENBERG IH, JACQUES PF (2002), `Folic acid intake from fortification in United States exceeds predictions', J Nutr 132, 2792±2798. CLEMENS TL, ZHOU X-Y, MYLES M, ENDRES D, LINDSAY R (1986), `Serum vitamin D2 and vitamin D3 metabolite concentrations and absorption of vitamin D2 in elderly subjects', J Clin Endocrinol Metab, 63, 656±660. CLEMENTS MR, CHALMERS TM, FRASER DR (1984), `Enterohepatic circulation of vitamin D: a reappraisal of the hypothesis', Lancet, 1, 1376±1379. CLEMENTS MR, DAVIES M, HAYES ME, HICKEY CD, LUMB GA, MAWER EB, ADAMS PH (1992), `The role of 1,25±dihydroxyvitamin D in the mechanism of acquired vitamin D deficiency', Clin Endocrinol, 37, 17±27.

166

Functional foods, ageing and degenerative disease

(1978), `Effect of 25± hydroxyvitamin D3 on intestinal absorption of calcium in normal man and patients with renal failure', Metabolism, 27, 745±753. COMMITTEE ON MEDICAL ASPECTS OF FOOD POLICY (COMA) (1991), Dietary reference values for food energy and nutrients for the United Kingdom, Department of Health. COMPSTON JE, SILVER AC, CROUCHER PI, BROWN RC, WOODHEAD JS (1989), `Elevated serum intact parathyroid hormone levels in elderly patients with hip fracture', Clin Endocrinol, 31, 667±672. COOPER C, MCLAREN M, WOOD PJ, COULTON L, KANIS JA (1989), `Indices of calcium metabolism in women with hip fractures', Bone Miner, 5, 193±200. COOPER C, CAMPION G, MELTON LJ (1992), `Hip fractures in the elderly: a world-wide projection', Osteoporosis Int, 2, 285±289. COUNCIL ON FOODS AND NUTRITION (1955), `Importance of vitamin D in milk', JAMA 159, 1018±1019. CREMIN FM, POWER P (1985), `Vitamins in bovine and human milk', ed. Fox PF, Developments in dairy chemistry: vol. 3, Elsevier Appl. Sci. Publ., Essex, p: 337±398. COLODRO IH, BRICKMAN AS, COBURN JW, OSBORN TW, NORMAN AW

CUMMINGS SR, NEVITT MC, BROWNER WS, STONE K, FOX KM, ENSRUD KE, CAULEY J, BLACK D,

(1995), `Risk factors for hip fracture in white women. Study of Osteoporotic Fractures Research Group', N Engl J Med, 332, 767±773. CUMMINGS SR, BROWNER WS, BAUER D, STONE K, ENSRUD K, JAMAL S, ETTINGER B (1998), `Endogenous hormones and the risk of hip and vertebral fractures among older women', N Engl J Med, 339, 733±738. DANISH FOOD AGENCY (1996), The Danish dietary survey 1995, Danish Food Agency, publication No. 235. DARNTON-HILL I, NALUBOLA R (2002), `Fortification strategies to meet micronutrient needs: successes and failures', Proc Nutr Soc, 61, 231±241. DAVIES M, MAWER EB, HANN JT, STEPHENS WP, TAYLOR JL (1985), `Vitamin D prophylaxis in the elderly: a simple effective method suitable for large populations', Age Ageing, 14, 349±354. DAWSON-HUGHES B, DALLA GE, KRALL EA, HARRIS S, SOKOL LJ, FALCONER G (1991), `Effect of vitamin D supplementation on wintertime and overall bone loss in healthy postmenopausal women', Ann Intern Med, 115, 505±512. DAWSON-HUGHES B, HARRIS SS, KRALL EA, DALLAL GE, FALCONER G, GREEN CL (1995), `Rates of bone loss in postmenopausal women assigned to one of two dosages of vitamin D', Am J Clin Nutr, 61, 1140±1145. DAWSON-HUGHES B, HARRIS SS, DALLAL GE (1997a), `Plasma calcidiol, season, and serum parathyroid hormone concentrations in healthy elderly men and women', Am J Clin Nutr, 65, 67±71. DAWSON-HUGHES B, HARRIS SS, KRALL EA, DALLAL GE (1997b), `Effect of calcium and vitamin D supplementation on bone density in men and women 65 years of age or older', N Engl J Med, 337, 670±676. DEHARVENG G, CHARRONDIERE UR, SLIMANI N, SOUTHGATE DAT, RIBOLI E (1999), `Comparison of nutrients in the food composition tables available in nine European countries participating in EPIC', Eur J Clin Nutr 42, 2449-2453. DE JONG N, ADAM SGM, DE GROOT LCPGM, DE GRAAF C (2000), `Variability of micronutrient content in enriched dairy and fruit products', Intern J Food Sci Technol 51, 247± 257. DELUCA HF, CANTORNA MT (2001), `Vitamin D: its role in immunology', FASEB J, 15, 2579±2585. VOGT TM

Vitamin D fortification and bone health È HRUNG È R ERNA DEUTSCHE GESELLSCHAFT FU

Heinrich, Frankfurt am Main.

167

(2000), ErnaÈhrungsbericht 2000, Druckerei

È HRUNG, O È STERREICHISCHE GESELLSCHAFT FU È R ERNA ÈR DEUTSCHE GESELLSCHAFT FU È HRUNG, SCHWEIZERISCHE VEREINIGUNG FU È HRUNG (2000), Referenzwerte È R ERNA ERNA

fuÈr die NaÈhrstoffzufuhr, Frankfurt am Main: Umschau/Braus. (1986), `The prevention of vitamin D deficiency in the elderly', Scott Med J, 31, 144±149. EID AM (1978), `Osteomalacia as a contributing factor in fracture of the femoral neck in the elderly in Qatar', Clin Orthopaed Rel Res, 132, 129±135. EUROPEAN COMMISSION (1998), Report on osteoporosis in the European Community, Action for prevention. FALCH JA, MOWe M, BONNER T, HAUG E (1992), `Serum levels of intact parathyroid hormone in elderly patients with hip fracture living at home', Acta Endocrinol, 126, 10±12. FAO/WHO (1994), General principles for the addition of essential nutrients to foods. CAC/ GL 09-1987, volume 4. FESKANICH D, SINGH V, WILLETT WC, COLDITZ GA (2002), `Vitamin A intake and hip fractures among postmenopausal women', JAMA, 287, 47±54. FESKANICH D, WILLETT WC, COLDITZ GA (2003), `Calcium, vitamin D, milk consumption, and hip fractures: a prospective study among postmenopausal women', Am J Clin Nutr, 77, 504±511. FINCH S, DOYLE W, LOWE C, BATES CJ, PRENTICE A, SMITHERS G, CLARKE PC (1998), National Diet and Nutrition Survey. People aged 65 years and over. Volume 1: Report of the diet and nutrition survey, HMSO, Norwich. FOOD AND AGRICULTURE ORGANIZATION (1996), Food Fortification: Technology and Quality Control. Report of an FAO Technical Meeting, Rome, 20±23 November 1995, FAO Food and Nutrition Paper. Rome: FAO. È LLER DJG, ROLAND V (2003), `Vitamins FLYNN A, MOREIRAS O, STEHLE P, FLETCHER RJ, MU and minerals: a model for safe addition to foods', Eur J Nutr, 42, 118±130. FUSS M, PEPERSACK T, GILLET C, KARMALI R, CORVILAIN J (1992), `Calcium and vitamin D metabolism in granulomatous disease', Clin Rheumatol, 11, 28±36. GANNAGEÂ-YARED MH, BRAX H, ASMAR A, TOHME A (1998), `Statut vitaminique D des sujets ages. Etude de la population libanaise', La Presse Medicale, 27, 900±904. GARLAND C, SHEKELLE RB, BARRETT-CONNOR E, CRIQUI MH, ROSSOF AH, PAUL O (1985), `Dietary vitamin D and calcium and risk of colorectal cancer: a 19-year prospective study in men', Lancet, 1, 307±309. GEZONDHEIDSRAAD (2000), Voedingsnormen: calcium, vitamin D, thiamine, riboflavine, niacine, pantotheenzur en biotine, Den Haag, Publicatie nr 2000/12. GILLESPIE WJ, AVENELL A, HENRY DA, O'CONNELL DL, ROBERTSON J (2003), `Vitamin D and vitamin D analogues for preventing fractures associated with involutional and postmenopausal osteoporosis (Cochrane Review)', In: The Cochrane Library, Issue 1, Oxford: Update Software. GLOTH FM, GUNDBERG CM, HOLLIS BW, HADDAD JG, TOBIN JD (1995), `Vitamin D deficiency in homebound elderly persons', JAMA, 274, 1683±1686. GRADY LT, THAKKER KD (1980), `Stability of solid drugs: degradation of ergocalciferol (vitamin D2) and cholecalciferol (vitamin D3) at high humidities and elevated temperatures', J Pharm Sci, 69, 1099±1102. GUYTON KZ, KENSLER TW, POSNER GH (2001), `Cancer chemoprevention using natural vitamin D and its synthetic analogs', Annu Rev Pharmacol Toxicol, 41, 421±442. HADDAD JG, WALGATE J (1976), `25±hydroxyvitamin D transport in human plasma', J Biol DUNNIGAN MG, FRASER SA, MCINTOSH WB, MOSELEY H, SUMNER DJ

168

Functional foods, ageing and degenerative disease Chem, 251, 4803±4809.

HANNAN MT, FELSON DT, DAWSON-HUGHES B, TUCKER KL, CUPPLES LA, WILSON PWF, KIEL DP

(2000), `Risk factors for longitudinal bone loss in elderly men and women: the Framingham Osteoporosis Study', J Bone Miner Res, 15, 710±720. È RMA È M, PARVIAINEN M, KOSKINEN T, HOIKKA V, ALHAVA E (1987), `Bone density, HA histomorphometry and biochemistry in patients with fractures of the hip or spine', Ann Clin Res, 19, 378±382. HARRIS S (2002), `Can vitamin D supplementation in infancy prevent type 1 diabetes?', Nutr Rev, 60, 118±121. HARTMAN AM, DRYDEN LP (1974), `Vitamins in milk and milk products', Fundamentals of dairy chemistry, 2nd edn, ed. Webb BH, Johnson AH, Alford JA, van Nostrand Reinhold, New York, pp. 325±401. HEALTH EDUCATION AUTHORITY (1998), Folic Acid Update, Health Education Authority, London, England. HEANEY RP, BARGER-LUX MJ, DOWELL MS, CHEN TC, HOLICK MF (1997), `Calcium absorptive effects of vitamin D and its major metabolites', J Clin Endocrinol Metab, 82, 4111±4116. HEANEY RP, ABRAMS S, DAWSON-HUGHES B, LOOKER A, MARCUS R, MATKOVIC V, WEAVER C

(2000), `Peak bone mass', Osteoporosis Int, 11, 985±1009. (2003), `Human serum 25± hydroxycholecalciferol response to extended oral dosing with cholecalciferol', Am J Clin Nutr, 77, 204±210. HICKS T, HANSEN AP, RUSHING JE (1996), `Procedures used by North Carolina dairies for vitamins A and D fortification of milk', J Dairy Sci, 79, 329±333. HOLICK MF (1995), `Environmental factors that influence the cutaneous production of vitamin D', Am J Clin Nutr, 61(suppl), 638S-645S. HOLICK MF (1998), `Redefining vitamin D insufficiency'. Lancet, 351, 805±806. HOLICK MF, SHAO Q, LIU WW, CHEN TC (1992), `The vitamin D content of fortified milk and infant formula', N Engl J Med, 326, 1178±1181. HOLLIS BW (1996), `Assessment of vitamin D nutritional and hormonal status: what to measure and how to do it', Calcif Tissue Int, 58, 4±5. HONEIN MA, PAULOZZI LJ, MATHEWS TJ, ERICKSON JD, WONG L-YC (2001), `Impact of folic acid fortification of the US food supply on the occurrence of neural tube defects', JAMA, 285, 2981±2986. HORDON LD, PEACOCK M (1990), `Osteomalacia and osteoporosis in femoral neck fracture', Bone Miner, 11, 247±59. HULSHOF KFAM, KISTEMAKER C, BOUMAN M (1998), De inname van energie en voedingsstoffen door Nederlandse bevolkingsgroepen Voedselconsumptiepeiling 1997±1998, TNO report V98.805. Zeist. HUTCHISON FN, BELL NH (1992), `Osteomalacia and rickets', Sem Nephrol, 12, 127±145. JACOBSON SJ, GOLDBERG J, MILES TP, BRODY JA, STIERS W, RIMM AA (1991), `Seasonal variation in the incidence of hip fractures among white persons aged 65 years and older in the United States', Am J Epidemiol Rev, 133, 996±1004. HEANEY RP, DAVIES KM, CHEN TC, HOLICK MF, BARGER-LUX MJ

JACOBUS CH, HOLICK M, SHAO Q, CHEN TC, HOLM IA, KOLODNY JM, FULEIHAN GE-J, SEELY EW

(1992), `Hypervitaminosis associated with drinking milk', N Engl J Med, 326, 1173±1177. JANSSEN HCJP, SAMSON M, VERHAAR HJJ (2002), `Vitamin D deficiency, muscle function, and falls in elderly people', Am J Clin Nutr, 75, 611±615. JOHANSSON L, SOLVOLL K (1999), Norkost 1997, Statens RaÊd for Ernñring og fysisk

Vitamin D fortification and bone health

169

aktivitet, Rapport nr. 2/1999, Oslo.

(2002), Treatment of established osteoporosis: a systematic review and cost-utility analysis, Health Technology Assessment 2002, 6. KEANE EM, ROCHFORT A, COX J, MCGOVERN D, COAKLEY D, WALSH JB (1992), `Vitamin-Dfortified liquid milk a highly effective method of vitamin D administration for house-bound and institutionalised elderly', Gerontology, 38, 280±284. KEANE EM, HEALY M, O'MOORE RO, WALSH JB (1998), `Vitamin D-fortified liquid milk: benefits for the elderly community-based population', Calcif Tissue Int, 62, 300± 302. KELLY PJ, NGUYEN T, HOPPER J, POCOCK N, SAMBROOK P, EISMAN J (1993), `Changes in axial bone density with age: a twin study', J Bone Miner Res, 8, 11±17. È HRING M, HOPFENMU È LLER W, HOLICK MF, SHARMA AM (1998), `Ultraviolet B KRAUSE R, BU and blood pressure', Lancet, 352, 709±710. KREUTLER PA (1980), `Fat soluble vitamins', Nutrition in perspective, ed. Kreutler PA, Prentice-Hall inc., Englewood Cliffs, NJ, pp. 216±253. KRISTAL-BONEH E, FROOM P, HARARI G, RIBAK J (1997), `Association of calcitriol and blood pressure in normotensive men', Hypertension, 30, 1289±1294. KUTSKY RJ (1981), Handbook of vitamins, minerals and Hormones, van Nostrand Reinhold Co., New York. LEBOFF MS, KOHLMEIER L, HURWITZ S, FRANKLIN J, WRIGHT J, GLOWACKI J (1999), `Occult vitamin D deficiency in postmenopausal US women with acute hip fracture', JAMA, 281, 1505±1511. LIPS P (2001), `Vitamin D deficiency and secondary hyperparathyroidism in the elderly: consequences for bone loss and fractures and therapeutic implications', Endocr Rev, 22, 477±501. KANIS JA, BRAZIER JE, STEVENSON M, CALVERT NW

LIPS P, VAN GINKEL FC, JONGEN MJM, RUBERTUS F, VAN DER VIJGH WJF, NETELENBOS JC

(1987), `Determinants of vitamin D status in patients with hip fracture and in elderly control subjects', Am J Clin Nutr, 46, 1005±1010.

LIPS P, WIERSINGA A, VAN GINKEL FC, JONGEN MJM, NETELENBOS JC, HACKENG WHL, DELAMS

(1988), `The effect of vitamin D supplementation on vitamin D status and parathyroid function in elderly subjects', J Clin Endocrinol Metab, 67, 644±650. LIPS P, GRAAFMANS WC, OOMS ME, BEZEMER D, BOUTER LM (1996), `Vitamin D supplementation and fracture incidence in elderly persons', Ann Intern Med, 124, 400±406. LIPS P, CHAPUY MC, DAWSON-HUGHES B, POLS HAP, HOLICK MF (1999), `An international comparison of serum 25±hydroxyvitamin D measurements', Osteoporosis Int, 9, 394±397. LUND J, DELUCA HF (1966), `Biologically active metabolite of vitamin D3 from bone, liver, and blood serum', J Lipid Res, 7, 739±744. MACLAUGHLIN J, HOLICK MF (1985), `Ageing decreases the capacity of human skin to produce vitamin D3', J Clin Invest, 76, 1536±1538. MARSHALL D, JOHNELL O, WEDEL H (1996), `Meta-analysis of how well measures of bone mineral density predict occurrence of osteoporotic fractures', BMJ, 312, 1254± 1259. PD, VAN DER VIJGH WJF

MARTINEZ ME, GIOVANNUCCI EL, COLDITZ GA, STAMPFER MJ, HUNTER DJ, SPEIZER FE, WING A, WILLETT WC (1996), `Calcium, vitamin D, and the occurrence of colorectal cancer among women', J Natl Cancer Inst, 88, 1375±1382.

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(2002), `Topical preparations for the treatment of psoriasis: a systematic review', Br J Dermatol, 146, 351±364. MAWER EB, BACKHOUSE J, HOLMAN CA, LUMB GA, STANBURY SW (1972), `The distribution and storage of vitamin D and its metabolites in human tissues', Clin Sci, 43, 413± 431. MCKENNA MJ (1992), `Differences in vitamin D status between countries in young adults and the elderly', Am J Med, 93, 69±77. MCKENNA MJ, FREANEY R (1998), `Secondary hyperparathyroidism in the elderly: means to defining hypovitaminosis D', Osteporosis Int, Suppl. 8, S3±S6. MASON J, MASON AR, CORK MJ

MCKENNA MJ, FREANEY R, BYRNE P, MCBRINN Y, MURRAY B, KELLY M, DONNE B, O'BRIEN M

(1995), `Safety and efficacy of increasing wintertime vitamin D and calcium intake by milk fortification', QJ Med, 88, 895±898. MELTZER HM, ARO A, ANDERSEN NL, KOCH B, ALEXANDER J (2002), `Risk analysis applied to food fortification', Publ Health Nutr, 6, 281±290. MEYER HE, SMEDSHAUG GB, KVAAVIK E, FALCH JA, TVERDAL AA, PEDERSEN JI (2002), `Can vitamin D supplementation reduce the risk of fracture in the elderly? A randomised controlled trial', J Bone Miner Res, 17, 709±715. MINISTEÁRE DES AFFAIRS SOCIALE, DE LA SANTEÂ PUBLIQUE ET DE L'ENVIRONMENT (2000) Apports quotidiens recommandeÂs pour les vitamines. MOWEÂ M, HAUG E, BéHMER T (1999), `Low serum calcidiol concentration in older adults with reduced muscular function', J Am Geriatr Soc, 47, 220±226. MURPHY SC, WHITED LJ, ROSENBERRY LC, HAMMOND BH, BANDLER DK, BOOR KJ (2001), `Fluid milk vitamin fortification compliance in New York state', J Dairy Sci, 84, 2813±2820. NATIONAL FOOD AGENCY (1996), The Composition of Foods, 4th edn, The National Food Agency. NATIONAL HEALTH AND MEDICAL RESEARCH COUNCIL (NHMRC) (1991), Recommended dietary intakes for use in Australia. NATIONAL PUBLIC HEALTH INSTITUTE (1998), The 1997 dietary survey of Finnish adults. National Public Health Institute: Helsinki. NESBY-O'DELL S, SCANLON KS, COGSWELL ME, GILLESPIE C, HOLLIS BW, LOOKER AC, ALLEN C,

(2002), `Hypovitaminosis D prevalence and determinants among African American and white women of reproductive age: third National Health and Examination Survey, 1988±1994', Am J Clin Nutr, 76, 187±192.

DOUGHERTLY C, GUNTER EW, BOWMAN BA

NIH CONSENSUS DEVELOPMENT PANEL ON OSTEOPOROSIS PREVENTION, DIAGNOSIS AND THERAPY

785±795.

(2001), `Osteporosis prevention, diagnosis and therapy', JAMA, 285,

(1972), `Binding of vitamin D to its human carrier plasma protein', Biochem Biophys Res Comm, 46, 1380±1387. NORDIC COUNCIL OF MINISTERS (1995), Addition of nutrients to food principles and practices. TemaNord, 643. NORDIC COUNCIL OF MINISTERS (1996), Nordic nutrient recommendations 1996, KoÈpemhamn: Nordiska MinisterraÊdet, Nord 1996:28. O'BRIEN MM, KIELY M, HARRINGTON KE, ROBSON PJ, STRAIN JJ, FLYNN A (2001), `The North/ South Ireland Food Consumption Survey: vitamin intakes in 18±64-year-old adults', Publ Health Nutr, 4, 1069±1079. È STBERG L, PETERSON PA NILSSON SF, O

OOMS ME, LIPS P, ROOS JC, VAN DER VIJGH WJF, POPP-SNIJDERS C, BEZEMER D, BOUTER LM

(1995a), `Vitamin D status and sex hormone binding globulin: determinants of

Vitamin D fortification and bone health

171

bone turnover and bone mineral density in elderly women', J Bone Miner Res, 10, 1177±1184. OOMS ME, ROOS JC, BEZEMER PD, VAN DER VIJGH WJF, BOUTER LM, LIPS P (1995b). `Prevention of bone loss by vitamin D supplementation in elderly women: a randomised, double-blind trial', J Clin Endocrinol Metab, 80, 1052±1058. OVESEN L, BROT C, JAKOBSEN J (2003), `Food contents and biological activity of 25± hydroxyvitamin D: a vitamin D metabolite to be reckoned with?', Ann Nutr Metab, 47, 107±113. PAPDIMITROPOULOS E, WELLS G, SHEA B, GILLESPIE W, WEAVER B, ZYTARUK N, CRANNEY A, ADACHI J, TUGWELL P, JOSSE R, GREENWOOD C, GUYATT G, THE OSTEOPOROSIS

(2002), `VII: meta-analysis of the efficacy of vitamin D treatment in preventing osteoporosis in postmenopausal women', Endocrine Rev, 23, 560±569. PARFITT AM, GALLAGHER JC, HEANEY RP, JOHNSTON CC, NEER R, WHEDON GD (1982), `Vitamin D and bone health in the elderly', Am J Clin Nutr, 36, 1014±1031. PARFITT AM (1998), `Osteomalacia and related disorders', In: Metabolic bone disease and clinically related disorders, eds, Avioli LV, Krane SM, pp. 327±86. San Diego, CA: Academic Press. PARK YK, SEMPOS CT, BARTON CN, VANDERVEEN JE, YETLEY EA (2000), `Effectiveness of food fortification in the United States: the case of pellagra', Am J Public Health, 90, 727±738. PEACOCK M, LIU G, CAREY M, MCCLINTOCK R, AMBROSIUS W, HUI S, JOHNSTON CC (2000), `Effect of calcium or 25OH vitamin D3 dietary supplementation on bone loss at the hip in men and women over the age of 60', J Clin Endocrinol Metab 85, 3011± 3019. PFEIFER M, BEGEROW B, MINNE HW, NACHTIGALL D, HANSEN C (2001), `Effects of short-term vitamin D3 and calcium supplementation on blood pressure and parathyroid hormone levels in elderly women`, J Clin Endocrinol Metab, 86, 1633±1637. PILAI S, BIKLE DD (1991), `Epidermal vitamin D metabolism, function, and regulation', Adv Lip Res, 24, 321±341. PRENTICE A (2002), `What are dietary requirements for calcium and vitamin D?' Calcif Tissue Int, 70, 83±88. QUINLIVAN EP, GREGORY JF (2003), `Effect of food fortification on folic acid intake in the United States', Am J Clin Nutr, 77, 221±225. RAPIN CH, LAGIER R, BOIVIN G, JUNG A, MCGEE W (1982), `Biochemical findings in blood of aged patients with femoral neck fractures: a contribution to the detection of occult osteomalacia', Calcif Tissue Int, 34, 465±469. RAY JG, MEIER C, VERMEULEN MJ, BOSS S, WYATT PR, COLE DEC (2002), `Association of neural tube defects and folic acid food fortification in Canada', Lancet, 360, 2047± 2048. REICHEL H, KOEFFLER HP, NORMAN AW (1989), `The role of vitamin D endocrine system in health and disease', N Engl J Med, 320, 980±991. REID IR, GALLAGHER DJA, BOSWORTH J (1986), `Prophylaxis against vitamin D deficiency in the elderly by regular sunlight exposure', Age Ageing, 15, 35±40. RENKEN SA, WARTHESEN JJ (1993), `Vitamin D stability in milk', J Food Sci, 58, 552±556. SCIENTIFIC COMMITTEE ON FOOD (2002), Opinion of the Scientific Committee on Food on the tolerable upper intake level of vitamin D, SCF/CS/NUT/UPPLEV/38 Final. SCRAGG R, KHAW K-T, MURPHY S (1995), `Life-style factors associated with winter serum 25±hydroxyvitamin D levels in elderly adults', Age Ageing, 24, 271±275. METHODOLOGY GROUP AND THE OSTEOPOROSIS RESEARCH ADVISORY GROUP

172

Functional foods, ageing and degenerative disease

(1989), `Reduced bone mass in daughters of women with osteoporosis', N Engl J Med, 320, 554±558. SHEPARD RM, DELUCA HF (1980), `Plasma concentrations of vitamin D3 and its metabolites in the rat as influenced by vitamin D3 or 25±hydroxyvitamin D3 intakes', Arch Biochem Biophys, 202, 43±53. SHIN M-H, HOLMES MD, HANKINSON SE, WU K, COLDITZ GA, WILLETT WC (2002), `Intake of dairy products, calcium, and vitamin D and risk of breast cancer', J Natl Cancer Inst, 94, 1301±1311. SLATOPOLSKY E, BERKOBEN M, KELBER J, BROWN A, DELMEZ J (1992), `Effects of calcitriol and non-calcemic vitamin D analogs on secondary hyperparathyroidism', Kidney Int, 42 (Suppl. 38), S43±S49. SLOVIK DM, ADAMS JS, NEER RM, HOLICK MF, POTTS JT JR (1981), `Deficient production of 1,25±dihydroxyvitamin D in elderly osteoporotic patients', N Engl J Med 305, 372±374. SNELL AP, MACLENNAN WJ, HAMILTON JC (1978), `Ultraviolet irradiation and 25±hydroxyvitamin D levels in sick old people', Age Ageing, 7, 225±228. SEEMAN E, HOPPER JL, BACH LA, COOPER ME, PARKINSON E, MCKAY J, JERUMS G

STANDING COMMITTEE ON THE SCIENTIFIC EVALUATION OF DIETARY REFERENCE INTAKES

(1997), Dietary reference intakes for calcium, phosphorus, magnesium, vitamin D and fluoride, National Academy Press, Washington, D.C. STEARNS G, JEANS PC, VANDECAR V (1936), `The effect of vitamin D on linear growth in infancy', J Pediatr, 9, 1. SUBAR AF, BOWERING J (1988). `The contribution of enrichment and fortification to nutrient intake of women', J Am Diet Assoc 88, 1237±1245. SUDA T, TAKAHASHI N, ABE E (1992), `Role of vitamin D in bone resorption', J Cell Biochem, 49, 53±58. SUDA T, TAKAHASHI N, MARTIN J (1995), `Modulation of osteoclast differentiation: update 1995', Endocrine Rev, 4, 266±270. TANGREA J, HELZLSOUER K, PIETINEN P, TAYLOR P, HOLLIS B, VIRTAMO J, ALBANES D (1997), `Serum levels of vitamin D metabolites and the subsequent risk of colon and rectal cancer in Finnish men', Cancer Causes Control, 8, 615±625. TANNER JT, SMITH J, DEFIBAUGH P, ANGYAL G, VILLALOBOS M, BUENO MP, MCGARRAHAN ET,

(1988), `Survey of vitamin content in fortified food', J Assoc Off Anal Chem, 71, 607±610.

WEHR HM, MUNIZ JF, HOLLIS BW, KOH Y, REICH P, SIMPSON KL

THOMAS MK, LLOYD-JONES DM, THADHANI RI, SHAW AC, DERASKA DJ, KITCH BT, VAMVAKAS

(1998), `Hypovitaminosis D in medical inpatients', N Engl J Med, 338, 777±783. 3 THOMPSON GR, LEWIS B, BOOTH CC (1966), `Absorption of vitamin D3- H in control subjects and patients with intestinal malabsorption', J Clin Invest, 45, 94±102. TRANG HM, COLE DEC, RUBIN LA, PIERRATOS A, SIU S, VIETH R (1998), `Evidence that vitamin D3 increases serum 25±hydroxyvitamin D more efficiently than does vitamin D2', Am J Clin Nutr, 68, 854±858. TRIVEDI DP, DOLL R, KHAW KT (2003), `Effect of four monthly oral vitamin D3 (cholecalciferol) supplementation on fractures and mortality in men and women living in the community: randomised double blind controlled trial', BMJ, 326, 469±474. UPRETI P, MISTRY VV, WARHESEN JJ (2002), `Estimation of fortification of vitamin D3 in pasteurised process cheeses', J Dairy Sci, 85, 3173±3181. EC, DICK IM, PRINCE RL, FINKELSTEIN JS

È WIK MRH, VAN DEN BERG H, DE GROOT LCPGM, HALLER J, MOREIRAS VAN DER WIELEN RPJ, LO

Vitamin D fortification and bone health

173

O, VAN STAVEREN WA (1995), `Serum vitamin D concentrations among elderly people in Europe', Lancet, 346, 207±210. VIETH R, MCCARTEN K, NORWICH KH (1990), `Role of 25±hydroxyvitamin D3 dose in determining rat 1,25±dihydroxyvitamin D3 production', Am J Physiol, 258, E780± E789. VIETH R, CHAN P-CR, MACFARLANE GD (2001), `Efficacy and safety of vitamin D3 intake exceeding the lowest observed adverse effect level', Am J Clin Nutr, 73, 288±294. Ã te Individuelle et Nationale sur les consommations VOLATIER J-L (2000), Enque Alimentaires, Editions TEC et DOC Lavoisier, Paris. WALTERS MR (1992), `Newly identified actions of the vitamin D endocrine system', Endocr Rev, 13, 719±764. WEATHERALL M (2000), `A meta-analysis of 25 hydroxyvitamin D in older people with fracture of the proximal femur, NZ Med J, 113, 137±140. WEISMAN Y, SCHEN RJ, EISENBERG Z, AMARILIO N, GRAFF E, EDELSTEIN-SINGER M, GOLDRAY D,

(1986), `Single oral high-dose vitamin D3 prophylaxis in the elderly', J Am Geriatr Soc, 34, 515±518. WHITING B, LEMKE SJ (1999), `Excess retinol intake may explain the high incidence of osteoporosis in northern Europe', Nutr Rev, 57, 192±198. WU T, LIU GJ, LI P, CLAR C (2003), `Iodised salt for preventing iodine deficiency disorders (Cochrane Review)', in: The Cochrane Library, Issue 2, Oxford: Update Software. È RFER R, STEHLE P (2003), ZITTERMANN A, SCHLEITHOFF SS, TENDERICH G, BERTHOLD HK, KO `Low vitamin D status: a contributing factor in the pathogenesis of congestive heart failure', J Am Coll Cardiol, 41, 105±112. HARELL A

7 Calcium citrate (TCC) and bone health S. Edelstein, The Weizmann Institute of Science, Israel

7.1

Introduction: bone formation and calcium fortification

Calcium is a major constituent of bone and teeth, and is the most abundant mineral in the body. Apart from being a major constituent of hard tissue, this mineral plays an important role in many physiological systems. Since the body cannot manufacture calcium, all calcium must be obtained from the diet. However, a very large percentage of populations fail to meet the recommendations for optimal calcium intake. Inadequate intake of calcium is associated with reduced bone mineral density, resulting in osteoporosis. Osteoporosis affects more than 25 million people in the United States only, resulting in 1.5 million fractures annually at a cost of over ten billion dollars per year to the health care system. Adequate intake of calcium is important in order to achieve maximal peak bone mass in the first three decades of life, and to minimize the rate of bone loss associated with ageing. The recommendations for optimal intake of calcium are shown in Table 7.1. Thus, the importance of adequate calcium intake is recognized during the whole life cycle of the human being; body growth, rapid skeleton development of children and teenagers, achievement of peak bone mass in adults, women at child-birth age and lactation, and elderly people, especially women at postmenopausal age who are most susceptible to osteoporosis.

7.1.1 The role of calcium in the calcification process of mineralized tissues The process of calcification of mineralized tissues differs from one bone to another. Morphologically, bones are of two kinds. Skull, vertebrae, mandibles and ribs are referred to as soft-bones and are rich in large areas of spongiosa type

Calcium citrate (TCC) and bone health Table 7.1

175

Optimal daily intake of calcium1

Age Birth ± 6 months 6±12 months 1±5 years 6±10 years 11±24 years 25±65 years (men) Over 65 25±50 years (women) Over 50 years (postmenopausal) Pregnant and nursing

Calcium (mg) 400 600 800 800±1200 1200±1500 1000 1500 1000 1500 1200±1500

of bone from which bone salts are easily withdrawn. The second type are the long-bones that consist of cortical bone which is of dense formation with greater resistance to mobilization of bone salts. In general, all bone formation is essentially a process of deposition of mineral elements into an organic matrix which consists mainly of collagen fibres. Dicalcium phosphate (CaHPO4) is central to the process. The events of bone formation involving calcium, are postulated to be as follows. First, dicalcium phosphate molecules aggregate into microscopic particles. Then, three molecules of dicalcium phosphate condense to form one molecule of tricalcium phosphate: 3CaPHO4 ÿ! Ca3(PO4)2 ‡ H3PO4

7.1

Tricalcium phosphate is an unstable molecule and other ions such as hydroxyl and fluorine attach to it to complete crystal formation of the apatite minerals: Fÿ,OHÿ Ca3(PO4) ÿ! Ca10(PO4)6OH2 and/or Ca10(PO4)6F

7.2

These bone salts, once formed, are less soluble than the salts formed in the process of its deposition. 7.1.2 Key issues in calcium fortification of foods The basic source of calcium is the diet. Yet, based on normal diet and especially with the increasing trends of using processed food, ready-prepared meals, `fastfood' and TV meals, only a part of the physiological need for calcium is supplied through food consumption.2 The question therefore arises, what are the best ways to attain optimal calcium intake? There are three main strategies. The first strategy is a change in dietary habits, namely, to include increased frequency of consumption of dairy products and calcium-rich vegetables. This approach of recommending the consumption of calcium-rich foods is consistent with the current dietary guidelines (the US Department of Agriculture Food

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Guide Pyramid), which includes 2±3 servings per day of dairy products and 3±5 servings of vegetables. However, persuading individuals to meet calcium requirements on a continuing daily basis by changing their food habits is a very difficult challenge. The second strategy, calcium supplementation, may be the preferred way for some individuals to attain optimal calcium intake. Various calcium salts are used in calcium supplements. Absorption of calcium supplements is most efficient at doses of 500 mg or less and when taken after meals. However, the long-term acceptance of taking daily calcium tablets as a measure to prevent bone loss is very poor. The third strategy, consumption of calcium-fortified food is probably the best way to achieve optimal calcium intake. By adopting this approach, there is no need to change food habits, and the added calcium is usually tasteless, odourless and is practically free of any caloric value. To maximize calcium absorption, the food selection decisions regarding fortification with calcium should include information on bioavailability. Bioavailability (absorption) of calcium from food depends on the food's total calcium content and the presence of components that enhance or inhibit absorption. For instance, oxalates, which are present at high levels in spinach, have been found to depress absorption of calcium. Phytates also depress calcium absorption. 7.1.3 Choosing a calcium fortifier There are several calcium salts that are available to food manufacturers for calcium fortification. From an economical point of view, the higher the calcium content of the salt is, so less will need to be added. The content of calcium in the various calcium salts is shown in Table 7.2. A responsible manufacturer, producing a successful brand, would consider a calcium fortifier with a high nutritional value and with low interference with absorption of other nutrients. In addition, cost effectiveness and minimal effects Table 7.2

Calcium content of the different salts

Calcium salt Calcium carbonate Tribasic calcium phosphate Bone meal Calcium chloride Dibasic calcium phosphate Dolomite Tribasic calcium citrate 4-hydrate Calcium lactate Calcium lactate gluconate Calcium gluconate

Calcium content (%) 40 38 31 27 23 22 21 14 13 9

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on consistency, mouthfeel and taste are also important considerations. Bioavailability and solubility are the main measures when comparing the nutritional value of calcium fortifiers. In general, organic salts of calcium, such as gluconate, lactate and citrate, are more bioavailable than the inorganic salts, such as carbonate and phosphate. The interaction between calcium and phosphate metabolism indicates that excessive phosphate content in the food would result in low calcium absorption. A prerequisite is that the calcium-to-phosphate ratio will exceed 1:1 on a weight basis. In some countries, including the US, health claims are allowed by food regulating bodies (FDA) provided the fortified food contains at least 200 mg of elemental calcium per 100 g of food, and that the calcium-to-phosphate ratio is >1:1.

7.2

Calcium citrate as a calcium supplement

When comparing calcium citrate (TCC) as a calcium supplement with other calcium supplements such as the widely used calcium carbonates, TCC is by far the most preferred supplement. The relative absorption of calcium from the different calcium sources is dose dependent in the normal dose range. Comparing TCC to calcium carbonate, for instance, suggests a higher absorption of calcium from 0.5 g calcium dose from TCC than a 2 g calcium dose from calcium carbonate.3,4 TCC was also found to be absorbed regardless of the levels of gastric acid secretion. Defective absorption of calcium exists in patients with achlorhydria. It was shown that absorption of calcium from calcium carbonate in patients with achlorhydria was significantly lower than in normal subjects, and was lower than absorption from calcium citrate in either group; absorption from citrate in those with achlorhydria was significantly higher than in normal subjects, as well as higher than absorption from carbonate in either group.5 Since achlorhydria is common in older persons, TCC may be the ideal dietary supplement. The solubility model for prediction of bioavailability was found to be a paradigm.6 This is in accordance with the supposition that there is an absorption mechanism for TCC complex rather than a single absorption mechanism of calcium ions.7 The unique mechanism of intestinal absorption of TCC as opposed to calcium in general is the reason that consumption of TCC has only a marginal effect interfering with absorption of other minerals, especially iron.8 Finally, concern has been raised as to the effect of long-term calcium supplementation on the formation of kidney and urinary duct stones. TCC has been shown to attenuate this risk since it enhances renal excretion of citrate, an inhibitor of crystallization of stone-forming calcium salts.9

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7.3

Measuring the effectiveness of TCC

Recent clinical studies have established that TCC is a superior calcium supplement in young people, elderly people and post-menopausal women, capable of suppressing parathyroid hormone (PTH) secretion and bone resorption, thus helping to stabilize bone mineral density. It has been shown that in young healthy volunteers,10 TCC enriched powdered milk improved calcium bioavailability, enhanced the inhibitory effect on serum PTH, and was superior to milk, yogurt or calcium-carbonate pills. In another study conducted on young men,11 an oral load of 1 g of elemental calcium, such as TCC, resulted in a very significant increase in PTH, and significant suppression of bone resorption as judged by urinary reduction of bone resorption markers. In recent years, numerous studies of the effects of calcium on postmenopausal women were performed, leading to parallel underlying conclusions. A study conducted at Mayo Clinic12 on 236 postmenopausal women with a mean age of 66, evaluated the effect of 1600 mg a day of TCC on calcium metabolism in a four-year randomized, double-blind, placebo-controlled trial. It was concluded that long-term administration of TCC to elderly women reverses age-related increases in serum PTH level and bone resorption, and decreases bone loss. Moreover, the investigators concluded that calcium supplements, with calcium salts like TCC, is a cost-effective preventive measure against bone loss because of its safety, high tolerance and low expense. The effect of TCC on bone density in the early and mid-post-menopausal period was also considered in a recent study13 in which early post-menopausal women were given 400 mg of TCC supplement twice daily for two years, and compared with a similar group of women who took a placebo. Bone density was tested at different skeletal sites. Supplementation with TCC was shown to have a protective effect on the existing bone mass, and was shown to slow the further loss of bone. Moreover, the investigators pointed out the fact that TCC is absorbed as readily by the body and may account for its success as a proved aid in the fight against bone loss.

7.3.1 Comparing the effectiveness of TCC with calcium carbonate Investigators addressed the question whether some form of calcium may be particularly advantageous in the management of post-menopausal osteoporosis.14 A comparison between TCC and calcium carbonate was carried out on 25 postmenopausal women who underwent three phases of study wherein they each took a single dose of TCC calcium-carbonate or placebo. Sera and urines were collected for several hours after the load of calcium was given, and the change in serum calcium, PTH, and urinary calcium, was monitored. TCC was found to be more bioavailable than calcium-carbonate when given with a meal in postmenopausal women. TCC load resulted in a higher increase in serum calcium when compared with calcium-carbonate or the placebo, and a higher cumulative increment in urinary calcium. These two parameters indicate

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higher intenstinal absorption of calcium from TCC than from calciumcarbonate. Morever, TCC treatment produced a greater suppression of parathyroid function than calcium-carbonate. Researchers at the University of Texas Southwestern Medical Center in Dallas,15 conducted a meta-analysis of calcium bioavailability. They evaluated 15 studies that were published on the bioavailability of two of the most common forms of calcium supplements, TCC and calcium-carbonate. All but one study showed significantly greater absorption of calcium from TCC than from calcium-carbonate, by an average of 22% to 27%, regardless of whether the candidate had eaten a meal or not. Thus, the meta-analysis confirmed that TCC provides superior bioavailability of calcium. 7.3.2 Comparing the effectiveness of TCC with other calcium salts In general, calcium when supplemented as salts of organic acids, such as TCC (21% calcium content) calcium gluconate (9% calcium content) or lactate (14% calcium content), is more bioavailable than when supplemented as inorganic salts, such as calcium phosphate or calcium carbonate. All calcium phosphates are poorly soluble in gastric juice, and like calcium carbonate, calcium phosphates are practically insoluble without acid secretion.6 When gastric effluent is neutralized by pancreatic secretion, reprecipitation of calcium phosphate is taking place, due to reduced acidity (pH>6.0). Therefore, the reduced solubility and reprecipitation contribute to the limited availability of calcium from calcium phosphate salts. When the absorption of calcium from TCC was compared with the absorption of calcium from calcium phosphate, a significantly higher absorption was observed with calcium citrate, almost three times more.12 When the absorption of calcium from TCC was compared with the absorption of calcium from calcium gluconate and lactate, or with the absorption from milk, very similar absorption was established.16,17 7.3.3 Summary In summary, TCC with its unique properties (Table 7.3), is an ideal calcium source for food fortification and for supplementation. Though improvements in calcium intake have been reported for most age groups of Americans, a large Table 7.3 The unique properties of TCC as a calcium supplement and as a calcium fortifier of food High content of elemental calcium. Excellent bioavailability of calcium. Calcium absorption is independent of gastric acid secretion. Only marginal interference with the absorption of other minerals, especially iron. No risk of renal stone formation.

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percentage still fail to meet currently recommended guidelines for calcium intake,7 including 6±11-year-old children. Calcium supplementation therefore should include practically all age groups, and this can be achieved easily by fortification with TCC of a large variety of products. Moreover, in postmenopausal women and the elderly in general, supplementation with TCC, as is evident from recent studies, seems to be the preferred means to prevent bone loss. Impaired intestinal absorption of calcium in these physiological states is practically restored due to the high bioavailability of calcium from this organic salt, and as a result, TCC yields in these states a remarkable suppression of parathyroid function, resulting in reduction of bone resorption, thus, preserving bone.

7.4

TCC fortification

Many calcium supplements, mainly in tablet form, are available. However, any additional intake of calcium would be better achieved by dietary fortification. Eating a meal with calcium salt will result in increased absorption of calcium due to stimulation of gastric acid secretion, better solubilization and delayed stomach emptying. When choosing a calcium source for fortification of food, many parameters should be taken into account; calcium content, bioavailability, sensory properties, solubility and price. Many people are prepared to pay more for a food product which has a health claim. In several countries nowadays, health claims for calcium fortification of food are allowed. These are functional claims that describe the physiological role of the nutrient during growth development and normal bodily functions. In the UK, the Health Claims Code allows the statement `a diet high in calcium combined with regular exercise is important for healthy bones'. In France, the CEDAP states that when a functional claim is made there must be a significant amount of calcium (at least 20% of the RDA). If this condition is fulfilled, the claims concerning the following properties are considered not to be misleading and can be used without the manufacturer having to justify them; bone construction, bone density, mineralization of bone tissue, strengthening the bones, and formation of the skeleton and teeth. In China, the 24 categories of function of foods recognized by Chinese legislation, include the category `prevention of osteoporosis and increase of bone density', which must be confirmed by the manufacturer in human and animal studies. Inorganic salts of calcium, such as calcium carbonate, tricalcium phosphate and calcium oxide, all contain a relatively high content of elemental calcium and are also relatively low-cost sources. However, they have to be weighed against sensory and formulation performance. Calcium carbonate, for instance, though of high calcium content and inexpensive, is sparingly soluble and can cause chalkiness. Moreover, calcium carbonate neutralizes gastric acid, which may interfere with the absorption of calcium which is more soluble in acidic pH. In general, organic calcium salts are more soluble than inorganic salts. Gluconates

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and lactate are invariably soluble, but the content of elemental calcium is relatively low. TCC, with its high content of elemental calcium (21%) is the salt of choice for food fortification. As calcium supplementation should include all age groups, a large variety of products can be easily fortified with TCC. These include cheeses, yogurts, baby foods, desserts, cereals, snacks, soy milk and beverages. In fruit drinks and juices, the pH is below 4.5, thus providing an acidic environment that increases the solubility of TCC and makes TCC an ideal salt for such fortifications. It may be also possible to maintain relatively insoluble calcium salts in solution by the use of stabilisers and emulsifiers. In calcium fortified jams, TCC has an additional role as a gelling agent.

7.5

Future trends

The calcium fortification market is increasing very rapidly, with more and more new products appearing on the shelves. Consumers want the health benefits of calcium, and manufacturers want the market benefits. In the US, for instance, 40% of new food products are fortified with calcium. Calcium fortification of food offers a great growth opportunity in an era when people care more and more for their nutrition and health. One of the fastest-growing areas is energy drinks fortified with calcium. Athletes lose calcium due to heavy transpiration in endurance sports, and have difficulty replacing it. There is also a very wide potential in `health foods' such as bars, snacks, diet foods and cereal products. Dairy products are popular, and are accepted as foods with high calcium content. In the fast-growing soy sector there are many fortified soy milks, blends and soy drinks. In Asia, calcium fortified noodles is also a fast-growing field of calcium fortification. The greatest challenge for the food industry in the field of calcium fortification is the development of new calcium sources that will be characterized by a high content of elemental calcium, good solubility, high bioavailability and good sensory properties.

7.6

Sources of further information

There are four main manufacturers of TCC in the world. These are: 1. Boehringer Ingelheim GmbH 55216 Ingelheim am Rhein Germany. www.boehringer-ingelheim.com 2. Gadot Biochemical Industries Ltd. Haifa Bay 26118 Israel. www.gadotbio.com

182 3.

4.

7.7 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

11. 12.

13.

Functional foods, ageing and degenerative disease Dr. Paul Lohmann GmbH Kg Hauptstr. 2 D-31860 Emmerthal Germany. www.lohman4minerals.com Jungbunzlauer Ladenburg GmbH Dr. Albert-Reimann Str 18 D-68526 Ladenburg Germany. www.jungbunzlauer.com

References (1994) Optimal calcium intake, JAMA, 272, 1942±1948. FLEMING KH and HEIMBACH JT (1994) Consumption of calcium in the U.S.: Food sources and intake levels, J Nutr, 124, 1426S±1430S. HARREY JA, ZOBITZ MM and PAK CYC (1988) Dose dependency of calcium absorption: A comparison of calcium carbonate and calcium citrate, J Bone Min Res, 3, 253±258. HARVEY JA, KENNY P, POINDEXTER J and PAK CYC (1990) Superior calcium absorption from calcium citrate then calcium carbonate using external forearm counting. J Am Coll Nutr, 9, 583±587. RECKER RR (1985) Calcium absorption and achlorhydria, N Engl J Med, 313, 70± 73. HENNEY RP, RECKER RR and WEAVER CM (1990) Absorbability of calcium sources: The limited role of solubility, Calcif Tiss Int, 46, 300±304. PAK CYC and AVIOLI LV (1988) Factors affecting absorbability of calcium from calcium salts and food, Calif Tiss Int, 43, 55±60. HALLBURG L, ROSSANDER-HULTEN L, BRUNE M and GLEERUP A (1992) Calcium and iron absorption: Mechanism of action and nutritional importance. Europ J Clin Nutr, 46, 317±327. PAK CYC (1994) Citrate and renal calcium: An update, Miner Electrolyte Metab, 20, 371±377. NIH CONCENSUS DEVELOPMENT PANEL ON OPTIMAL CALCIUM INTAKE

TALBOT JR, GUARDO P, SOCCIA S, GEAR L, LUBARY DR, SAAD S, ROBERTS ML,

FRADINGER E, NARISO A and ZANCHETTA JR (1999) Calcium bioavailability and parathyroid hormone acute changes after oral intake of dairy and non-dairy products in healthy volunteers, Osteop Int, 10, 137±142. GUILLEMENT J, LE H, MOIRA A and GUILLEMENT S (2000) Acute effect of oral calcium load on parathyroid function and bone resorption in young men, Am J Nephal, 20, 48±52. RIGGS BL, O'FALLON WM, MUHS, J, O'CONNOR MK, KUMAR R and MELTON LJ (1998) Long term effects of calcium supplementation on serum parathyroid hormone level, bone turnover and bone loss in elderly women, J Bone Miner Res, 13, 168± 174. RUML LA, SAKHAEE K, PETERSON R, ADAMS-HUET B and PAK CYC (1999) The effect of

Calcium citrate (TCC) and bone health

14. 15. 16. 17.

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calcium citrate on bone density in the early and mid-postmenopausal period; a radomized placebo-controlled study, Am J Ther, 6, 303±311. HELLER HJ, GREEN LG, HAYNES SD, POINDEXTER JR and PAK CYC (2000) Pharmacokinetic and pharmacodynamic comparison of two calcium supplements in postmenopausal women, J Clin Pharmacol, 40, 1237±1244. SAKHAEE K, BHUKET T, ADAMS-HUET B and RAO DS (1999) Meta-analysis of calcium bioavailability: a comparison of calcium citrate with calcium carbonate, Am J Ther, 6, 313±321. SHEIKH MS, SANTA ANA CA, NICAR M J, SCHILLER LR and FORDTRAN JS (1987) Gastrointestinal absorption of calcium from milk and calcium salts, 317, 532±536. HANSEN C, WERNER E, ERBES HJ, LARRAT V and KALTWASSER JD (1996) Intestinal calcium absorption from different calcium preparations: Influence of anion and solubility, Osteoporosis Int, 6, 386±393.

8 Diet, functional foods and oral health M. Edgar, formerly The University of Liverpool, UK

8.1

Introduction: key dietary factors in oral health

During eating, the oral structures have multiple functions ± food processing, sensing of food composition and consistency, temporary storage prior to deglutition (swallowing), and food transport during swallowing. Unlike other tissues, in the mouth essentially unmodified food components interact directly with the gums and teeth, and it is not surprising that foods themselves (not necessarily individual food components) have been the focus of studies to explain the major oral and dental diseases. Periodontal (gum) disease is prevalent in well-nourished individuals, and its aetiology is essentially bacterial. Detersive foods ± those which were believed to cleanse the teeth by physically removing food remnants and bacterial plaque ± have been shown not to influence the development of periodontal disease, probably because they do not cleanse the teeth at the gum margin. In addition, the development of non-reversible periodontal disease (a chronic bacterial destruction of the periodontal ligament between the root of the tooth and the bony socket) is not dependent upon, or an inevitable consequence of, the presence of disease at the gum margin (gingivitis). Although periodontal disease is a feature of classical scurvy, and the periodontal ligament is known to be the site of the most rapid collagen turnover in the body, the disease is not attributable to Vitamin C deficiency in the great majority of cases. Thus there are few well-founded instances of direct local interactions between foods and either the bacteria causing periodontal disease, or the tissues themselves, resulting in either an increase or decrease in severity. Oral carcinoma may be associated with chronic irritation of the tongue and cheeks by contact with ingested agents ± some examples are betel nut, alcohol

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and tobacco smoke components (the latter two agents probably exerting a synergistic effect). So far as protective interactions between foods and oral cancer have been studied, there has been some interest in the role of antioxidants, but no well-established relationship has been conclusively demonstrated. In contrast with the tenuous nature of the relationships between food, periodontal disease and oral carcinoma, dental caries, which is the most prevalent dental disease, demonstrates very clear links with diet and foods in terms both of causation and of prevention. These links result mainly from local effects of food in the mouth, and with the exception of fluoride, there is scant evidence of post-absorptive effects of diet on the dental tissues leading to changes in caries susceptibility. 8.1.1 Caries aetiology The dental tissues mainly involved in carious destruction are enamel and dentine. Enamel forms a surface cap of hard, highly mineralised tissue, which acts to resist abrasion of the teeth during mastication. Beneath the enamel, and making up the bulk of the tooth crown and root is the less highly mineralised dentine, which acts as a shock-absorber. Both tissues are mainly composed of mineral crystals known as `biological apatite', a form of calcium phosphate similar in structure to the mineral hydroxyapatite (Ca10(PO4)6(OH)2). In acid conditions, calcium phosphates such as apatite are more soluble than in neutral or alkaline conditions, because the constituent phosphate and hydroxyl ions react with hydrogen ions to form other ionic species. The apatite crystals in teeth are no exception. In the process of caries formation, tooth tissues dissolve in acids, which are produced by bacteria residing in dental plaque, by the fermentation of carbohydrates derived from food. `Fermentable carbohydrates' in foods are those that can diffuse into plaque and be taken up by the bacterial calls for energy metabolism. Bacterial cells have transport systems which are specific for dietary mono- and disaccharides, but larger oligo- and polysaccharides in foods, especially cooked starch and its products, can be hydrolysed to glucose and maltose (by salivary or bacterial amylases) sufficiently rapidly to be taken up by the plaque bacteria before the foods are swallowed. Foods are known as `cariogenic' if during and after ingestion the carbohydrates they contain give rise to a sufficient amount of acid to lead to dissolution. Dental plaque is a loosely-structured deposit of bacteria within a matrix derived from salivary glycoproteins and bacterial extracellular polysaccharides. The bacteria of plaque are described as commensal they are neither parasitic nor symbiotic. There are multitudinous species present, some of which are pathogens which give rise to periodontal disease and are mainly fastidious anaerobes. Others, including many gram-positive rods and cocci, produce mainly lactic acid as a result of anaerobic glycolysis of fermentable food carbohydrate, although other pathways and end products are involved also. The

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species most closely associated with caries are of the genus Streptococcus, and are described as the `mutans group' after the type species. Besides acid formation, the mutans streptococci also form a characteristic extracellular glucan from sucrose which may contribute to the plaque matrix and assist in their retention on the tooth. Saliva is usually neutral or alkaline due to its content of bicarbonate ions, and it also contains calcium and phosphate ions in solution at levels sufficient to saturate the chemical environment of the teeth. As a result, the teeth do not dissolve in saliva unless enough acid is formed to overcome these neutralising and saturating conditions and allow the apatite crystals to begin to dissolve. Indeed, partially dissolved crystals in the initial phases of caries can be repaired (remineralised) by the acquisition of calcium and phosphate ions from saliva. The presence of food in the mouth, the associated taste stimuli and the action of the muscles of mastication, lead to activation of reflex salivation. Stimulated saliva contains higher levels of bicarbonate and calcium ions, and thus has greater neutralising and remineralising properties than unstimulated saliva. By stimulating salivation, the natural defences against dental caries can be enhanced. Saliva also contains fluoride ions, albeit at very low concentrations. These levels are however sufficient to promote remineralisation especially when they are elevated after ingestion of fluoride-containing foods, brushing with fluoride toothpaste or using a fluoride mouthrinse. 8.1.2 Food cariogenicity The principal characteristic of foods that determines their cariogenicity is the amount of fermentable carbohydrate they contain. There is not a `dose response' relationship between cariogenicity and, say, sucrose content; if after consuming a food the concentration in saliva is high (in excess of about 30%) it can exert an antibacterial effect, analogous to the preservative effect of sugar in conserves and confections. In addition to their carbohydrate content, properties of foods that affect the duration, timing and intensity of salivary stimulation (such as taste, texture and consumption in relation to other foods) have to be taken into account when considering food cariogenicity. Foods may contain intrinsic acids, which are in sufficient concentrations to dissolve enamel directly, without formation of bacterially derived acids from carbohydrate. In doing so, the enamel is softened, leaving it more susceptible to mechanical tooth wear from foods, or practices such as tooth-brushing. This form of tooth tissue loss is often referred to as dental erosion, although it is usually difficult to distinguish between acid erosion and mechanical wear. The general term `tooth wear' is preferred to avoid unwarranted assumptions about aetiology. Foods may also contain protective substances ± those that interfere with the `acidogenicity' of the bacterial plaque, or with the solubility of the tooth tissues. Fluoride in tea or fish is one such protective substance, and many others have been investigated ± examples include phytates, glycyrrhizinic acid in liquorice, and unidentified antibacterial factor(s) in cocoa.

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The effects of ageing on oral health

The ageing mouth exhibits a number of characteristic changes that are well established. `Getting long in the tooth' is an expression of the effect of gingival recession away from the crown of the tooth towards the root apex, exposing the dentine (covered by a thin layer of mineralised cementum, which is part of the attachment of the tooth to the bone, and which is soon worn off). This is not however an inevitable consequence of ageing; rather it is due to loss of tooth support by chronic periodontitis, destroying the periodontal ligament and adjacent bone. This process is irreversible, and after each episode of periodontal destruction the gingival margin migrates down the root. The exposed dentine often becomes sensitive to changes in temperature and osmotic pressure in the mouth. It also becomes susceptible to carious attack; root caries occurs only in older individuals whose roots have been exposed. The teeth themselves become darker in colour with age. This is in part due to staining: extrinsic staining from food pigments (typically red wine, tea, etc.), and intrinsic staining, which may derive from food pigments diffusing into the enamel, or from systemically derived chromophores from the pulp chamber. In addition, darkening of the teeth can be caused by tooth wear ± as the enamel becomes thinner, the dentine which is darker in colour shows through. Use of abrasive toothpastes in an attempt to `clean' the teeth will only exacerbate this ageing effect. Loss of teeth with age is normally the result of extraction of teeth which have lost their bony support through periodontal disease, or have become irretrievably decayed, perhaps leading to infection of the root canal and the formation of an abscess at the root apex. Because the dental tissues, once formed, are not constantly remodelled like bone, they retain the evidence of each pathological event that occurs over a life-time. These changes then are not true age effects, in that in the absence of caries, periodontal disease or tooth wear they are not seen. One of the few true age effects in the mouth is the deposition within the pulp chamber and root canal of `secondary dentine'. This is deposited very slowly as a result of continued action of the cells (odontoblasts) responsible for the primary dentine during tooth formation. The result of this deposition is the narrowing of the pulp chamber and root canal, usually without any consequence unless root canal therapy is required. Secondary dentine is formed rather rapidly if there is a threat to the tooth from penetrating carious destruction, but this is a pathological event. Salivary gland dysfunction, with accompanying xerostomia or dry mouth, is a feature of ageing. Again, there is no evidence of a true reduction in salivary function with age in healthy individuals ± the volume of saliva secreted per minute is virtually the same for all age groups. Histologically, the number of functional secretory units per unit volume of gland tissue falls in older people, but it would appear that there is a functional excess of secretory cells, not all of which are activated. The observed age-related incidence of salivary gland dysfunction is attributable to two causes: there is a well-defined relationship

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between age and the number of medications taken by an individual, many of which have the side effect of reducing salivary flow (xerogenic). The second cause is salivary gland disease, especially Sjùgren's Syndrome ± an autoimmune destruction of secretory tissue in the salivary and lachrymal glands, often occurring together with an inflammatory arthritis. The sense of taste is said to decline with age ± again, tests of the threshold concentration at which a sapid (taste) substance like salt or sugar can be detected show no decrease in acuity with age in normal individuals, but the loss of enjoyment of foods in older people may be due to other factors ± the presence of dentures, or changes in central processing of sensory information.

8.3

Dietary strategies for oral health

As discussed above, the principal action of foods in oral health is the local action in the causation of dental caries, and there are few effects of age in the mouth that are not due to the accumulation of pathological effects. The development of dietary strategies for oral health does not therefore require special consideration for different age groups, except where individual cases demand individual treatment ± for example, for denture wearers, and where other age-related conditions require special dietary regimes, whose impact on oral health should be taken into consideration ± an example being type II diabetes. Caries arises from an interaction between food carbohydrates, dental plaque and susceptible teeth, and saliva can play a major role in modifying this interaction. It is clear therefore that optimising the diet for oral health can involve a reduction in the carbohydrate challenge, a reduction in the cariogenicity of the plaque, a reduction in the susceptibility of the teeth, and an enhancement of the protective salivary effect. 8.3.1 Reduction in the carbohydrate challenge Reduction in the exposure of cariogenic bacteria to fermentable carbohydrate can be achieved in several ways. A reduction in the amount of all carbohydrates can lead to an increase in fat intake, with probably undesirable implications. Selective reduction in the intake of sugars may have the same effect, and also may be ineffective, e.g., if the reduction is achieved by reducing the concentration of sugar in a food there may be no reduction in its cariogenicity. The contribution of oligo- and polysaccharides to the cariogenicity of the diet must also be taken into account. The nature of the sugar has been regarded in the United Kingdom as a key indicator for elimination, with `non-milk extrinsic sugars' (mainly added sugar, at the table or in manufacture) being a target for elimination, and intrinsic sugars (present in fruit and vegetables, within the cellular structure of the plant, together with lactose) being regarded as noncariogenic (Department of Health, 1989). The evidence for this distinction is rather equivocal.

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Two ways of reducing the carbohydrate challenge have been shown to be effective clinically. One way is to substitute all sugars with a non-fermentable sweetener (see Chapter 9). In a series of studies carried out in Turku, Finland, the sweetener was xylitol (Scheinin and MaÈkinen, 1975). The subjects were the staff and students of the Turku Dental School, who were allocated randomly to three groups and given a range of foods, drinks and snacks made with sucrose, fructose or xylitol for sole consumption over two years; those in the xylitol group experienced virtually no new caries, while those in the sucrose and fructose groups developed normal levels of new caries. The other way is the reduction in the frequency of exposure to carbohydrates, demonstrated in a classical experiment conducted around 50 years ago in Sweden (Gustavson et al., 1954). The experimental design was not optimal, and the ethics of inducing caries in the subjects, who were long-term residents of a mental institution, would nowadays be regarded as untenable. Nevertheless, the conclusion was sufficiently robust to be still applicable ± when subjects consumed carbohydrates including sweetened bread, caramels and toffees between meals, the development of caries was marked, and was related to the number of intakes, while consumption of the same amount or more of the `snack foods' at mealtimes resulted in no increase in caries. A third way, not tested in a large-scale clinical experiment but supported by several lines of experimental evidence, is the ordering of a sequence of foods, as in a meal of more than one course. In a pioneer study (Rugg-Gunn et al., 1975), a `meal' consisting of a meat pie, a portion of cheese and canned pears in syrup was eaten while the pH of the plaque was monitored as a measure of the cariogenic potential. The pH was found not to fall until the end of the eating sequence after the pears in syrup. If however the cheese followed the pears, no terminal pH fall was seen. This suggests that the carbohydrate challenge can be reduced by altering eating sequence without a reduction in the amount of carbohydrate. In this particular experiment, the effect was attributed to the salivary stimulus provided by the cheese, but there is other evidence showing that cheese may itself inhibit enamel dissolution. These mechanisms are discussed further below. 8.3.2 Reduction in plaque cariogenicity The fall in pH, indicating the severity of the potential demineralisation of the tooth surface following ingestion of a given carbohydrate challenge, is a function of the ecological balance of the bacterial flora. Those species which are most capable of lowering the pH of plaque because of (a) the rate at which they form acid, and (b) their ability to continue to carry out glycolysis at a low environmental pH to produce further acid, are the most cariogenic. A high level of fermentable carbohydrate present habitually in the diet can select a highly cariogenic flora; conversely, a reduction in dietary carbohydrate intake or substitution with non-fermentable substitutes leads to the development of a lesscariogenic flora. In practice, however, changes in dietary carbohydrate need to

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be substantial if they are to affect the balance of the plaque flora, and minor variations such as are seen in free-living individuals are largely without effect. Some plant food components have been shown to reduce the potential cariogenicity of the flora independently of dietary carbohydrate. Examples include tannins and other carbohydrate and phenolic components (Kashket et al., 1985). The nature of these effects is not well understood; it is possible that lectin-like activity is responsible for some of the actions of these components. Cheese can modify plaque composition. For example, when rats were fed small helpings of cheese interspersed among a sugary diet, they developed less caries and had fewer numbers of mutans streptococci in their plaque than controls without cheese (Edgar et al., 1982). This could be due to the lipids in cheese; lipids are known to affect bacterial colonisation of the teeth. Alternatively, the effect on the flora could be due to anti-microbial agents in cheese, or to the raised plaque pH on eating cheese. Other actions of cheese will be discussed later. Xylitol is known to have an anti-microbial effect on plaque with prolonged administration (SoÈderling et al., 1989). This pentitol is present in fruit and other plant foods and commonly used as a sugar substitute especially in the Nordic countries after the Turku experiment. Xylitol is taken up by the bacterial cell, is phosphorylated, and in doing so both wastes ATP energy and blocks further glycolysis (Trahan et al., 1985). Thus it is not merely a non-fermentable sweetener, but it also inhibits fermentation of sugars. The effect of xylitol on levels of mutans streptococci in plaque when the sweetener is present in chewing gum has been well documented. In contrast to gum sweetened with sorbitol, the numbers of these organisms in plaque and saliva fell, the plaque formed less acid from sucrose or glucose, and the amount of plaque was reduced (Edgar, 1998). 8.3.3 Reduction in tooth susceptibility The protective effects of dietary fluorides ± mainly from drinking water, but also present in useful concentrations in tea, and perhaps fish ± are well established, and for a full discussion the reader is referred to standard reviews (Murrayet al., 1991). Dietary fluoride can be incorporated into developing teeth in children, in which form it is said to reduce the solubility in acid of the formed tissues after eruption of the teeth. However, fluoride from foods can interact with the teeth after eruption into the mouth, either by direct contact, or after absorption by resecretion into the saliva. This `ambient fluoride' is thought to be the most important source of fluoride for caries prevention. Although the concentrations of fluoride in the environment of the teeth are normally low (around 1 nmol/l), small elevations such as are seen after ingestion of fluoride are sufficient to reduce the rate of dissolution of the tissues, and perhaps more importantly to increase the remineralisation of initial caries formation by enhancing apatite crystal growth. Addition of fluoride to drinking water, or administration of fluoride tablets to increase fluoride intake, are of course major components of the armamentarium of preventive dentistry.

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Other food additives besides fluoride have been used in foods with the purpose of protection of the teeth. The most obvious additives are the ions involved in the dissolution of apatite ± namely calcium and phosphate ions. In animal feeding experiments, calcium or orthophosphate supplements to foods have shown promising results but these supplements have not always proved beneficial in humans. For example, addition of phosphates to breakfast cereals proved ineffective in reducing caries in children, although they were effective food supplements in rat studies (Rugg-Gunn, 1993). The discrepancy can be explained by the fact that rat saliva is lower in phosphate content than human saliva, so the effect in rats of phosphate supplementation on the concentration of phosphate in the mouth is greater. However, phosphates ± especially polyphosphates ± have proved effective as additives to chewing gum in clinical studies. The widespread use of polyphosphates in chewing gum has been inhibited by the fact that the reduction in caries achieved is similar to that found with the substitution of sucrose with sugar substitutes (Finn and Jamieson, 1967; Finn et al., 1978). Addition of phosphoric acid to soft drinks has been shown in vitro to reduce the erosive action of the drink. Calcium supplements such as dicalcium phosphate, calcium lactate, and calcium glycerophosphate have received extensive study and have proved effective in caries reduction (Rugg-Gunn, 1993). The effect of naturally occurring calcium and phosphate in milk and milk products on caries has received considerable study. Milk itself is generally regarded as non-cariogenic, despite its lactose content, because of the presence of calcium and phosphate (Jenkins and Ferguson, 1966), but the levels of the ions in milk are low. Most work has been done on the effects of cheese and cheese extracts on levels of calcium and phosphate in plaque, and on intra-oral changes in enamel mineralisation (Jenkins and Hargreaves, 1989; Silva et al., 1986). Certain phosphate-containing peptides from casein have been shown to have important protective effects in rat experiments (Reynolds and Black, 1989). These properties of milk products are discussed further below. Some studies have shown that xylitol has a protective effect in reduction of tooth susceptibility by promoting remineralisation (Smits and Arends, 1988), although this has been convincingly demonstrated only at high concentration, and in vitro. 8.3.4 Enhancement of salivary protective effect: stimulation of salivary flow As discussed above, stimulated saliva contains elevated levels of bicarbonate, which can neutralise and buffer acids in plaque. Increased saliva flow also helps to dilute and wash away food residues and plaque acids, thus assisting the plaque pH raising effect of bicarbonate. Stimulated saliva also contains elevated levels of the ions responsible for remineralisation ± calcium and phosphate ± and it provides enough fluoride to enhance this process. Salivary stimulation on eating occurs while food is in the mouth, but for caries prevention the optimum time for saliva to

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be stimulated is in the period following a carbohydrate food intake, as the acid attack occurs during 30 minutes or so immediately after the food is swallowed. Many foods have been suggested for consumption after meals, although the rationale initially was to promote oral food clearance, an effect found not to be important. Apples were recommended by dentists for after-meal consumption for many years, backed by a study which showed a small but significant reduction in caries in children who were given a piece of apple to eat after school meals (Slack and Martin, 1958). Vegetables such as carrots have also been suggested for after-meal use. However, if the reason for the recommendation is to stimulate salivary flow, fruit and vegetables are less effective as they are rapidly consumed and the stimulation of saliva is therefore brief. Moreover, they themselves may contain sugars and are often acidic. More suitable saliva stimulants include cheese which, as has been shown above can reverse dramatically the fall in plaque pH following a sugary food (Rugg-Gunn et al., 1975). As a saliva stimulant however, cheese also is consumed rapidly, and is therefore short-lived. Also, too much cheese will lead to unacceptable increases in calorie and lipid intakes. Chewing sugar-free gum has been shown to raise plaque pH after an acidogenic challenge (Jensen and Wefel, 1989 ; Manning and Edgar, 1993). Sugar-free gum is an ideal saliva stimulant, as in normal use it is chewed over a prolonged period (up to 30 minutes), yet it neither adds significantly to calorie intake nor contributes any sugars or acidity. Sugar-free gum has also been used as a vehicle for beneficial agents such as fluoride, and urea (which helps to neutralise plaque due to the action of ureolytic plaque bacteria with the release of ammonia). Furthermore, many gums contain xylitol, with its beneficial effects on plaque cariogenicity. Sugar-free chewing gums can have value in restoring some function in patients with salivary dysfunction by the prolonged activation of residual gland tissues, leading to a functional hypertrophy.

8.4

Functional foods for promoting oral health

A food may be regarded as functional if: · it affects beneficially one or more target functions in the body other than through nutritional effects, so as to improve health or reduce disease risk · it is a true food, and consumed in normal amounts · it is not a pill or capsule but is part of a normal food pattern. So far as oral health is concerned, post-absorptive nutritional effects of foods are of only marginal importance, and so any foods having beneficial or protective effects can perhaps be described as functional. Functional foods are normally regarded as products manufactured with a specific health aim in mind, but this could perhaps be extended to the use of normal foods in particular ways to produce a health benefit ± for example, the use of cheese, and raw fruit and vegetables as salivary stimulants. It would not perhaps be stretching the

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definition too far to include normal foods with protective effects when consumed normally ± an example is tea, which through its fluoride content has been shown to give protection against caries if taken without sugar. 8.4.1 Sugar-free chewing gum as a functional food A true example of a functional food, which was manufactured to have a specific health benefit, is sugar-free chewing gum. The initial health aim was that of substitution of sugar, in gum mostly sucrose, and the usual sugar substitute was sorbitol. Subsequently, the demonstration of the effect of sugar-free gum in raising plaque pH, and the remineralising powers of stimulated saliva, led to these actions becoming additional health gains. The special role of xylitol is indicated by the fact that not only does it stimulate saliva as do other sweeteners, but in addition it has a well-proved beneficial effect in reducing the cariogenicity of the plaque microflora, and has some interesting potential in the area of remineralisation (see above). The clinical benefits of xylitol gum were first demonstrated as part of the xylitol sugar substitution experiment carried out in Turku, referred to above. Over 12 months, volunteers chewed sugared gum or xylitol-sweetened gum. The amount of caries increased in the sucrose group, but actually decreased slightly in the xylitol group; the latter finding suggested that the xylitol gum was having a therapeutic effect, reducing the caries that might have been expected to occur from the fermentable carbohydrates in the rest of the diet (Scheinin et al., 1975) (see Chapter 9). The efficacy of xylitol gum chewing has been demonstrated conclusively in at least three separate studies, in Canada, Finland and most recently and clearly in Belize in South America (Kandelman and Gagnon, 1990; Isokangas et al., 1989; MaÈkinen et al., 1995a). In the latter study, the development of caries was examined over periods of 24±40 months in schoolchildren who were given gum sweetened with sucrose, sorbitol, sorbitol/xylitol mixtures or pure xylitol. The gum was either in slab or pellet form. The results showed clearly the reduction in caries experience in children chewing sugar-free gum compared with sucrose gum. However, children in the no-gum group had an increment of caries (measured as decayed, missing or filled tooth surfaces) of around 4.5. Sorbitol gum was effective in reducing the caries increment by about 30%, in confirmation of other studies that have shown reductions in caries with sorbitol gum compared with no-gum controls varying between 7 and 40% (Beiswanger et al., 1998; Sjoke et al., 2001). With the xylitol group there was a much greater reduction compared with the no-gum group ± in this group there was even an apparent repair of carious lesions present at baseline, presumably by remineralisation of early lesions from salivary ions. In other data, it was found that the beneficial effects were greatest in those teeth erupting during the administration of xylitol gum (Hujoel et al., 1999). The reduced caries experience was maintained long after the gum administration ceased, confirming other findings in Finland that indicated a lower plateau of

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caries and a reduction in the treatment need of the xylitol gum chewers (Virtanen et al., 1997). In a group of infants with advanced untreated caries in their primary teeth, the xylitol group exhibited a stabilisation of the caries, with marked re-hardening of the soft, carious dentine at the floor of the cavities (MaÈkinen et al., 1995b). The benefits of xylitol gum have even been demonstrated in an indirect experiment (Isokangas et al., 2000) in which the caries development was monitored in infants whose mothers chewed xylitol gum during pregnancy and during the first years of childhood. The caries experience of the children in the xylitol group was lower than in the controls. The suggested explanation is that the oral microbiota in the mothers in the xylitol group was of low cariogenicity, and it is known that infants' mouths are initially colonised by plaque organisms derived from their mothers. Sugar-free chewing gums have been used as vehicles for other anti-caries agents as well as xylitol. Urea-containing gum causes a rise in plaque pH when chewed which is larger than that resulting from gum alone, due to the formation of ammonia from the hydrolysis of urea by plaque organisms. Supplementation by fluoride has also been used as an alternative form of systemic fluoride administration, but this is only obtainable as a prescription medicine. The addition of very low levels of fluoride to raise ambient levels to increase remineralisation, together with calcium and phosphate addition, could provide further benefits. 8.4.2 Milk and milk products Milk is commonly thought of as `good for the teeth' on the basis of its provision of dietary calcium and phosphate. As a food for neonates and infants, during the period of tooth development, milk ensures a good supply of calcium, although in a deficiency the teeth have priority over the bones in the acquisition of calcium, and signs of disturbance of dental development only occur in combined deficiencies of calcium, phosphate and Vitamin D with gross skeletal effects. Milk, as stated above, is generally regarded as non-cariogenic. Lactose is slowly fermented by plaque organisms, but the resulting levels of acids are too low to lower plaque pH significantly, especially in the presence of casein and lactalbumin, whose combined buffering effects further reduce the pH fall. In addition, milk can be regarded as a protective food, because of the local action of its calcium and phosphate ions (Jenkins and Ferguson, 1966), although their levels in milk are low. Human milk, with its higher lactose and lower calcium content, may be slightly more cariogenic (Rugg-Gunn et al., 1985), and evidence exists of caries developing in infants who are demand-fed on breast milk. Milk fats have been shown to reduce plaque development in laboratory experiments, although the importance of this action in the mouth is uncertain. The studies of mineral supplements derived from whey, and added to fruit juice, have shown beneficial effects on enamel demineralisation in intra-oral experiments (Thompson, 1990), and large reductions in enamel demineralisation

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were found in similar experiments with a popular sugary snack containing milk solids (Higham et al., 1991). The effects of cheese and cheese extracts on the levels of calcium and phosphate in plaque, and on intra-oral changes in enamel mineralisation have already been discussed. This effect of cheese seems to be specific, as other foods with the same or greater calcium content do not raise the level of calcium in plaque so effectively. Finally, it has been shown that calcium-binding phosphopeptides from casein have a major effect in raising plaque calcium and preventing tooth dissolution (Reynolds, 1987). Feeding trials in rats have also demonstrated impressive reductions in caries development (Reynolds and Black, 1989; Reynolds et al., 1995), and clinical trial data in human subjects are keenly awaited. The possibility of using these components as food supplements presents an interesting area of development of functional foods aimed at preventing dental disease.

8.5

Future trends

As discussed above, many foods and food components have been shown to possess protective effects. However, few have been tested in clinical trials, mainly because dental caries is a chronic disease whose clinical development requires it to be followed for at least two years in groups of several hundred susceptible subjects to give statistically powerful data. Thus, properly conducted, randomised double-blind trials for dental caries are very difficult to administer and are often prohibitively expensive to carry out. Examples have been given above where short-term measurements of intra-oral de-and remineralisation of tooth tissue, and plaque pH and other caries-related measurements, have been of value in testing new formulations, but these data may not be regarded as sufficiently reliable to predict actual clinical benefit. Only two classes of foods regarded as functional have been examined in this chapter. The opportunity for food manufacturers to extend the range of foods having oral health benefits has hardly been breached, but they will not wish to make the necessary investment unless they can make valid claims about their products. It is important therefore that agreed tests of food benefits are developed, with the full backing of regulatory agencies concerned with food advertising, in order to permit accurate advertising claims. Dental professional bodies (the American Dental Association, the British Dental Association, the British Dental Health Foundation) already have schemes for acknowledging the validity of claims relating to oral hygiene products, and the British Dental Association has also approved claims for foods. The American Dental Association Health Foundation has sponsored Consensus Conferences to develop agreed ways to measure food cariogenicity, and similar tests could be applied to protective foods. These bodies could assist further by approving well-based claims for functional foods. The expertise they can call on should be made use of by agencies

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regulating advertising to stimulate industry to innovate while protecting the public.

8.6

Sources of further information

For detailed accounts of the anatomy, physiology and biochemistry of the oral structures, readers are referred to standard texts (Scott and Symons 1982; Jenkins, 1978; Ferguson, 1999). The modern classics on diet and dental health are the textbooks by Rugg-Gunn (1993) and Rugg-Gunn and Nunn (1999). For a full account of the formation, composition and properties of saliva in relation to oral health, a small text published by BDJ Publications (Edgar and O'Mullane, 1996) is invaluable. The subject of age changes in the oral cavity is well covered in a collection of papers on `The Ageing Mouth' (Ferguson, 1989). The aetiology and pathogenesis of dental caries is covered by textbooks on cariology (e.g. Fejerskov and Kidd, 2003). Educational material published in the UK by the Health Education Authority and regarded as the `gold standard' for dental health education in this country (Health Education Authority, 1992) has a section on diet and dental caries. This deals only with the traditional, long-established recommendations, and focuses on younger age groups, although a new edition is in preparation. Industry-based publicity material dealing with diet and oral health can be of value. An example is the information and publicity service to general dental practitioners by the Wm Wrigley Jr Co under the title `Oral Health Care in Action'. However, such a service could not be expected to cover all aspects of the subject with equal weight. The major national dental associations (e.g .the British Dental Association; other national associations can be reached via its website www.bdadentistry.org.uk) provide information services which are unbiased and comprehensive, and the umbrella organisation, the Federation Dentaire Internationale (www.fdiworldental.org) also provides well-researched information on recent trends. For on-going research concerns, the Nutrition Group of the International Association for Dental Research (www.dental research.org) holds frequent symposia at the annual meetings of the Association on all aspects of nutrition in relation to oral health.

8.7

References

and STOOKEY GK (1998), `The effect of chewing sugar-free gum after meals on clinical caries incidence', Journal of the American Dental Journal, 129, 1623±6. DEPARTMENT OF HEALTH (1989), Dietary sugars and human disease. Report on health and social subjects 37. HMSO, London. EDGAR W M (1998), `Sugar substitutes, chewing gum and dental caries a review', British BEISWANGER BB, BONETA AE, MAU MS, KATZ BP PROSKIN HM

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Dental Journal, 184, 29±32. and O'MULLANE D M (1996) (eds) Saliva and oral health, London, BDJ Publications. EDGAR WM, BOWEN W H, AMSBAUGH S, MONELL-TORRENS S and BRUNELLE J (1982), `Effects of different eating patterns on dental caries in the rat', Caries Research, 16, 384±9. FEJERSKOV O and KIDD, E A M (2003) (eds), Dental caries: the disease and its clinical management, London, Blackwell. FERGUSON DB (1989) (ed.), The ageing mouth, Basel, Karger. FERGUSON DB (1999), Oral biosciences. Edinburgh, Churchill-Livingstone. FINN SB and JAMIESON HC (1967), `The effect of a calcium phosphate chewing gum on caries incidence in children: 30 month result', Journal of the American Dental Association, 74, 987±95. FINN SB, FREW RA, LEIBOWITZ R, MORSE W, MANSON-HING L and BRUNELLE J (1978), `The effect of sodium trimetaphosphate (TMP) as a chewing gum additive on caries increments in children', Journal of the American Dental Association, 96, 651±5. GUSTAVSON B E, QUENSEL C E, LANKE L S, LUNDQUIST L, GRAHNEN H, BONOW B E and KRASSE B (1954), `The Vipeholm dental caries study. The effect of different levels of carbohydrate intake on caries activity in 436 individuals observed for five years', Acta Odontologica Scandinavica, 11, 232±364. HEALTH EDUCATION AUTHORITY (1992), The scientific basis of dental health education (4th edition), London, HMSO. HIGHAM SM, SMITH PW, WALSH P and EDGAR WM (1991), `In situ de- and remineralisaion studies of snack food use', Caries Research, 25, 233 (abstract). È KINEN KK, BENNETT CA, ISOTUPA KP, ISOKANGAS PJ, ALANEN P and MA È KINEN PHUJOEL PP, MA L (1999), `The optimal time to initiate habitual xylitol gum-chewing for obtaining long-term caries prevention', Journal of Dental Research, 78, 797±803. È KINEN KK (1989), `Long-term effect of xylitol ISOKANGAS P, TIEKSO J, ALANEN P and MA chewing gum on dental caries', Community Dentistry and Oral Epidemiology, 17, 200±3. È DERLING E, PIENIHAKKINEN K and ALANEN P (2000), `Occurrence of dental ISOKANGAS P, SO decay in children after maternal consumption of xylitol chewing gum, a follow-up from 0±5 years of age', Journal of Dental Research, 79, 1885±9. JENKINS GN (1978), The physiology and biochemistry of the mouth (4th edition), London, Blackwell. JENKINS GN and FERGUSON DB (1966), `Milk and dental caries', British Dental Journal, 120, 472±7. JENKINS GN and HARGREAVES JA (1989), `Effect of eating cheese on Ca and P concentrations of whole mouth saliva and plaque', Caries Research, 23, 159±64. JENSEN ME and WEFEL J S (1989), `Human plaque pH responses to meals and the effects of chewing gum', British Dental Journal, 167, 204±8. KANDELMAN D and GAGNON G (1990), `A 24-month study of the incidence and progression of dental caries in relation to consumption of chewing gum containing xylitol in school preventive programmes', Journal of Dental Research, 69, 1771±5. KASHKET S, PAOLINO VJ, LEWIS DA and VAN HOUTE J (1985), `In vitro inhibition of glucosyl transferase from the dental plaque bacterium Streptococcus mutans by common beverages and food extracts', Archives of Oral Biology, 30, 821±6. È KINEN KK, BENNETT CA, HUJOEL PP ISOKANGAS PJ, ISOTUPA KP, PAPE HR and MA È KINEN P-L MA (1995a), `Xylitol chewing gum and caries rates: a 40-month cohort study', Journal of Dental Research, 74, 1904±13. EDGAR W M

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and ISOTUPA KP (1995b), `Stabilisation of rampant caries: polyol gums and arrest of dentine caries in two long-term cohort studies in young subjects', International Dental Journal, 45, 93± 107. MANNING RH and EDGAR WM (1993), `pH changes in plaque after eating snacks and meals and their modification by chewing sugared or sugar-free gums', British Dental Journal, 174, 241±4. MURRAY JJ, RUGG-GUNN AJ and JENKINS GN (1991), Fluorides in caries prevention (3rd edition), Oxford, Butterworth-Heinemann. REYNOLDS EC (1987), `The prevention of sub-surface demineralisation and change in plaque composition by casein in an intra-oral model', Journal of Dental Research, 66, 1120±7. REYNOLDS EC and BLACK CL (1989), `Cariogenicity of a confection supplemented with sodium caseinate at a palatable level', Caries Research, 23, 368±70. REYNOLDS EC, CAIN CJ, WEBBER FL, RILEY PF, JOHNSON IH and PERICH JW (1995), `Anticariogenicity of tryptic casein- and synthetic-phosphopeptides in the rat', Journal of Dental Research, 74, 1272±9. RUGG-GUNN AJ (1993), `Dental caries milk, cheese and other protective factors', in Nutrition and dental health, A J Rugg-Gunn, Oxford, Oxford University Press. RUGG-GUNN AJ and NUNN J (1999), Nutrition, diet and oral health, Oxford, Oxford University Press. RUGG-GUNN AJ, EDGAR WM, GEDDES DAM and JENKINS GN (1975), `The effect of different meal patterns upon plaque pH in human subjects', British Dental Journal, 139, 351±6. RUGG-GUNN AJ, ROBERTS GJ and WRIGHT, WG (1985), `The effect of human milk on plaque in situ and enamel dissolution in vitro compared with bovine milk, lactose and sucrose', Caries Research, 19, 327±34. È KINEN KK, (1975) (eds), `Turku sugar studies', Acta Odontologica SCHEININ A and MA Scandinavica 33, Supplement 70. È KINEN KK, TAMMISALO E and REKOLA M (1975), `Turku sugar studies XVIII. SCHEININ A, MA Incidence of dental caries in relation to 1-year consumption of xylitol chewing gum', Acta Odontologica Scandinavica, 33, 269±78. SCOTT JH AND SYMONS NBB (1982), Introduction to dental anatomy (9th edition), Deinburgh, Churchill-Livingstone. SILVA MFA, JENKINS GN, BURGESS RC and SANDHAM HJ (1986), 'Effects of cheese on experimental caries in man', Caries Research, 20, 263±9. SJOKE J, BANOCZY J and PROSKIN HM (2001), `Effect of after-meal sucrose-free gumchewing on clinical caries', Journal of Dental Research, 80, 1725±9. SLACK GL and MARTIN WJ (1958), `Apples and dental health', British Dental Journal, 105, 366-71. SMITS MT and ARENDS J (1988), `Influence of extraoral xylitol and sucrose dippings on enamel demineralisation in vivo', Caries Research, 22, 160±5 È DERLING E, MA È KINEN KK, CHEN C-Y, PAPE HR, LOESCHE W and MA È KINEN P-L (1989), `Effect SO of sorbitol, xylitol and xylitol/sorbitol chewing gums on dental plaque', Caries Research, 23, 378±84. THOMPSON ME (1990), `Effect of fruit juice with or without 1% whey mineral on bovine dental enamel in intra-oral experiments', Caries Research, 24, 334±6 TRAHAN L, BAREIL M, GAUTIER L and VADEBONCOEUR C (1985), `Transport and phosphorylation of xylitol by a fructose phosphotransferase system in È KINEN KK, MA È KINEN P-L, PAPE HR, ALANEN P, ISOKANGAS PJ MA

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Streptococcus mutans, Caries Research, 19, 55±63. and LARMAS MA (1997), `Longitudinal analysis of the effect of xylitol chewing gum trials on the number, timing and cost of dental fillings', Acta Odontologica Scandinavica, 54, 211±6.

VIRTANEN JI, BLOIGU RS

9 Sweeteners and dental health K. K. MaÈkinen, University of Turku, Finland

9.1 Introduction: the relationship between dental caries and dietary carbohydrates Dental caries is a diet-associated, infectious and transmissible disease process affecting the dental health of humans at all ages. In spite of advances made in preventive strategies, dental caries continues to be a serious health problem in both industrialised and developing countries, causing enormous economic losses, pain, and unnecessary impairment of the quality of life. The reasons for the continued increase of dental caries incidence in many populations include, for example · the `deceitful' progress of the disease process itself (i.e. dental caries develops slowly and is often diagnosed at an advanced state), · hedonistic, behavioural factors related to consumption ± by most human populations of ± sweet, fermentable, acid-forming carbohydrates that nourish the caries-inducive oral flora, · belated timing or total neglect of caries prevention in infants and young subjects. Nevertheless, dental caries can be considered fully preventable and treatable. The reasons of the failure successfully to curb the spread of dental caries are partly related to the three factors mentioned above. Furthermore, dental caries is normally not a fatal disease. This situation may prevent some patients from considering dental caries with sufficient seriousness. Dental caries is a multifactorial disease. Factors associated with the development of dental caries include several physiological and behavioural phenomena, some of which are listed in Table 9.1. Several factors deal with diet

Sweeteners and dental health Table 9.1

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Factors that affect the development of dental caries infection

Factor

Possible relationship to dietary sweeteners and caries

Diet Eating habits Oral clearance

Fermentable, aciduric carbohydrates (sugars) are cariogenic. Frequent consumption of fermentable sugars increases caries. Carbohydrate residues such as those derived from starch may remain on tooth surfaces and increase acid formation. Poor oral hygiene increases plaque growth and acid production. Rapid plaque growth and high acidity of plaque are premonitory symptoms (risk factors) of dental caries. Certain orthodontic situations and tooth form may increase susceptibility to dental caries; retention of food is possible. The flow rate and composition of saliva are affected by diet. Saliva is perhaps the most important anticaries factor. Fluoride(s) can be regarded as anticariogenic. Only small quantities are needed in the diet (i.e. ca. 1 ppm concentration level); detrimental and even toxic at gradually higher levels. Availability of adequate dental care services is key to successful caries prevention.

Oral hygiene Dental plaque Tooth anatomy and heredity Saliva Fluoride Dental care

± such as the microbiologic and chemical composition of dental plaque ± while others are associated with the relative efficacy of innate defence mechanisms present in saliva. Owing to the multifaceted nature of dental caries, its prevention should rely also on several concerted strategies. Sole application of fluoride-based strategies and use of fissure sealants cannot be considered sufficiently effective since both are frequently exploited belatedly or may not protect all tooth surfaces. It is self-evident, however, that effective caries prevention must include all the above strategies complemented by dietary and other approaches. This chapter deals with the specific role that some dietary, predominantly carbohydrate-like sweeteners, have assumed in the prevention of dental caries during the past quarter of a century. Special consideration will be given to xylitol, a sugar alcohol of the pentitol type, which is currently widely used in the prevention of dental caries and in other medical and techno-chemical applications. Placing emphasis on xylitol in this chapter stems from the performance of a larger number of clinical trials with this sweetener. Data from caries studies with other dietary carbohydrates are also presented. Discussion of synthetic and natural intense sweeteners has been excluded from this chapter owing to inadequate clinical information on the caries effects of these substances as a whole, and because the chemical levels of most intense sweeteners added to food are normally too low to cause significant oral biological effects.

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9.2

Xylitol and the prevention of dental caries

9.2.1 Brief chemical characterisation of the xylitol molecule Xylitol is a natural carbohydrate which is classified in the systemics of organic chemistry as a sugar alcohol (polyol, polyhydric alcohol). Because the molecule consists of a skeleton of five carbon atoms and comprises a total of five hydroxyl groups (OH groups; Fig. 9.1), xylitol is one of the so-called pentitols. The molecule is symmetric and hence no D- or L-prefix is used. Of great importance to the chemistry of xylitol is the presence of a tridentate ligand, (H-C-OH)3 in the molecule. This arrangement can react with various polyvalent cations such as Ca(II). The interaction between Ca(II) and xylitol may be important in vivo in processes such as the transport of Ca(II) through the gut wall and in the remineralisation of early dental caries lesions. It is necessary to compare xylitol with hexitols (sugar alcohols of the hexitol type; Fig. 9.1) of which D-glucitol (sorbitol) and D-mannitol may be considered important from the point of view of dietary and medical applications. The hexitols differ decisively from the pentitols regarding their metabolic fate and their biochemical and nutritional significance to organisms. The specific clinical and nutritional effects of xylitol are associated with its `non-glucose' nature (i.e. with its pentitol structure) and are, consequently, impossible to observe with Dglucitol and D-mannitol which are `glucose polyols'. The latter quoted term refers to the close relationship between the structural configurations of Dglucitol and D-glucose molecules; the configurations are relatively similar. The structural configuration of xylitol, however, differs from that of D-glucitol and D-glucose. Several important metabolic features of xylitol ensue from this

Fig. 9.1 The structure formula of four dietary sugar alcohols. Xylitol and D-glucitol (sorbitol) are perhaps the most commonly used dietary sugar alcohols and also Dmannitol has been used in various medical and dietary applications. The formulae look similar, but differ from each other decisively with regard to important details that are in turn reflected in the metabolic behaviour of these molecules. The pentitol nature of the xylitol molecule (i.e. the presence of five OH-groups) has been used to partly explain the advantageous effects of xylitol in caries limitation. Galactitol has not been tested in clinical caries studies.

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specific configuration: once the xylitol molecules are present in the circulation, they can rapidly diffuse into the cells. This diffusion, for example, into the muscle cells, is not facilitated by insulin. The net result is a large distribution volume of xylitol in the body. A more detailed profile of the xylitol molecule, and of its biochemistry and metabolic aspects, has appeared elsewhere (Georgieff et al., 1985; MaÈkinen, 2000a). 9.2.2 Short-term and laboratory caries studies of xylitol Short-term laboratory and clinical studies of xylitol have most frequently focused on the elucidation of premonitory symptoms of dental caries. Such symptoms, or early caries markers, include the quantity and quality of dental plaque and saliva (normally `whole-mouth saliva'). The `quality' of dental plaque and saliva denote their microbiological and chemical composition. Shortterm tests have normally lasted six months or less. Such studies can be considered necessary since their performance normally requires much less economic and material resources than full-length caries trials that should preferably extend over two or more years and comprise larger patient cohorts. The strength of the premonitory symptoms of the disease (dental caries) normally determines the course of the disease process itself. For example, continued presence of a large number of mutans streptococci in dental plaque and the presence of larger amounts of sticky microbial plaque on tooth surfaces, are normally associated with a high rate of dental caries. The seriousness of both symptoms tends to increase as a result of increased consumption of certain fermentable sugars, notably sucrose and related six-carbon carbohydrates. A total of more than 300 short-term clinical or laboratory tests on the dental effects of xylitol have been carried out. Almost without exception, the use of xylitol in chewable products or oral hygiene adjuvants (most often toothpastes) has been associated with a reduction in the mass of dental plaque. At the same time, the numbers of caries-conducive organisms in dental plaque and whole mouth saliva have also decreased, and the acidity and adhesiveness of dental plaque have diminished. Compared with fermentable, hexose-based carbohydrates such as D-glucitol, D-fructose, D-glucose, and sucrose, xylitol has in most studies been more effective in weakening the strength of the cariespremonitory symptoms (MaÈkinen, 1985, 1989, 2000a,b; Trahan, 1995). Some short-term studies on the premonitory symptoms of dental caries have provided dubious results owing to the application of inadequate treatment, i.e., using too `mild' or too low xylitol levels, or erroneous analysis methods of dental plaque. For example, relying only on the determination of the nitrogen content of dental plaque as a measure of plaque mass (Scheie et al., 1998) can be regarded as questionable. This is because research has shown that dental plaque bacteria deprived of their required nutrients (i.e. normal, hexose-based dietary sugars) convert their normally carbohydrate-dominant metabolism into one where various reactions of nitrogen metabolism predominate (Scheinin and MaÈkinen, 1975; MaÈkinen and Scheinin, 1975, 1982). Consumption of xylitol

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increases the nitrogen content of dental plaque. However, at the same time the mass, the volume, and the adhesiveness of plaque tend to decrease. Plaque assessments based on nitrogen analyses thus fail to reveal the true and clinically more important changes in plaque quality and quantity. 9.2.3 Biochemical and bioinorganic mechanisms Based on the current view, the mechanism of action of xylitol in caries limitation is based on the following `crude' effects (more detailed information is available in review articles referred to in this chapter): 1. 2. 3.

4.

5.

6.

The five-carbon structure of the xylitol molecule (i.e. the `non-glucose' nature of xylitol. The presence of a tridentate ligand in the molecule, rendering an interaction between xylitol and Ca(II). A corollary of (1): the inability of most oral bacteria to utilise xylitol as an energy source. This results in smaller plaque growth, less adhesive plaque (the extracellular polysaccharides formed tend to be soluble), and in plaque that is less acidic and contains a smaller amount of insoluble polysaccharides. Under a xylitol regimen, the metabolism of dental plaque shifts towards one where the role of nitrogen-containing substances increases (compared with `sucrose plaque' where the metabolism of carbohydrate substrates dominates). A consequence of this is increased production of ammonia in plaque. Special effects of xylitol on the detailed chemical structure of the cell wall of mutans streptococci (less lipoteichoic acids and other, less adhesive biopolymers are formed; the adherence of the cells onto the enamel surface weakens). Special effects of xylitol on the growth and intermediary metabolism of several strains of mutans streptococci: formation of inhibitory xylitol (and/ or D-xylulose) phosphates.

Regarding the chemical mechanism of action of erythritol in caries limitation, it is likely that it differs from that of xylitol as far as the effects on mutans streptococci are concerned. However, in several cases where a dietary carbohydrate to a certain extent reduces the incidence of dental caries, the explanation may simply be found in the general deprivation of caries-inducive organisms of their required nutrients (i.e. D-glucose, sucrose, etc.) rather than the involvement of specific metabolic effects. The mechanistic aspects related to caries limitation by sugar alcohols have been discussed elsewhere (MaÈkinen, 1989, 2000a,b). 9.2.4 Use of xylitol in foods; suitable xylitol products Most dental studies on xylitol have focused on testing the caries-preventive effects of chewing gum, hard caramels, lozenges, and toothpastes. Clinical trials

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and laboratory studies have provided evidence that regardless of the vehicle used for xylitol, the caries-preventive effect of these products has been almost similar. Field experience indicates that the daily frequency of intake of xylitol and the amount of xylitol consumed per day should exceed a certain minimum level. The most important requirement seems to be the introduction of xylitol to the oral cavity in a form that releases xylitol molecules from the product over a longer period of time. It also seems likely that an initial, relatively high xylitol pulse of one to three minutes duration improves the dental effect of the product. Most studies suggest that 3 to 5 chewing episodes per day and 5 to 7 g daily doses of xylitol should produce a significant caries-limiting effect. Consequently, from the oral physiological point of view, a typical, `ideal' xylitol product is a full-xylitol hard caramel, lozenge, gum, or similar chewable product with no fermentable bulking agent present. Since some all-xylitol products may be unfeasible (owing to crystallisation and hygroscopicity of xylitol, and factors related to cost and shelf life of some products), use of bulking agents is frequently necessary. Substances that have been used for this purpose include polydextrose, maltitol syrups, starch, etc. Clinical studies suggest that an all-xylitol chewing gum made into a pellet that is coated with xylitol, is both physiologically, organoleptically, and dentally an almost ideal caries-preventive product. Table 9.2 shows a list of currently available and some other potential xylitol products with comments on their possible dental benefits or disadvantages. 9.2.5 Prevention of intrafamiliar transmission of dental caries The intrafamiliar transmission of mutans streptococci and other oral microorganisms can be regarded as a natural consequence of the mother-child relationship. It is normally the mother who `infects' her newborn child with micro-organisms. Transmission of bacteria takes place in the form of normal daily chores, such as kissing and other physical contacts. Transmission can, of course, also take place between the child and other persons who are in intimate contact with the infant. In addition to dental caries infection, also other infections of the upper respiratory tract can be transmitted. In an ongoing Finnish mother-child programme (Ylivieska study; Table 9.4) mothers received xylitol chewing gum during the 3±24 months following delivery. Control mothers received treatments with either a fluoride varnish or a chlorhexidine varnish. The children received none of these treatments. Compared with fluoride and chlorhexidine treatments, the use of xylitol chewing gum by mothers resulted in a significant reduction in the children's plaque levels of mutans streptococci, as evidenced by tests carried out when the children were two and six years old (SoÈderling et al., 2000, 2001). Owing to the existence of a relatively effective, community-supported caries prevention, Finnish children, as a group, do not normally have significant dental caries during their first years of life. Therefore, in such mother-child studies statistically meaningful caries data will normally become available first when

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Table 9.2 Examples of dentally relevant food items and other products containing xylitol or other saliva-stimulating, non- or anticariogenic agents listed in a subjectively estimated order of preference Product

Remarks

1. Chewing gum

Renders effective mastication and saliva stimulation possible; xylitol-containing saliva may effectively get pumped into the crevices and fissures of the teeth. Chewing of the bolus renders mechanical saliva stimulation possible; an advantage. Use of gum is regarded by most people as a pleasurable experience. Patients with certain occlusal problems may not like to use gum. Render effective sucking and mastication functions possible; advantages as above. Suitable for persons with occlusal problems and for those who dislike gum chewing. A further advantage: fully soluble. Renders simultaneous mechanical cleansing (tooth brushing) possible. Normally non-caloric use (unless swallowed in larger quantities). Advantages: makes possible the simultaneous use of detergents, fluorides, phosphate salts, etc. Normally render non-caloric use possible, but the time when dental plaque is exposed to xylitol may be short. The amount of xylitol derived from sprays may be too small. An experimental `slow-release' mechanism (the device includes a perforated nipple from which saliva dissolves the desired substance inserted in the nipple). Successfully tested with xylitol. Used to apply xylitol syrup on tooth surfaces in infants.

2. Chewable tablets, hard caramels, etc, 3. Toothpaste

4. Mouthwashes, sprays 5. Pacifier

6. Cotton swabs, toothbrushes

the children are about five years old. Up till now, there is published cariologic data on five-year-old children, demonstrating that maternal use of xylitol chewing gum can indeed prevent dental caries in children (Isokangas et al., 2000), most likely by prohibiting the transmission of mutans streptococci from mother to child. These findings indicate that the protective effect of xylitol is passed from parent to child. 9.2.6 Early prevention of dental caries The mother-child approach described above is a suitable example of early caries prevention; the preventive agent or procedure is introduced directly or indirectly to the infant at an early age. Early prevention of dental caries is important since once the deciduous teeth become seriously damaged by caries infection, the eruption of permanent teeth will take place under circumstances that may further accelerate the caries process. There is a special chemical relationship between the hydroxyapatite surface of the dental enamel and the cell wall structure of mutans streptococci. The eruption of the first deciduous teeth (normally at the

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age of 4±11 months), opens a so-called `window of infectivity', i.e., the caries infection may begin at this age (Caufield et al., 1993). In addition to hydroxyapatite surfaces, these organisms may also grow on glass surfaces and on dentures. Consumption of noncariogenic and anticariogenic carbohydrate sweeteners by infants is limited in many countries owing to the lack of suitable products on the market. Use of xylitol-containing pastilles and chewing gum have been recommended in Finland already starting at the age of two years, under parental guidance. Xylitol-containing toothpaste can also be used from an early age on. Special xylitol-delivering pacifiers have been tested (Aaltonen et al., 2000). In another application that is now being investigated, xylitol syrup is systematically swabbed with cotton swabs or with a tooth-brush on the tooth surfaces of deciduous teeth as they erupt. 9.2.7 Long-term effects of xylitol Among the potential sugar substitutes, only xylitol has been investigated in long-term trials that a priori have included re-examinations of subjects several years after systematic usage of xylitol had been discontinued. The rationale behind such trials has been to elucidate the possible long-term protective effect of xylitol in dental caries. Two such trials have been conducted (Ylivieska study and Belize studies; Table 9.4). Both trials were originally planned to include reexaminations several years after the discontinuation of the xylitol gum programme. At Ylivieska, the subjects received xylitol gum over a period of two to three years (in 1982±1985). The re-examinations were conducted two to three years and four to five years, respectively, following the end of the treatment period (Isokangas et al., 1993). The re-examinations convincingly demonstrated that the xylitol gum programmes produced a long-term benefit: a significant caries-preventive effect was still observable in 1987 and 1989 even though habitual use of xylitol gum had been discontinued several years earlier. The preventive effect was greater in erupting teeth than other teeth (erupting teeth are normally more vulnerable to caries infection than are mature teeth). One of the objectives of the Belize re-examination trial (Danriga trial; Table 9.4) was to elucidate the optimum time to initiate habitual xylitol gum-chewing for obtaining long-term caries prevention (Hujoel et al., 1999). Children, initially six years old, chewed for two years gums containing xylitol, D-glucitol, or their mixtures. Five years after the two-year programme ended, the available children were re-examined. Compared with the no-gum control group, Dglucitol gums had no significant long-term effect on the number of caries onsets in the permanent dentition of the subjects. Xylitol gum and to a lesser extent a xylitol/D-glucitol mixture gum exerted a long-term preventive effect. It appeared that for long-term caries-preventive effects to be maximised, habitual xylitol gum-chewing should be commenced at least one year before permanent teeth erupt.

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9.2.8 Combinations of xylitol and other adjuvants The available scientific evidence suggests that `full-xylitol' products (chewing gum, other confectionery, dentifrice, etc.) where xylitol is the only carbohydrate sweetener and bulking agent, occasions an effective caries prevention. Owing to techno-chemical and cost-related aspects, it has sometimes been necessary to use combinations of xylitol with other carbohydrates. While combinations of xylitol with regular, dietary hexose-based sugars must be regarded dentally detrimental, some combinations of xylitol and other polyols may provide limited caries-preventive effects. Most studies where the dental or oral microbiologic effects of polyol mixtures were tested have shown, however, the effects increasing with increasing ratio of xylitol to D-glucitol in the used product. Table 9.3 lists carbohydrates and certain dentally important substances that have been tested in the presence of xylitol, with comments on their mutual compatibility. It is desirable that in future clinical and laboratory studies, combinations of xylitol with these and other substances will receive attention. 9.2.9 Prevention of dental caries in orthodontic patients Orthodontic treatment almost always increases the risk of dental caries. Although the normal recommendation by most clinicians has been to discourage the use of chewing gum during treatment, the situation can be considered different if the gum's sweetener is not cariologically harmful. The first study in this area (Isotupa et al., 1995) clearly indicated that habitual usage of xylitol chewing gums significantly reduced the risk of dental caries (measured in terms of plaque and saliva growth of mutans streptococci). The use of the gum did not damage the orthodontic appliances and did not stick to them. It is expected that this integration of a xylitol gum programme will gain acceptance among orthodontists and patients.

9.3 The relationship between sucrose consumption and dental caries Perhaps the most impressive clinical study on the causal relationship between sucrose consumption and dental caries was conducted some fifty years ago in Sweden (Table 9.4). The study showed that frequent consumption of sucrose, especially in sticky (toffee) form, was associated with high caries activity. The purpose of Table 9.4 is to list caries studies with sucrose substitutes and mentions the Vipeholm study because of its historic importance. The Vipeholm study and other clinical studies have clearly vindicated the close relationship between sucrose (and related hexose-based sugars) and the development of dental caries. Since mere restriction of sugar consumption ± without suggesting alternatives ± must be regarded as unfeasible, scientists have turned their attention to other sweet, natural carbohydrates. Following the above Swedish study, several clinical trials have been carried out on the cariologic effects of

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Table 9.3 Observations on the compatibility of xylitol with other substances in products aimed at improving dental health Substance

Comments

Glucose and sucrose

Combinations with xylitol are discouraged owing to the pronounced acidogenic nature of these hexose-based sugars; the latter also accelerate the formation of insoluble polysaccharides in dental plaque. Maltose Preliminary information suggests that maltose per se may inhibit the synthesis of polysaccharides by S. mutans. Fructose Combinations are discouraged owing to the possibility that, in the presence of D-fructose, xylitol and D-fructose can be incorporated into the cells of mutans streptococci by means of the same phosphotransferase system present in the cell wall. Invert sugar may cause similar effects. Sorbitol Combinations of high D-glucitol:xylitol ratios are discouraged owing to the growth-stimulating effect of Dglucitol in cultures of several strains of S. mutans. For the same reasons, maltitol syrups with a high D-glucitol content may be questionable. Larger quantities are cathartic. D-Mannitol Behaves almost like D-glucitol, but can have an even lower oral tolerance. Maltitol, lactitol Combinations have not been tested, but may have dental advantages. Erythritol Based on theoretical evaluations and existing (still meagre) scientific information, combinations with other sweeteners (e.g. xylitol) may be caries-preventive. Polydextrose Used as a bulking agent in some xylitol products. Larger quantities are cathartic. Sodium lauryl sulphate Anionic detergent. Can prevent the beneficial effects of xylitol on mutans streptococci (combinations in oral hygiene products not recommended). Inorganic fluorides Current information suggests the existence of positive, additive, or even synergetic effects in combinations with xylitol. _______________________________________________________________________

sugar substitutes. Most later studies have dealt with xylitol. These trials have provided essentially similar findings. The usage of xylitol has been associated with a significant reduction in dental caries (MaÈkinen, 1985, 1989, 2000a,b; Birkhed, 1994; Tanzer, 1995). The results have been similar regardless of the type of human population studied. Since almost identical results have been obtained with various chewable products and dentifrice, or using a special dietary xylitol regimen, it is evident that the most important prerequisite for the manifestation of positive xylitol effects is to introduce xylitol to the oral cavity and to expose dental plaque to xylitol, obviously regardless of the method of administration, or of the vehicle used for xylitol.

Table 9.4 Human caries studies on dietary carbohydrates. The reduction percentages are given compared with a control group that received normal diet, fluoride treatment, or sucrose productsa. Non-dietary (dentifrice) applications are included Study location

Sweeteners tested (reference)

Duration (years)

Dose (g/day)

Caries reduction

Sweden (Vipeholm study)

Sucrose in various forms (Gustafsson et al., 1953)

4

Up to 300

Sucrose was highly cariogenic.

Denmark

Sorbitol gum vs. normal diet (MoÈller and Poulsen, 1973)

2

3.6

10%

Sweden (Roslagen study)

LycasinÕ candies vs. sucrose (Frostell et al., 1974)

Up to 2.5

Not given

25% (the authors were cautious as to results).

England

Sorbitol tablets vs. normal diet (Slack et al., 1964)

2

Not given

`Small but significant benefit' (quote).

Island of Crete (Greece)

Sucrose chewing gum (Slack et al., 1972)

3

Five sticks daily No significant effect

Finland (Turku study)

Fructose, xylitol, and sucrose (full substitution; Scheinin and MaÈkinen, 1975)

2

67

>85% (with xylitol)

Finland (Turku study)

Xylitol gum vs. sucrose gum (Scheinin and MaÈkinen, 1975)

1

6.7

>82%

Soviet Union (Kazan study)

Xylitol candies vs. sucrose candies (Galiullin, 1981)b

2

30

Up to 73%

Xylitol-sorbitol gum vs. fluoride (Barmes et al., 1985)

2.3±2.7

20

Preventive effect noted.

WHO studies Thailand

French Polynesia

Xylitol gum vs. fluoride (Kandelmann et al., 1988)

3

Up to 20

58±62%

Hungary

Xylitol gum, candies, dentifrice vs. fluoride (Scheinin et al., 1985a,b)

2±3

14±20

37±45%

Canada (Montreal study)

Xylitol gum vs. normal diet (Kandelmann and Gagnon, 1990)

1±2

1.0±3.9

52%

Sweden (Gustavsberg)

Invert sugar vs. sucrose (candies, jam, etc.; Frostell et al., 1981)

2

Not given

35%

Finland (Ylivieska)

Xylitol vs. normal diet (all subjects; 2 Isokangas et al., 1988; MaÈkinen et al., 1989)c Xylitol gum vs. normal diet (high-risk 3 subjects; Isokangas et al., 1988)c

7±10

30±57%

7±10

59±84%

USA (Boston)

Sorbitol gum vs. normal diet (Glass, 1983)

2

Not given (2 sticks)

Not significant.

Costa Rica

10% Xylitol in dentifrice (silica-NaF) (Sintes et al., 1995) 10% Xylitol in dentifrice (NaFPO3ÿ dicalcium phosphate dihydrate; Sintes et al., 2002)

3

Twice daily

Up to 12.3%.

3

Twice daily

Up to 10%.

3.3

Up to 10.7

Xylitol reduced by up to 73%d.

2

Up to 10.7

Xylitol reduced by up to 63%e .

Belize

Xylitol, sorbitol, and sucrose gums vs. no gum (permanent teeth; MaÈkinen et al., 1995, 1996c) Xylitol and sorbitol gum vs. no gum (deciduous teeth; MaÈkinen et al., 1996a)

Table 9.4

Continued

Study location

Sweeteners tested (reference)

Duration

Dose

Caries reduction

USA (Dayton, OH)

Xylitol vs. sorbitol (gum, pastilles; root caries; MaÈkinen et al., 1996b)

1.8

Up to 8.5

80%

Estonia

Xylitol gum and hard caramels vs. normal diet; 200 days/year; Alanen et al., 2000)

2±3

5

50±60%

Finland (Ylivieska)

Xylitol gum vs. fluoride or chlorhexidine 21 mo. treatments for mothers only. Children did not receive treatmentsf (Isokangas et al., 2000)

6±7

70% (in children).

a The studies are shown in approximate chronological order. Clinical caries trials on xylitol began in 1972 in Turku, Finland, and the last one on the list, at Ylivieska, Finland, continues. New trials have been commenced in Finland, Brazil, China, U.S.A., and elsewhere. b The original paper is in Russian. English summaries have been published (MaÈkinen, 1985, 1989). c These trials included re-examinations on long-term effects (Isokangas et al., 1993). d A continuation programme of 16 months (comparison between sucrose and xylitol gums) demonstrated a remarkable difference in caries activity between treatments (MaÈkinen et al., 1998). e A re-examination of the permanent dentition of the same subjects five years later showed that, compared with the no-gum group, sorbitol had no significant long-term effect, whereas xylitol gum and to a lesser extent xylitol/sorbitol gum, exerted a long-term preventive effect (Hujoel et al., 1999). f The children of xylitol-using mothers showed also reduced levels of mutans streptococci (SoÈderling et al., 2000, 2001). The programme continues.

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213

Among the sugar alcohols, D-glucitol has evoked interest owing to its low production costs and its suitability in various techno-chemical applications. Direct comparisons between xylitol and D-glucitol have shown xylitol to be more effective in caries limitation. The true cariologic benefit of D-glucitol has been questioned owing to the suitability of this polyol as a growth substrate of several strains of mutans streptococci. Table 9.4 indicates, however, the caries reduction percentage achieved in D-glucitol studies have ranged from zero to about 33%. It is evident that D-glucitol is less cariogenic than sucrose. A confirmation of this was also obtained in the Belize studies where xylitol, however, was most effective against dental caries (Table 9.4). D-Mannitol obviously behaves in the human dental plaque system almost like D-glucitol. The use of these hexitols in dietary products is to a certain extent restricted by their slower absorption rate. This results ± when larger quantities of D-glucitol and D-mannitol are consumed ± to osmotic diarrhoea and flatulence that are more severe than experienced with equimolar levels of xylitol. These cathartic differences between xylitol and the above hexitols are partly attributed to the size of the polyols molecules in relation to the size of the pores present in the gut wall; the larger molecular mass of D-glucitol and D-mannitol delay their absorption. The structural moieties of the sucrose molecule, i.e., D-glucose and Dfructose, have also turned out to be highly cariogenic, owing to their six-carbon structure and suitability as energy sources in bacterial metabolism. Theoretically, the monosaccharides D-fructose and D-glucose should be less cariogenic than sucrose. In fact, one two-year study suggested about 30% reduction in caries incidence in fructose-using subjects compared with sucrose consumption (Turku study; Table 9.4). This caries reduction is relatively similar to the 35% reduction achieved in another study with invert sugar (Gustavsberg study; Table 9.4). In both studies the comparison was made with normal sucrose-consuming subjects. The current view is, however, that D-glucose, Dfructose, and their mixtures, must be regarded as grossly cariogenic carbohydrates; the cariologic difference between sucrose and its constitutive monosaccharides may be subtle. The cariologic evidence on other common dietary carbohydrates is scant. Although maltose and lactose are not as cariogenic as sucrose, their fermentation in dental plaque produces monosaccharides that are cariogenic. Erythritol is a sugar alcohol of the tetritol type, i.e., the erythritol molecule comprises four carbon atoms and four OH-groups. No true clinical caries trials on erythritol have been conducted, but short-term tests and laboratory studies suggest that erythritol may turn out to have a role as a sugar substitute in certain dietary applications (Kawanabe et al., 1992; MaÈkinen et al., 2001, 2002). These studies have suggested that the mechanism of the biochemical effect of erythritol on mutans streptococci differs from that of xylitol. It is possible, therefore, that erythritol-xylitol combinant saliva stimulants could have additive effects on these bacteria. An indication of the involvement of different mechanisms in the growth inhibition of mutans streptococci in the presence of

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Fig. 9.2 An example of the selective effects of dietary sugar alcohols on the growth of cariogenic streptococci. The selectivity clearly extends beyond the cases shown, since the growth of each cariogenic bacterial species and of each bacterial strain can be affected by sugar alcohols to a varying degree and by different mechanisms. The examples shown are those obtained with Streptococcus sobrinus ATCC 33478 (A) and S. mutans (strain 267-S-2) (B), grown in the presence of 6% polyols. Unpublished data from the author's laboratory.

various polyols is illustrated in Fig. 9.2. The growth curves show that the presence of erythritol in the broth caused an inhibition that was manifested at a later growth phase compared with the inhibition caused by xylitol. This result speaks for the existence of selective effects of sugar alcohols on mutans streptococci. Potential sugar substitutes from the cariologic point of view are the disaccharide sugar alcohols maltitol and lactitol. Neither has been tested in long-term studies, but at least maltitol (owing to its higher sweetening power compared with lactitol) possesses potential as a cariologically interesting sweetener. Lactitol has been recommended as a functional ingredient in pre- and probiotic products. Lactulose may to some extent provide similar effects. Both

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215

lactulose and lactitol may be regarded as dentally safe, although this matter must be elucidated in clinical tests. The food and confectionery industry exploits maltitol syrups as bulking agents. From the dental point of view the presence of D-glucose and D-glucitol in some manufactured syrups may be considered to a certain extent questionable. Inadequate design of some published caries trials has led to invalid conclusions which may be difficult to correct owing to the long duration of caries trials and the sluggish scientific publication processes involved. Serious inadequacies in caries trials on chewing gums containing sugar substitutes can include the following: (a) inadequate (too low) dose level of the substitute, and (b) unnecessarily long chewing time (say, 10 to 20 min. or longer). Consequently, the findings of Machiulskiene et al., (2001) of five different chewing regimens (that included xylitol and D-glucitol) showing no difference in caries prevention, cannot be regarded as valid owing to the involvement of the above two inadequacies. Prolonged gum chewing nullifies the possible chemical or pharmacological effect of the active principles present in the gum. Observing such a study design may equalise the sweetener effects; the tested substances will eventually behave similarly in the study model. A recommended chewing time in such studies is less than ten min., preferably five min. This requirement is based, for example, on observations that the minimum pH value is often reached in dental plaque within the first one to three min. of chewing. Sometimes also the interpretation of the caries data has been executed erroneously. In a critical review (Hayes, 2002) of the above paper (Machiulskiene et al., 2001) which claimed no differences existing between the five treatments, it was demonstrated ± despite the shortcomings of the study design ± that xylitol chewing gum was actually the only gum that lowered the DMFS increment rate compared with the no-gum control after three years. A similar problem caused by prolonged gum chewing may have occurred in a study where a D-glucitol gum was chewed for 20 min. after meals three times a day (SzoÈke et al., 2001). The control group did not receive gum as part of the study. Effective and long-term chewing may explain the 38.7% reduction in caries incidence measured between the groups. The chewing time was five min. in the Belize studies. 9.3.1 Animal experiments Animal studies were popular during the earlier days of caries research and a large number of caries studies have been carried out especially in rats and hamsters. The animal model can be considered to have generated a wealth of important cariologic information. These studies have generally shown sucrose to be extremely cariogenic and profound differences to exist between the cariogenicity of sucrose and xylitol. With only one exception, studies in rats have demonstrated an extremely low caries rate in relation to a xylitolcontaining diet (the above study, published only in the form of a congress abstract, and other older literature were reviewed in MaÈkinen and Scheinin,

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1982). Despite certain limitations that prevent direct extrapolation of animal data into human caries, ingenious models have contributed to the understanding of cariostatic mechanisms.

9.4

Future trends

Development of alternatives to sugar continues on several fronts. On academic grounds, future clinical studies in the field of caries prevention and sugar substitutes should include detailed comparison between xylitol and other polyols such as erythritol and maltitol, and combinations of all these polyols. Regarding xylitol, the use of which as a caries-limiting dietary substance can now be regarded as established, it will be necessary to focus on the elucidation of the following issues: (a) What is the exact chemical mechanism of the xylitol effect? (b) At which age exactly should a xylitol programme be employed to maximise protection against dental caries? (c) To what extent exactly do bacterial cells in a xylitol environment differ structurally and physiologically from those present in a sugar environment? (d) What is ± under different conditions ± the minimum level of xylitol that still brings forth an effective caries-preventive effect? (e) What is the most effective delivery mechanism of xylitol? Clarification of these issues may be considered important since the number of patients suffering unnecessarily from dental caries is increasing. Important target groups of effective caries prevention do not include children only, since also other patient cohorts, such as functionally dependent subjects (housebound or institutionalised people) deserve adequate caries prevention.

9.5

References

and INKILAÈ-SAARI I (2000), `Efficacy of a slowrelease device containing fluoride, xylitol and sorbitol in preventing infant caries', Acta Odontol Scand, 58, 285±292. ALANEN P, ISOKANGAS P and GUTMANN K (2000), `Xylitol candies in caries prevention: results of a field study in Estonian children', Community Dent Oral Epidemiol, 28, 218±224. BARMES D, BARNAUD J, KHAMBONANDA S and SARDO-INFIRRI J (1985), `Field trials of preventive regimes in Thailand and French Polynesia', Int Dent J, 35, 66±72. BIRKHED D (1994), `Cariologic aspects of xylitol and its use in chewing gum: a review', Acta Odontol Scand, 52, 116±127. CAUFIELD PW, CUTTER GR and DASANAYAKE AP (1993), `Initial acquisition of mutans streptococci by infants: evidence for a discrete window of infectivity', J Dent Res 72, 37±45. AALTONEN AS, SUHONEN JT, TENOVUO J

Ê , HENRIKSON CO, È F L, BLOMQVIST T, DAHL HM, EDWARD S, FJELLSTRO ÈM A FROSTELL G, BLOMLO

and NORDENVALL KJ (1974), `Substitution of sucrose by LycasinÕ in candy. The Roslagen study', Acta Odontol Scand, 32, 235±254.

LARJE O, NORD CE

Ê , HENRIKSON C-O, LARJE O, FROSTELL G, BLOMQVIST T, BRUNEÂR P, DAHL GM, FJELLSTROÈM A

Sweeteners and dental health

217

and WIK O (1981), `Reduction of caries in pre-school children by sucrose restriction and substitution with invert sugar. The Gustavsberg study', Acta Odontol Scand, 39, 333±347. GALIULLIN AN (1981), `Evaluation of the caries-preventive action of xylitol', Kazan Med J, 67, 16±18 (in Russian). GEORGIEFF M, MOLDAWER LL, BISTRIAN BR and BLACKBURN GL (1985), `Xylitol, an energy source for intravenous nutrition after trauma', Parenter Enteral Nutr, 9, 199±209. GLASS RL (1983), `A two-year clinical trial of sorbitol chewing gum', Caries Res, 17, 365±368. GUSTAFSSON BR, QUENSEL CE, LANKE LS, LUNDQVIST C, GRAHNEÂN H, BONOW BE and KRASSE B (1953), `The Vipeholm dental caries study. The effect of different levels of carbohydrate intake on caries activity in 436 individuals observed for five years', Acta Odontol Scand, 11, 232±364. HAYES C (2002), `Xylitol gum decreases the decayed, missing, and filled surfaces (DMFS) score over a 3±year period by an average of 1.9', J Evid Base Dent Pract, 2, 14±15. È KINEN KK, BENNETT, CA, ISOTUPA KP, ISOKANGAS PJ, ALLEN P and MA È KINEN P-L HUJOEL PP, MA (1999), `The optimum time to initiate habitual xylitol gum-chewing for obtaining long-term caries prevention', J Dent Res, 78, 797±803. È KINEN KK (1988), `Xylitol chewing gum in caries ISOKANGAS P, ALANEN P, TIEKSO J and MA prevention: A field study in children at caries-active ages', J Am Dent Assoc, 117, 315±320. È KINEN KK, TIEKSO J and ALANEN P (1993), `Long-term effect of xylitol ISOKANGAS P, MA chewing gum in the prevention of dental caries: a follow-up 5 years after termination of a prevention program', Caries Res, 27, 495±498. È DERLING E, PIENIHA È KKINEN K and ALANEN P (2000), `Occurrence of dental ISOKANGAS P, SO decay in children after maternal consumption of xylitol chewing gum, a follow-up from 0 to 5 years of age', J Dent Res, 79, 1885±1889. È KINEN KK (1995), `Effect of polyol gums ISOTUPA KP, GUNN S, CHEN C-Y, LOPATIN D and MA on dental plaque in orthodontic patients', Am J Orthod Dentofac Orthop, 107, 497± 504. KANDELMAN D and GAGNON G (1990), `A 24±month clinical study of the incidence and progression of dental caries in relation to consumption of chewing gum containing xylitol in school preventive programs', J Dent Res, 69, 1771±1775. È R A and HEFTI A (1988), `Collaborative WHO xylitol field study in KANDELMAN D, BA French Polynesia. I. Baseline prevalence and 32-month caries increment', Caries Res, 22, 55±62. KAWANABE J, HIRASAWA M, TAKEUCHI T, ODA T and IKEDA T (1992), `Noncariogenicity of erythritol as a substrate', Caries Res, 26, 358±362. MACHIULSKIENE V, NYVAD B and BAELUM V (2001), `Caries preventive effect of sugarsubstituted chewing gum', Community Dent Oral Epidemiol, 29, 278±288. È KINEN KK (1985), `New biochemical aspects of sweeteners', Int Dent J, 35, 23±35. MA È KINEN KK (1989), `Latest dental studies on xylitol and mechanism of action of xylitol MA in caries limitation', in Grenby TH, Progress in Sweeteners, London and New York, Elsevier, 331±362. È KINEN KK (2000a), `Can the pentitol-hexitol theory explain the clinical observations MA made with xylitol?', Med Hypotheses, 54, 603±613. È KINEN KK (2000b), `The rocky road of xylitol to its clinical application', J Dent Res, MA 79, 1352±1355. È KINEN KK and SCHEININ A (1975), `Turku sugar studies. VII. Principal biochemical MA NORD C-E, NORDENVALL K-J

218

Functional foods, ageing and degenerative disease

findings on whole saliva and plaque', Acta Odontol Scand, 33 (Suppl. 70), 129± 171. È KINEN KK and SCHEININ A (1982), `Xylitol and dental caries', Ann Rev Nutr, 2, 133±150. MA È KINEN KK, SO È DERLING E, ISOKANGAS P, TENOVUO J and TIEKSO J (1989), `Oral MA biochemical status and depression of Streptococcus mutans in children during 24to 36-month use of xylitol chewing gum', Caries Res, 23, 261±267. È KINEN KK, BENNETT CA, HUJOEL PP, ISOKANGAS P, ISOTUPA K, PAPE HR JR and MA È KINEN P-L MA (1995), 'Xylitol chewing gums and caries rates: a 40±month cohort study', J Dent Res, 74, 1904±1913. È KINEN KK, HUJOEL PP, BENNETT CA, ISOTUPA KP, MA È KINEN P-L and ALLEN P (1996a), MA `Polyol chewing gums and caries rates in primary dentition: a 24±month cohort study', Caries Res, 30, 408±417. È KINEN KK, PEMBERTON D, MA È KINEN P-L, CHEN C-Y, COLE J, HUJOEL PP, LOPATIN D and MA LAMBERT P (1996b), `Polyol-combinant saliva stimulants and oral health in Veterans Affairs patients ± an exploratory study', Spec Care Dentistry, 16, 104±115. È KINEN KK, CHEN C-Y, MA È KINEN P-L, BENNETT CA, ISOKANGAS PJ, ISOTUPA KP and PAPE HR MA JR (1996c), `Properties of whole saliva and dental plaque in relation to 40±month consumption of chewing gums containing xylitol, sorbitol or sucrose', Caries Res, 30, 180±188. È KINEN KK, HUJOEL PP, BENNETT CA, ISOKANGAS P, ISOTUPA K, PAPE HR JR and MA È KINEN P-L MA (1998), `A descriptive report of the effects of a 16-month xylitol chewing gum programme subsequent to a 40-month sucrose gum programme', Caries Res, 32, 107±112. È KINEN KK, ISOTUPA KP, KIVILOMPOLO T, MA È KINEN P-L, TOIVANEN J and SO È DERLING E MA (2001), `Comparison of erythritol and xylitol saliva stimulants in the control of dental plaque and mutans streptococci', Caries Res, 35, 129±135. È KINEN KK, ISOTUPA KP, KIVILOMPOLO T, MA È KINEN PL, MURTOMAA S, PETA È JA È J, TOIVANEN J MA and SOÈDERLING E (2002), `The effect of polyol-combinant saliva stimulants on S.

mutans levels in plaque and saliva of patients with mental retardation', Spec Care Dentist 22, 187±193. È LLER IJ and POULSEN S (1973), `The effect of sorbitol-containing chewing gum on the MO incidence of dental caries, plaque and gingivitis in Danish schoolchildren, Community Dent Oral Epidemiol, 1, 58±67. SCHEIE AA, FEJERSKOV O and DANIELSEN B (1998), `The effects of xylitol-containing chewing gums on dental plaque and acidogenic potential', J Dent Res, 77, 1547± 1552. È KINEN KK (1975), `Turku sugar studies I-XXI', Acta Odonol Scand, SCHEININ A and MA 33(Suppl 70), 1±349.

 NO  CZY J, SZO È KE J, ESTA  RI I, PIENIHAÈKKINEN K, SCHEININ U, TIEKSO J, SCHEININ A, BA

and HADAS E (1985a), `Collaborative WHO xylitol field studies in Hungary. I. Three-year caries activity in institutionalized children', Acta Odontol Scand, 43, 327±347. È KKINEN K, TIEKSO J, BA Â NO Â CZY J, SZO È KE J, ESTA Â RI I, ZIMMERMANN P and SCHEININ A, PIENIHA HADAS E (1985b), `Collaborative WHO xylitol field studies in Hungary. VII. Twoyear caries incidence in 976 institutionalized children', Acta Odontol Scand, 43, 381±387. SINTES JL, ESCALANTE C, STEWART B, MCCOOL JJ, GARCIÂA L, VOLPE AR and TRIOL C (1995), `Enhanced anticaries efficacy of a 0.243% sodium fluoride/10% xylitol/silica dentifrice: 3±year clinical results', Am J. Dent, 8, 231±235. ZIMMERMANN P

Sweeteners and dental health

219

and LOVETT J (2002), `Anticaries efficacy of a sodium monofluorophosphate dentifrice containing xylitol in a dicalcium phosphate dihydrate base. A 30-month caries clinical study in Costa Rica', Am J Dent, 15, 215±219. SLACK GL, MILLWARD E and MARTIN WJ (1964), `The effect of tablets stimulating salivary flow on the incidence of dental caries', Br Dent J, 116, 105±108. SLACK GL, DUCKWORTH R, SCHEER B, BRANDT RS and AILIANOU MC (1972), `The effect of chewing gum on the incidence of dental diseases in Greek children. A 3±year study', Br Dent J, 133, 371±377. È DERLING E, ISOKANGAS P, PIENIHA È KKINEN K and TENOVUO J (2000), `Influence of SO maternal xylitol consumption on acquisition of mutans streptococci by infants', J Dent Res, 79, 882±887. È DERLING E, ISOKANGAS P, PIENIHAÈKKINEN K, TENOVUO J and ALANEN P (2001), `Influence SO of maternal xylitol consumption on mother-child transmission of mutans streptococci: a 6±year follow-up', Caries Res, 35, 173±177. È KE J, BA Â NO Â CZY J and PROSKIN HM (2001), `Effect of after-meal sucrose-free gumSZO chewing on clinical caries', J Dent Res, 80, 1725±1729. TANZER J (1995), `Xylitol chewing gum and dental caries', Int Dent J, 45 (Suppl 1), 65± 76. TRAHAN L (1995), `Xylitol: a review of its action on mutans streptococci and dental caries ± its clinical significance', Int Dent J, 45 (Suppl 1), 77±92. SINTES JL, ELIÂAS-BONETA A, STEWART B, VOLPE AR

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Part II Obesity

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10 Nutrient-gene interactions in the control of obesity C. Verdich, Copenhagen University Hospital, Denmark, Karine Clement, INSERM, France and T. I. A. Sùrensen, Copenhagen University Hospital, Denmark

10.1

Introduction

By the year 2000, being overweight or obese was more common than being normal weight among the adult population in many European countries and in the United States. Also among children and adolescents the prevalence of obesity is increasing (Strauss and Pollack 2001; Reilly and Dorosty 1999; Thomsen et al. 1999; Sorensen et al. 1997). The World Health Organisation has classified this as a global epidemic of obesity and emphasised that it is not restricted to the industrialised countries (World Health Organisation 1997). The epidemic of obesity has major costs on the individual level. The costs encompass excess mortality and morbidity from cardiovascular diseases, type II diabetes, certain forms of cancers, osteoarthritis and sleep apnoea. Moreover, obese subjects tend to suffer from various forms of social stigmatisation and discrimination contributing to low quality of life. The medical-care costs burden of obesity is considerable, and increasing along with the epidemic. Obesity and its related co-morbidities are estimated to account for 5.5±7% of the total health care expenditure in the United States, and 2.0±3.5% in other Western countries (Thompson and Wolf 2001). Obesity causes a considerable increase in sick leave, and risk of early retirement (Seidell 1998). Unfortunately, current strategies for prevention and treatment of obesity have failed to reverse the epidemic of obesity, and therefore a continued search for modifiable causes is imperative. The development of obesity is determined by both genetic and environmental factors. A considerable proportion of the between-subject variation in body weight is determined by genetic differences, but part of the variation must also

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be attributed to differences in environment. Whereas changes in the environment must be responsible for the increasing prevalence of obesity, genetic factors together with environmental factors are expected to determine who will become obese, and to what degree obesity will develop. Thus, genetic factors influence the distribution of obesity in a given environment, in a given population, at a given time. It is generally assumed that the genes and the environment interact in some way, but there is considerable uncertainty about how this interaction takes place. Obviously, there must be a tight interaction between genes and the environment fully integrated in the biological system that constitutes the organism of any species. This type of interaction does not in itself contribute to the interindividual differences, and particularly not to the explanation of why some become and stay obese and others do not. If gene-environment interaction contributes to this kind of difference, there must be between-subject variations that cannot be attributed to the genetic differences and/or the environmental differences as such. Thus, this type of gene-environment interaction implies that the response to a certain environmental exposure depends on the particular genotype, and vice versa, that the effect of a particular genotype depends on the environmental exposures. The aim of the present chapter is to present an overview of the topics related to studying the role of genetic and environmental factors, and especially the role of nutrient-gene interaction, in the control of obesity. The strategies for identifying `nutrient sensitive genes' and the current knowledge on nutrientgene interactions with a putative role in obesity will be presented.

10.2

Genetic influences on obesity

10.2.1 The role of genetic factors in obesity The estimated quantitative role of genetic factors varies dependent of study type. In family studies, the heritability has been estimated to 20±40% (Maes et al. 1997). The correlations between full siblings are higher than between parents and their offspring, which suggest non-additive genetic influences, possibly due to intra- and inter-locus gene-gene interactions, or a higher degree of shared environment between siblings than between parents and their offspring. However, family studies do not allow separation of genetic effects and effects of shared family environment, which may be achieved in studies of adopted children and their biological and adoptive families and by twin-studies. In adoption studies, the resemblance between the adoptee and the biological family members, i.e., parents, full and half-siblings, can be ascribed solely to genetic factors. This approach has suggested that genetic factors account for 20±40% of between-subject differences in obesity and associated phenotypes (Maes et al. 1997; Stunkard et al. 1986; Sorensen et al. 1989). Studies of monozygotic and dizygotic twins have revealed a much greater resemblance in the degree of obesity between monozygotic twins than between

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dizygotic twins, indicating that the resemblance is related to their similar genetic background rather than shared environment. These types of studies indicate a higher heritability, indicating that up to 60±80% of the between-subject differences can be ascribed to genetic factors. It might be argued that monozygotic twins may tend to share more environmental factors than dizygotic twins, which would lead to an overestimation of the heritability. However, twins raised apart show the same resemblance in body weight as twins raised together (Stunkard et al. 1990). Also, adoption and twin studies suggest that there are non-additive genetic influences, but generally they are difficult to disentangle from shared environmental influences, which naturally make studies of gene-environment interactions in this setting difficult. Both twin studies and adoption studies have indicated that the childhood family environment plays a minor ± if any ± role in adult obesity and associated phenotypes, whereas the rearing environment may have some influence while the child lives in the parents' home (Sorensen 1996). This indicates that the within family resemblance in BMI in adults can be almost exclusively ascribed to genetic background (Sorensen et al. 1992; Vogler et al. 1995). Selecting the optimal obesity phenotype for genetic research Obesity represents merely the extreme in continuously distributed phenotypes. Although standardised categorisation of subjects as normal-weight and obese may be relevant in relation to treatment and prevention of obesity, arbitrary classification may hamper the identification of genetic and environmental factors contributing to the between-subject variation in obesity and related traits. Studies aiming to elucidate the role of genetic components and nutrient-gene interactions in obesity should ideally involve detailed characteristics of the obesity state, including a broad range of obesity-related and intermediate phenotypes (Comuzzie and Allison 1998). Specification such as body fat percentage, or body fat distribution, and the use of intermediate phenotypes such as energy expenditure, fat oxidation, and plasma levels of hormones expected to be involved in the regulation of energy balance, has several advantages. Firstly, assessment of body composition gives a more refined measure of the degree of fat accumulation, as compared to body weight and BMI. Secondly, assessment of parameters related to adipose tissue metabolism, energy expenditure, and appetite regulation offers the possibility of studying genetic factors involved in the regulation, of energy balance, and exploring the mechanisms of action. Thirdly, it is conceivable that intermediate phenotypes, such as energy expenditure or fat oxidation, may be less influenced by environmental factors than BMI per se. Indeed, when addressing the role of specific candidate genes, the phenotypic profile should include intermediate phenotypes presumed to be closely linked to the function of the candidate genes. Finally, recognising obesity as a complex heterogeneous phenotype it is of importance to address the common traits, i.e., the high body weight, as well as the heterogeneity with regard to, for example, abdominal fat accumulation, insulin sensitivity, lipid

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metabolism, etc. Changes in the phenotype by environmental manipulation, e.g., changes in body weight, body composition, or abdominal obesity by changes in energy balance, is another potentially profitable approach to study the effect of different genotypes, and it may be particularly suitable to the study of geneenvironment interaction. 10.2.2 Candidate genes and the mechanism behind the role of genetic factors A major aim in obesity research is to identify single gene variants involved in the development of obesity and to explore and clarify the interaction between specific gene variants and specific environmental factors, with the prospect of directly transforming this knowledge into techniques for identification of individuals at risk for developing obesity, and developing strategies for specific prevention and treatment. As judged from the phenotypic segregation pattern in the families, the general between-subject variations in body weight and other obesity-related phenotypes undoubtedly involve a complex oligo- or polygenic non-Mendelian pattern of inheritance. Challenges in identifying obesity genes There are two strategic approaches for identifying potential candidate genes. The first one is to study the association between obesity or obesity-related phenotypes and already identified candidate genes selected on the basis of their known or presumed biological function, and the second one is to search for regions in the genome which appear to be linked to the obese phenotypes (Clement et al. 2002a). A great number of research groups have contributed to this field by studying candidate genes of interest in cohorts of obese patients. For this purpose they have constituted banks of clinical data and DNA in large cohorts of obese patients and controls. Group of patients and their families have been characterised with regard to clinical and biological parameters related to obesity. In association studies, the frequency of DNA variations between groups of subjects (i.e. obese vs. non-obese) is compared, or a measurable phenotype (body mass index, fat mass, skin folds, waist/hip ratio) in subjects carrying or not carrying the given polymorphism is compared. Such association studies have been conducted in many populations collected in Europe and in North America. They have provided a huge number of putative susceptibility genes, but with small or uncertain effects (Chagnon et al. 2003; Swarbrick and Vaisse 2003). This strategy has been used both for adults (Clement et al. 2002a) and children (Clement and Ferre 2003). For the candidate genes that have shown association to the obese phenotype in one population, the general situation is lack of replication in independent populations. Association studies encounter many pitfalls. They include doubtful links between the physiological roles of the candidate genes and body weight regulation. Selecting candidate genes based on rodent models of monogenic forms of obesity have been considered for genetic studies of human obesity.

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Although previous studies have led mainly to the discovery of rare forms of obesity in humans, it is likely that key regulatory genes discovered in animal forms of monogenic obesity may reveal genetic factors involved in the common forms of obesity in humans. There are also difficulties related to statistical aspects including too small sample sizes of obese and controls, non-representative control groups, biased population stratification, false positive results due to multiple testing, and suppression of negative results. This state of affairs has led to development of recommendations that should secure more robust results; sufficient sample size, necessity of replication in independent groups, statistical correction for multiple testing, and functional assessment of the gene variant. However, only few published studies have met these criteria. Whereas the candidate gene approach may be successful in addressing the genetic factors influencing mono- or oligogenic traits, this approach seems destined to fail when studying polygenic inheritance where many different genes contribute to the phenotype in interaction with environmental factors and other genes (Sorensen and Echwald 2001; Comuzzie et al. 2001; Comuzzie and Allison 1998). One set of problems is related to the identification of relevant candidate genes as described. Further, genes identified as major obesity genes in family-based linkage studies may turn out to be of major importance only in coexistence with other obesity genes, which are present in the selected populations, and have only minor influence on the common forms of obesity. Another set of problems is related to the study size and statistical power, which in these settings are even more demanding. In very large studies it may be possible to study the gene-gene interactions, whereas collection of detailed information regarding the environment, e.g., the habitual lifestyle, and thorough phenotypic profile including the response to dietary interventions, is feasible only in smaller studies. Moreover, the success of addressing the gene-gene and gene-environment interaction may greatly depend on the inclusion of genes and environmental factors with major effects on the model, since even relatively large effects of `minor' genes will become evident only after adjustment for major effect (Williams 1984). Thus, the `major effect' factors will need to be clarified before the `minor-effect' factors can be addressed. The other approach for identifying obesity genes does not involve any a priori hypothesis about the genes and their function. Linkage analyses in families offer the possibility to study the co-segregation of chromosomal markers with obesity or related phenotypes. The technique of genome-wide scans offers a new way of identifying candidate genes, which that can then be examined further using the candidate gene approach. Genome-wide scans have been performed in populations originating from Europe (France, Germany, Finland, Denmark), United States and Canada (Chagnon et al. 2003; Swarbrick and Vaisse 2003; Clement et al. 2002a). In North America, the genome-wide scan has been performed either in Caucasian families, or in selected populations with less admixture such as Pima Indians, Amish, Mexican, Indian or African Americans. Usually, families in which

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obesity-related traits segregate are analysed using 400 to 600 polymorphic markers regularly spanning the genome, with the goal of finding the genes and pathways underlying these complex traits. The genome-wide scan approach has provided more than 25 genome-wide scans for obesity and related phenotypes. The validity of chromosomal loci identified in genetic linkage is also here increased if the association between the loci and the phenotype has been replicated in other studies. Seven regions of the human genome harbour quantitative trait loci (QTL) replicated in two to five studies with high lod score (Chagnon et al. 2003). Some of the QTLs could explain a significant part of the variance of obesity-related phenotypes. In mice, hundreds of QTLs have been linked to body weight or body fat. Among them at least six different chromosomal loci (DO1-6) have been identified by genetic mapping studies after crosses of mice strain differentially sensitive to diet-induced obesity (e.g. the AKR/J being the most sensitive strain and the SWR/J, the less sensitive strain to high fat diet). However, the corresponding genes explaining the linkage have not been found in mice or in humans, even in chromosomal regions showing high and replicated statistical linkage. The multi-factorial nature of obesity with a polygenic, non-Mendelian inheritance is probably responsible for the lack of success of gene identification. Several years will probably be needed to clone the genes located in the regions of linkage but the time needed for gene identification will possibly be considerably reduced thanks to the use of strategies combining analysis of genome scans and gene expression (see below). Monogenic forms of human obesity Although studying the role of single gene variants may not solve the enigma of obesity, this approach has led to the discovery and classification of a series of rare monogenic types of obesity that might contribute to the understanding of the molecular basis of a number of well known rare syndromes in which obesity is a main feature, such as Bardet-Biedl syndrome, Prader-Willi syndrome, and Alstrom syndrome. However, for several of the identified genes it remains unclear what the role of these genes is in the complex pathogenesis of the disease. The discovery and characterisation of these rare monogenic forms of obesity provide a valuable insight into the complex physiological pathways involved in the control of size of fat tissue and energy balance. Such discoveries may pave the way for developing new pharmacological aids for treating obesity, irrespective of the cause. During the last decade, several rare monogenic forms of obesity have been described, involving the genes encoding for the fat cell hormone leptin and its receptor, pro-opiomelanocortine (POMC) and its converting enzyme, pro hormone convertase 1, and finally the melanocortin 4 receptor (Clement et al. 2002a). The examples below are monogenic forms of obesity, in which the discovery of the underlying genetic cause has led to new insights into the pathways involved in the regulation of body weight.

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In 1994, the product of the ob-gene, the 16 kd peptide hormone, referred to as `leptin', was described for the first time (Zhang et al. 1994) followed by the description of the rodent leptin receptor (Lee et al. 1996). Mice lacking either functional leptin (ob/ob mice) or leptin receptor (db/db mice) are severely obese. Rare homozygous loss of function mutations in the human leptin and leptin receptor genes have been shown to lead to symptoms similar to those seen in ob/ ob and db/db animals, including early onset of severe obesity, abnormal eating behaviour, and hypogonadotropic hypogonadism (Clement et al. 1998; Montague et al. 1997; Ozata et al. 1999). Several studies have suggested a possible association between more common polymorphisms in the human leptin and leptin receptor gene and obesity, but it has not been possible to confirm these associations. The role of genetic variants of the Melanocortin-4 receptor (MC4-R), the receptor for alpha-melanocyte-stimulating hormone, in the regulation of body weight and obesity in humans has been addressed in several studies (Vaisse et al. 1998, 2000; Sina et al. 1999). The frequency of rare heterozygous MC4-R missense and frameshift mutations has been found to be 4% in a population of morbidly obese subjects (Vaisse et al. 2000) but very low in normal-weight subjects. Among the obese subjects, phenotypic characteristics did not differ between carriers and non-carriers of the mutations, but carriers tended to have a higher prevalence of childhood obesity (Vaisse et al. 2000). In addition, a recent study has suggested binge eating as a major phenotypic trait in obese carriers of MC4-R mutations (Branson et al. 2003). Where other known forms of monogenetic obesity are recessive, and associated with other endocrine abnormalities, functional polymorphisms of the MC4-R gene appear to be associated with a dominant non-syndromic form of obesity, and it is the most frequent genetic cause of obesity described to date (Vaisse et al. 2000; Sina et al. 1999). Different types of obesity-mutations Until recently, mutations in the coding regions have been the major focus in the research addressing obesity genes. Localising the coding region of a gene is far less complicated than localising all of the regulatory elements. In addition, knowing the structure and the function of the gene product, it is possible to predict the potential effect of changes in a specific area of the coding region. However, mutations in the non-coding region have gained increasing attention. Addressing the regulatory regions of putative obesity genes may lead to the discovery of gene variants involved in obesity. In addition, combining genotyping with studies of gene expression in specific tissues and in response to specific exposures, such as changes in fat intake or calorie restriction, will improve understanding of the specific mechanisms of gene regulation and the mechanism for regulatory gene variants. In the past, mutations and polymorphisms in the promoters of several putative obesity genes including 5HT receptor, CART, UCP2, UCP3, TNFalpha, resistin and leptin, have been suggested to be associated with obesity-related phenotypes

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(Rosmond et al. 2000; Yamada et al. 2002; Halsall et al. 2001; Esterbauer et al. 2001; Mammes et al. 2000; Hoffstedt et al. 2000; Engert et al. 2002). However, several of these observations are challenged by negative findings, and still need to be replicated in additional studies. Physiological mechanisms behind the genetic effects in obesity Development of obesity is very slow, taking several years, and is typically considered to be a result of inappropriate adaptation of the systems involved in the control of energy balance to either a primarily increased energy intake or reduced energy expenditure, leading to a passive accumulation of surplus energy as body fat. Studies addressing the heritability of intermediate phenotypes have suggested that 30±50% of between-subject differences in metabolic variables, and 25±50% of between-subject variation in energy intake can be ascribed to genetic factors (Bouchard et al. 1989; de Castro 1993). These findings suggest that the search for specific genetic effects should encompass both components of the energy balance. However, the paradigm of obesity as passive storage of surplus energy may be insufficient. Active accumulation of fat in the adipose tissue, due to dys-regulation of the adipose tissue balance between release of fat and fat accumulation followed by a subsequent corresponding regulatory adjustment of the energy balance, should be considered (Sorensen 2003b). Table 10.1 illustrates the pathways in which genetic polymorphisms may affect the physiological pathways involved in the regulation of energy balance, hereby increasing the susceptibility to developing obesity in a given environmental setting. Examples of putative candidate genes are given for each pathway. 10.2.3 The epidemic of obesity: the role of environment and the interaction between genes and lifestyle As mentioned, there is no doubt that obesity is strongly influenced by environmental factors and that changes in the environment with which our genetic background interacts must be the direct cause of the rapid increase in the prevalence of obesity world-wide. This implies that prevention of obesity may be achieved through modification of environmental factors. However, knowing the basis for the gene-environment interactions in obesity may pave the way for more targeted prevention strategies, and thereby better success in the prevention of obesity. Given that genetic factors mediated the susceptibility to obesity in a given environmental setting and that different genes provide susceptibility to different types of environmental influences, the context of environment will be a critical factor in determining which genes will be identified (Barsh et al. 2000; Leibel 1997). In a restrictive environment characterised by low food resources and high demand for physical activity, obesity may be a rare phenomenon. Since the restrictive environment may have prevailed throughout the development of mankind, it can be speculated that the physiological defence against weight loss

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Table 10.1 The different mechanisms by which genetics are expected to play a role in the development of obesity; examples of putative candidate genes are given for each category. The genes are annotated with the approved gene symbol (Human Genome Nomenclature Database) Level Adipose tissue Auto regulation Metabolic function

Endocrine function

Energy intake Central Peripheral Food preferences

Energy expenditure Central

Physiological mechanism

Candidate genes

Adipocyte differentiation, fat accumulation Balance between fat release and fat accumulation

FOXC2, PPARA, PPARD, PPARG, RXRA, RXRB VLDLR, LIPE, LPL, SCD, UCP2, ADRB1, ADREB2, ADRB3, ADRA2A, ADRA2B, INSR Signals from adipose tissue to LEP, LEPR, NPPA, SPARC, central regulation of energy TNF, IL6, AMP1, balance Hypothalamic neurotransmitters or receptors Hormones or other signalling compounds involved in appetite regulation Preference for sweet, fat, aversion to certain fruits and vegetables due to high sensitivity to bitter taste.

Mediator

Hypothalamic neurotransmitters or receptors Symphatoadrenergic system

Effector

EE as such, fat oxidation

NPY, NPYR, POMC, MC4R, LEPR, CART, 5HT2C, CCKAR, AGRP CCK, APOA4, GHRL, PPY, GCG TAS1R, TAS2R

(MC-4R), (Dopamin 2R), (NPY-R) ADRB1, ADREB2, ADRB3ADRA2A, ADRA2B UCP1, UCP2, UCP3

and under-nutrition are stronger than the defence mechanisms protecting against weight gain. As hypothesised by Neel in the 1960s, gene variants now associated with type 2 diabetes and obesity helped our early ancestors survive in a restrictive environment (Neel 1999). This theory, commonly referred to as `the thrifty gene hypothesis', states that what is now seen as a susceptibility to obesity and diabetes may indeed be a conserved mechanism of economical management of body energy resources. Basically, weight gain will occur only when energy intake exceeds energy expenditure, and it may therefore be stated that the reason why more and more people are becoming obese is that they either eat too much or have too low a level of physical activity. This highly simplified statement may capture the essence of the mechanism by which our environment and our behaviour promote obesity, although there is no convincing evidence available to support the

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contention. Thus, there is no evidence precluding the alternative hypothesis that the epidemic is due to an increased primary accumulation of fat in the adipose tissue that subsequently leads to a corresponding adjustment of the energy intake relative to the energy expenditure. The prevailing opinion does not hold any other key to prevention than telling the public to eat less and increase their physical activity; which has not succeeded in reducing the prevalence of obesity. Obviously, a more refined strategy for prevention is needed, and one of the major challenges in obesity research is to identify the environmental factors responsible for the increasing prevalence and reveal the interaction between environmental and genetic factors. Macronutrient composition of the diet and obesity Although fat intake is one of the potential obesity promoting factors that has gained more attention in obesity research, the role of fat intake in the development of obesity is still controversial, and the findings are not consistent. In animal models, changing from a low-fat diet to a high-fat diet leads to an increase in body fat and an increase in the inter-individual and inter-strain variation in body fat, suggesting a genetic susceptibility to become obese on a high-fat diet (West et al. 1995; Salmon and Flatt 1985). Epidemiological studies have not so far led to any clear conclusions on the role of fat intake and obesity in humans (Lissner and Heitmann 1995). In general, cross-sectional studies have indicated positive associations between dietary fat energy per cent and body weight, but such studies cannot distinguish between the possible effects of obesity on fat intake, vice versa or common effects on both obesity and fat intake of an underlying third factor. There are some paradoxical observations regarding the relationship between reported fat intake and obesity that raise the suspicion that there is no simple relation between the two. In the US and in many European countries, fat intake has decreased during the last decade, whereas the prevalence of obesity has increased. This may be interpreted as an indication that reducing dietary fat may not lead to a concomitant reduction in obesity. However, increased under-reporting of fat intake may bias these observations (Heitmann et al. 2000), and subgroups of the population may have increased their fat intake and become obese, whereas others may have reduced their fat intake. In line with the findings from animal models, a high habitual fat energy percentage has been shown to be associated with a higher mean BMI, as compared to low fat consumers (Macdiarmid et al. 1996). Another common feature between animal and human studies is that between-subject variation is higher in high-fat consumers, and that some individuals appear to be protected from developing obesity even when consuming a high-fat diet (Macdiarmid et al. 1996). Results from prospective observational studies do not support the hypothesis that high fat intake leads to later obesity. However, these studies are inconsistent and may be confounded by people modifying food intake in order to prevent changes in body weight or that for other reasons the baseline food recording

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does not reflect food habits during the follow up period (Lissner and Heitmann 1995). Intervention studies generally support the hypothesis about a relationship between fat intake, energy intake and eventual weight change. Ad libitum intake of a low-fat diet has been shown to induce a mean weight loss of 1±4 kg over a period of 1±12 months, and has further shown a dose-response relationship between the reduction in fat intake and weight loss (Astrup et al. 2000a,b). Although a weight loss of 0.5±1 kg per month may occur during the first month of such an intervention, weight loss will in many cases tend to level off, which may be due to either adaptation to the diet or cessation of compliance. If the dietary effect holds over longer periods of time, then reduction of fat intake may be effective in prevention of further development of obesity. Fat content and energy density of the diet are highly correlated, and only a few studies have targeted the effect of energy density and fat content separately. These studies have indicated that energy density rather than fat content per se may favour an increase in energy intake. However, it might be hypothesised that in subjects with a genetic predisposition to low fat oxidation and high fat accumulation in adipose tissue, fat intake per se could be expected to promote obesity. Further, genetic factors may influence the satiety effect of fat and the preference for fat, and may cause some people markedly to increase their food intake in response to a high-fat diet. Taken together, both animal studies, epidemiological observational studies, and intervention studies offer substantial support to the hypothesis that high fat energy percentage or high energy density may play a role in the development of obesity. Increase in dietary carbohydrate and protein energy percentage will cause a reduction in dietary fat energy percentage, and vice versa. Epidemiological studies have suggested a positive association between fat-sugar ratio in the diet and BMI (Bolton-Smith and Woodward 1994). With respect to the type of carbohydrates, intervention studies have indicated no differences in weight loss during intake of a low-fat diet rich in either simple or complex carbohydrates (Saris et al. 2000). However, there is evidence that high intake of simple carbohydrates in liquid form (soft drinks) may predispose to weight gain (Raben et al. 2002; DiMeglio and Mattes 2000; Ludwig et al. 2001). Animal studies have indicated inverse relationships between dietary protein content and energy intake with between-strain differences in response, which suggest a nutrient-gene interaction (West et al. 1995). Intervention studies have suggested that a low-fat high-protein diet may lead to a larger weight reduction compared with a low-fat high-carbohydrate diet (Skov et al. 1999; Baba et al. 1999). Recent intervention studies suggest that low carbohydrate-high protein diets may be superior to the low-fat high-carbohydrate diet in terms of weight reduction (Samaha et al. 2003; Foster et al. 2003). However, large-scale intervention studies are required to determine long-term safety and efficacy of these dietary strategies for both prevention and treatment.

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Other dietary factors During later years, several lines of studies have indicated a relationship between dairy calcium and regulation of body weight (Zemel 2003). Observational studies have shown a lower prevalence of obesity and rate of weight gain for subjects reporting a high calcium intake (Heaney 2003; Teegarden 2003). Several intervention studies have indicated that high intake of dairy calcium may aid intentional weight loss and prevent weight gain (Davies et al. 2000), although other studies have not indicated such effect (Bowen et al. 2003).

10.3

Nutrient-sensitive genes

10.3.1 Definition of nutrient-sensitive genes Naturally, numerous genes are involved through their gene products in the entire biological system regulating and responding to food intake and processing and metabolisation of nutrients. In the present context, the nutrient-sensitive genes are defined as genes of which the transcriptional activity is influenced, enhanced or reduced or even turned on or off by reduced or increased energy intake (calorie restriction and overfeeding, respectively) or by specific nutrients. Thus, this is a general biological phenomenon common to individuals of a species, and it should be distinguished from nutrient-gene interaction, which refers to interindividual differences between members of a species. 10.3.2 Nutrient-sensitive candidate genes The study of expression of specific candidate genes during changes in the nutritional environment, e.g., during fasting or intake of food enriched in fat, is an alternative approach to finding genetically defined pathways. Several candidate genes have been tested to decipher whether or not their level of expression in key tissues involved in body weight regulation (in humans, mostly adipose tissue and muscle) is modulated by change of environmental conditions. Most of the human studies have aimed at analysing the changes of expression after drastic caloric restriction in obese subjects. Most of the genes studied encode proteins involved in three different functional groups: (i) metabolic enzymes and related signalling proteins or receptors such as the hormonesensitive lipase (HSL), the lipoprotein lipase (LPL) involved in triglyceride hydrolysis and synthesis, respectively, adrenoreceptor genes and uncoupling proteins; (ii) factors involved in adipogenic process such as the transcription factors C/EBP, PPAR and SREBP1c; (iii) proteins secreted by adipose tissue such as leptin, TNF and inflammation-related proteins such as interleukine 8. As expected, severe caloric restriction leading to increased lipolysis and decreased lipid synthesis and improvement of insulin sensitivity in adipose tissue was associated with increased expression of HSL (Kolehmainen et al. 2002; Richelsen et al. 2000) and alpha-2-adrenergic receptor (Stich et al. 1997, 2002) and decreased expression of the LPL (Richelsen et al. 2000), leptin, and

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TNF genes (Bastard et al. 1999). The adipose tissue transcription factors are also mobilised in this situation (Bastard et al. 1999; Kolehmainen et al. 2002; Redonnet et al. 2002). Some of the changes in gene expression induced by weight loss have not been consistently reproduced, and the discrepancies may be attributed to the time of adipose tissue sampling over the course of weight loss. In some studies, tissue biopsies were obtained during active weight loss, whereas in others the biopsies were performed after weight stabilisation, which is probably associated with a new metabolic status. 10.3.3 Expression profiling of nutrient-sensitive genes The possibilities of studying multiple gene expression patterns in response to various nutritional conditions opened a new era for seeking pathways that could be implicated in the response to nutritional changes. The key issue is to dissect and characterise the regulatory pathways and networks involved in energy balance and to define the resulting signalling patterns in gene expression. This approach will facilitate a more integrative picture of the complex biologic process. The technology for the study of many RNA at the same time, i.e., micro-array, is now available (Copland et al. 2003). Micro-array technology is based on the simple concept of dot blot and northern blot analysis, where you reverse the hybridisation, put the probes on a filter and label the bulk RNA. One can study very large numbers (~100,000) of cDNA sequences or synthetic DNA oligomers on a glass slide (or other substrate like filters) in known locations on a grid. The target RNA sample is labelled and hybridised. The measured amount of RNA bound to each square in the grid reflects the level of expression of the gene. While initially used for simple organisms (e.g., yeast), this approach now indexes thousands of known and newly discovered genes into various large groups defined by expression similarities in terms of physiological pathways, for example respiration, cell division, and response to chemical or thermal stress. Micro-array DNA screening is now applied for the understanding of complex diseases including cancer and ageing. This technique can be applied to many other basic and clinical research problems, including the consequence of nutritional changes on the modification of gene transcripts. In humans, these techniques have been used to define molecular signatures of the physiology of insulin and thyroid hormone action. Variations in gene expression in muscle of healthy men treated with triiodothyronine (T3) has been investigated using cDNA micro-arrays representing 24,000 human genes (Clement et al. 2002b). It showed up-regulated genes encoding for proteins involved in a wide range of cellular functions including transcriptional control, mRNA maturation, protein turnover, signal transduction, cellular trafficking and energy metabolism. A lot of these genes were new targets of T3 action (Clement et al. 2002b; Viguerie and Langin 2003). Similar findings were made regarding the transcription consequences of insulin action in muscle. By analysing the global changes in mRNA levels after a hyper-insulinemic euglycemic clamp in

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healthy subjects, it was observed that a large number of transcripts were significantly modified. Most of the genes with known function are novel targets of insulin, and may define a transcriptional signature of insulin action in human skeletal muscle (Rome et al. 2003). Although information and resources are growing, it should be kept in mind that there are difficulties and pitfalls in the interpretation of microarray data, and progress still has to be made. One aspect is the tremendous source of variability at many levels. Thus, mRNA measurements are inherently highly variable (biological variability). The variability also depends on the level of expression of the gene (low vs. highly expressed genes). The methods by themselves induce variability: mRNA extraction, hybridisation (variability due to temperature, time, mixing), probe labelling (the chemistry of the fluorescent label is different), image analysis and scanning (laser and detector). In addition, measurement of thousands of values will find large differences that are only attributable to the random normal distribution of the data, and adequate procedures for multiple testing have to be applied. The goal of future projects in this field should aim at characterising clusters of genes that are recruited or modified by the given nutritional conditions, their links in biological families, their co-regulation in different tissues, gene markers specific for some nutrients, differences/similarities in different models, and eventually the patterns of tissue expression in individuals with different genetic polymorphisms in these genes.

10.4 Nutrient-gene interaction and the development of obesity Nutrient-gene interaction deals with the differential functional and eventual phenotypic effects of different doses ± ranging from zero to excessive amounts ± of total energy intake or specific nutrients, in combination with different gene variants. Nutrient-gene interaction addresses inter-individual differences within a species, for example, the biological basis for development of obesity in some but not in other individuals. Genetic differences that induce different risks and degrees of obesity without modifying the response to differences in the diet, e.g., its fat content, may not be considered to contribute to nutrient-gene interactions. Similarly, dietary differences that induce different risks and degrees of obesity, without modification of the effects of genetic differences, do not contribute to nutrient-gene interactions. The nutrient-gene interaction can, of course, operate at the level of modifications of the transcriptional activity of nutrient-sensitive genes, but not necessarily so. The nutrient-gene interaction may as well take place at any downstream post-transcriptional step in the function of nutrientsensitive genes and of other genes of which the transcriptional activity is not nutrient-sensitive. On the other hand, the search for polymorphisms in genes involved in nutrient-gene interaction may become very profitable, and the technology now makes this feasible on a genome-wide basis.

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The classical example and model of nutrient-gene interaction is phenylketonuria (Fùlling's disease), which leads to severe mental retardation in the carriers of a mutation in the gene for the enzyme phenylalanine hydroxylase if the subjects are fed a diet containing the amino acid phenylalanine while the brain is growing. If the individuals with this mutation avoid phenylalanine in the diet during this period, then the brain develops normally, and they may return to a normal diet, containing phenylalanine, when they are grown up (women with the mutation who get pregnant should, however, return to the restricted diet in order to protect the brain development of their unborn child). 10.4.1 Strategies for analysing the interaction between genetic background and diet There are several strategies for addressing the possible interaction between genetic background and diet composition in the development of obesity. Both animal models and human studies allow for the study of the interaction between genotype and diet and between diet and gene expression. Animal studies can be designed to address the effect of a specific genetic modification on susceptibility to the obesity-promoting effect of a high-fat diet, and the effect of diet on gene expression in various tissues and organs. However, only human studies will reveal the complex interaction between genetic and environmental factors responsible for human obesity and hence for the current epidemic of obesity. Human studies addressing the nutrient-gene interaction in obesity can be divided into epidemiological observational studies and intervention studies, addressing the interaction of various environmental factors with either overall genetic predisposition for development of obesity or for specific gene variants. Epidemiological observational studies Interaction between overall change in environment and genetic background may be revealed in studies of subjects of different ethnic origin who have been exposed to a so-called Westernised environment. One example is the increase in the prevalence of obesity in Japanese migrating to Hawaii or the USA (Curb and Marcus 1991). In some ethnic groups, such as the Pima Indians living in Arizona, and the population of Western Samoa, adoption of a Western lifestyle has led to a prevalence of obesity exceeding that in the USA and other Western countries (Knowler et al. 1991; Krosnick 2000; Hodge et al. 1994). Finally, the recent dramatic increase in the prevalence of obesity in Mauritius has been shown to be more pronounced among Creole than in Indian, and less pronounced in Chinese Mauritians (Hodge et al. 1996). Although cultural differences may still play a role, such findings suggest an interaction between a `thrifty genotype' prevailing in some ethnic groups and the Western lifestyle (Hodge et al. 1996). The apparently specific genetic predisposition for obesity of the Pima Indians in Arizona has been studied thoroughly, but so far the specific genetic background has not been identified.

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Prospective studies can address the association between habitual diet and weight changes over time for subjects with different degrees of genetic predisposition. Using this approach, Heitmann and colleagues investigated the interaction between genetic predisposition to obesity and fat intake in the Prospective Study of Women in Gothenburg, Sweden (Heitmann et al. 1995). Baseline fat intake adjusted for total energy intake was found to be positively related to subsequent six-year weight gain only in women who were already overweight, who had at least one obese parent, and whose fat intake at baseline exceeded 40% of the total energy. This observation suggests the existence of a genetic predisposition for gaining weight on a high fat diet, but confirmation in other studies is needed. The finding is in accordance with clinical studies showing a reduced fat oxidation rate following a high-fat test meal in post-obese subjects with a genetic predisposition to obesity, compared with never obese subjects (Astrup et al. 1994; Raben et al. 1994). A few epidemiological observational studies have addressed the interaction between lifestyle factors and specific gene variants in relation to obesity and related phenotypes. The EPIC-Heidelberg study is a large European prospective study of the potential interaction between dietary fatty acid intake, assessed by food frequency questionnaire, and common allelic variants of candidate genes on the obesity phenotypes (Nieters et al. 2002). They found that the Pro12Ala variant of one of the nuclear receptor peroxisome proliferator-activated receptor genes, the PPAR 2 gene, as well as common polymorphisms in the leptin, and tumour necrosis factor (TNF) gene may interact with the intake of linoleic acid and aracchidonic acid (Nieters et al. 2002). Subjects carrying the Ala allele of the PPAR 2 gene had a higher odds ratio for obesity with high intake of arachidonic acid compared with subjects homozygous for the Pro allele. The analysis initially showed that total fat intake as well as intake of n-6 polyunsaturated fatty acids were significant predictors of obesity, but when linoleic and arachidonic acid were included in the analysis the effect of total fat intake was no longer significant. This study therefore suggests a specific nutrient-gene interaction between common polymorphisms in candidate genes and dietary intake of n-6 fatty acids in obesity. Another prospective population-based cohort study also suggested genenutrient interaction involving the PPAR 2 gene and fatty acids. Based on a study including about 600 non-diabetic subjects, Luan and colleagues have shown an interaction between the ratio of polyunsaturated fat to saturated fat (P:S ratio) and the Pro12Ala polymorphism on both BMI and fasting insulin. With a low P:S ratio the Ala carriers had a higher BMI than subjects homozygote for the Pro allele, whereas the opposite was seen in subjects with a high P:S ratio (Luan et al. 2001). These studies emphasise the interaction of this gene with inter-individual differences in nutritional habits, but the studies may also provide explanations of the discrepancies found in usual association studies that neglect the role of the environment. On the other hand, the methodological issues pertaining to the search for candidate genes are even more problematic in the study of nutrient-gene

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interaction. In both of the above-mentioned studies on fatty acids, habitual dietary intake was estimated by food frequency questionnaires, which implies considerable uncertainty. As stated by Luan and colleagues, the absence of an interaction between total fat intake or fat energy percentage may be due to the difficulties in assessment of absolute intakes of fat and total energy intake by this technique (Luan et al. 2001). Indeed, dealing with large populations in which the environment is well controlled is generally very difficult. The dilemmas of observational epidemiology, which the methods of the randomised clinical trials aim at coping with, are close to the crucial problems in genetic association studies. Intervention studies Studies on controlled overfeeding and calorie restriction in monozygotic twins have been taken to suggest a relatively strong genetic component in the effect of change in energy intake, i.e., a nutrient-gene interaction (Bouchard et al. 1990; Bouchard and Tremblay 1997; Hainer et al. 2000). In the overfeeding study, twelve pairs of monozygotic lean male twins were fed a 4.2 MJ per day energy surplus on 6 out of 7 weekdays during a 100-day period (Bouchard et al. 1990). Both BMI as well as the degree of visceral obesity increased in most twins, but there was a considerable inter-individual variation. However, the intra-pair resemblance was much greater than the between-pair resemblance. This phenotypic correlation may suggest that the genetic identity of the twin pairs also make them more similar in the response to the enforced dietary changes, whereas the genetic differences between the twin pairs are responsible for the greater differences in response to the same dietary challenge. This seems plausible, but, on the other hand, the similarity within the twin pairs can also be due to preceding shared environment that influences the response to the dietary change. The distinction between the genetic and the environmental interpretation of the phenotypic correlation among monozygotic twins requires, as in the classical twin study design, a corresponding investigation of dizygotic twins under the assumption that the shared environmental influences are the same for mono- and dizygotic twins. The fact that the phenotypic correlation among these monozygotic twin pairs is less than 1.0, unambiguously demonstrates (assuming negligible measurement errors) the role of environmental influences ± preceding or during the experiment ± that are not identical within the twin pairs. Similar considerations apply to the study of the effects of caloric restriction on weight loss among monozygotic twins pairs (Hainer et al. 2000). Further, the study of lean subjects not necessarily predisposed to develop obesity and of already obese subjects may limit applicability of the results to the process of development of obesity. The twin overfeeding study has been used as a platform for the investigation of the effects of several common polymorphisms in genes assumed to play a role in regulation of body weight. Ukkola and colleagues have published several analyses showing that gene variants in genes coding for the uncoupling proteins

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UCP1, UCP2, and UCP3, the IGF2, the IGF binding protein 1, the beta2 adrenergic receptor, the glucocorticoid receptor, and the Na+- K+ ATPase alpha2 are associated with the initial change in body weight, body composition, and metabolic parameters in response to overfeeding, and to recovery from the weight gain induced by overfeeding (Ukkola et al. 2001a,b,c,d; 2003). However, these analyses may have some limitations that must be taken into account in the interpretation. The 24 subjects included in the study represented 12 pairs of monozygotic twins, but were handled in one set of analyses as if they were unrelated persons, and by using only the phenotypic means of the 12 pairs in another set of analyses. Although significant findings are reported, uncertainty remains considerable due to the small sample size. Several other studies have addressed the effect of candidate gene variants on response to weight loss intervention in humans. Carriers of the C allele of the Ser(T) 343 Ser(C) polymorphism of the leptin receptor gene have been shown to experience a greater weight loss during calorie restriction than subjects homozygous for the T allele (Mammes et al. 2001). In a study including 163 overweight and obese subjects, Fumeron and colleagues addressed the possible effect of the Bcl 1 restriction polymorphism of the UCP gene and the Trp64Arg polymorphism of the beta 3 adrenergic receptor on weight loss achieved during four months of calorie restriction (Fumeron et al. 1996). Carriers of the UCP polymorphism lost more weight than non-carriers. Further, subjects homozygous for the polymorphism had the greatest weight loss, indicating a dose-response effect of this gene variant on weight loss (Fumeron et al. 1996). Whereas Fumeron and colleagues did not find any effect of the Trp64Arg polymorphism of the beta 3 adrenergic receptor, other studies have indicated that the Arg allele is associated with an impaired response to weight reduction in Japanese subjects (Sakane et al. 1997; Shiwaku et al. 2003; Xinli et al. 2001). A German study has indicated that subjects carrying the Arg allele of Trp64Arg, and the Arg allele of the Gly972Arg polymorphism in the insulin receptor substrate 1 (IRS-1) gene, experience low weight reduction in response to calorie restriction (Benecke et al. 2000). Several studies have addressed the possible effect of the Pro12Ala variant of the PPAR 2 gene in intervention trials. Nicklas and colleagues have addressed its possible effect on weight loss, metabolic response to the weight loss and weight regain following a six-month dietary intervention in women (Nicklas et al. 2001). Weight loss did not differ between women carrying the Ala allele and women homozygous for the Pro allele. However, fat oxidation was significantly decreased following the weight loss intervention only in Ala carriers. In addition, carriers of the Ala allele presented a larger reduction in insulin response to an oral glucose tolerance test compared with women homozygous for the Pro allele. Finally, 12±month weight regain was greater in Ala carriers. Lindi and colleagues have studied the effect of the Pro12Ala PPAR polymorphism on long-term weight change in response to energy and fat restriction and increased physical activity. The weight loss in the subjects homozygous for the Ala allele was significantly greater than in subjects

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homozygous for the Pro allele, and the weight loss for subjects with the Pro/Ala genotype was close to that of the Pro/Pro subjects (Lindi et al. 2002). Further, Lindi and colleagues have reported that carriers of the Ala allele have a greater reduction in serum triacylglycerol in response to long-term fish oil supplementation as compared to subjects with the Pro12Pro genotype (Lindi et al. 2003). A French group recently carried out an intervention study (called RIVAGE) to investigate the interactions between diets (Mediterranean or low-fat types v. standard Western type), risk factors for cardiovascular disease and gene polymorphisms in about 300 patients who were randomised into two groups over periods of three and 12 months. They studied several genes encoding for proteins that are known to be modulated in response to diet. In a first analysis performed in a subgroup of 100 subjects, they found, for example, that several common SNPs located in the Apolipoprotein E, ApoA VI, micosomal transfer protein (MTP), intestinal fatty acid protein (FABP2) might be associated with the metabolic response to diet (Vincent et al. 2002). The study has not yet provided results pertaining to obesity or weight change. In addition, a recent systematic review has indicated that genetic variations in several of the apolipoproteins may contribute to the heterogeneity in the lipid response to dietary intervention (Masson et al. 2003). Combining genome wide scanning for gene polymorphisms and expression The advances in biotechnology now allow combining the search for gene polymorphisms, possibly generating a basis for nutrient-gene interaction, and the gene expression profiling on a genome-wide basis. Thus, the overlap between gene profiling studies, the whole genome scan and the candidate gene map available in humans and rodents will constitute important steps. Obviously, the development of data mining tools is essential to develop and fully exploit these new opportunities. A proof-of-concept of this new approach was recently provided by a study in which gene expression studies and genome wide scan was combined in standard inbred mice strains (C57BL/6J and DBA/2j) (Schadt et al. 2003). The strains were crossed together and the F2 generation yielded by these crosses was fed a high-fat diet for four months. All animals were carefully phenotyped with regard to obesity-related traits and metabolic parameters. Subcutaneous fat pad mass was measured and animals were categorised as either lean or obese if they were below or above the first or last quartile of fat amount, respectively. In the first step, using micro-arrays, the authors compared the differential gene expression levels in the liver of obese and lean animals. The gene profiling study showed that 30% of the genes differentially mobilised in the animals could represent molecular signatures of the thin and the obese status, respectively. Among the 300 highly-ranked genes with a differential expression in thin and obese animals, two different profiles of gene expression were observed in the liver of the obese animals, thus separating these animals in different obese categories based on their gene expression levels in the liver. The second step aimed at

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identifying genes or regions that contributed to the increased fat mass. Genomewide scans were utilised to detect chromosomal regions showing linkage with obesity-related phenotypes in these animals. The variation of liver gene expression in the animal strains was also used as a quantitative trait (eQTL). The linkage study not only identified chromosomal regions involved in the control of adiposity, but also in the control of the liver gene expression. Specific chromosomal regions discriminated the two types of obese mice with different patterns of liver expression. Some genes or chromosomal regions that may be involved both in the regulation of genes expressed in the liver (with a different pattern of expression in lean and obese) and in adiposity will probably be identified. The future will tell us if they are good targets for intervention (Schadt et al. 2003). Current large-scale international studies Large multi-centre studies are currently addressing the nutrient±gene interaction in obesity. One of these is the NUGENOB study `Nutrient Gene Interaction in Human Obesity ± Implications for Dietary Guidelines' which is funded by a European Commission grant under the 5th Framework Programme. The NUGENOB study aims to improve the understanding of the interaction between nutrition, especially fat intake, and genetic variations and functions in obesity. The outline of the study is illustrated in Fig. 10.1. Approximately 750 obese subjects and 115 normal weight reference subjects are included in the study. All subjects complete a three-day food diary to assess habitual diet, and complete questionnaires addressing dietary habits and life-style, and family history of obesity and related diseases. All subjects complete a one-day clinical investigation programme including assessment of metabolic responses to a high-fat test meal. Obese subjects are then randomised to a hypocaloric dietary intervention with either high or low fat content. Novel candidate genes possibly involved in pathogenesis of obesity are identified based on linkage results obtained in data outside the project, as well as bioinformatics. Candidate gene variants are identified through scanning of candidate genes and bioinformatics. All subjects are genotyped for a large number of gene polymorphisms with a putative role in the regulation of body fat accumulation. Quantitative gene expression analyses on selected genes, and microarray-based gene expression profiling are conducted on adipose tissue samples taken in obese subjects before and after dietary intervention. The study specifically addresses the nutrient-gene interactions by investigating the effect of genotype on the metabolic response to a high-fat test meal, the effect of genotype on weight loss, including the interaction between genotype and diet, and finally the changes in adipose tissue gene expression in response to energy restriction, and changes in dietary fat content. In addition to improving the understanding of nutrient-gene interaction, the project aims to improve understanding of the specific mechanisms underlying the well-documented genetic predisposition to obesity, and the specific pathways involved in the regulation of adipose tissue fat accumulation and metabolism.

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Outline of the NUGENOB project. EU grant QLK1-CT-2000-00618.

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Another multi-centre project is the `Diet and Obesity' project, which is also funded by the European Commission within the 5th Framework Programme. This project addresses the causes of weight gain with particular focus on an energy-dense, high-fat diet, including the mechanisms of susceptibility to weight gain and diet preferences, using both animal models and human epidemiological and interventional models (Mercer 2001). The project aims at addressing the mechanism and defining the factors that predispose some individuals to prefer a high-fat diet and to gain weight on this type of diet. Nutrient-gene interaction will be addressed by attempting to identify the genes and the gene variants responsible for the differences in susceptibility to the obesity-promoting effect of a high-fat diet. The project will aim at defining diagnostic biomarkers of susceptibility or resistance to excess weight gain on a high-fat diet, and to identify the mechanistic basis of post-dieting weight gain, addressing both the central and the peripheral regulatory systems.

10.5

Managing obesity: dietary and other strategies

There are several strategies for inducing weight loss in overweight and obese subjects. However, in most cases long-term success is limited as the majority of subjects regain all lost weight within 3±5 years. Obesity is a chronic condition, and it therefore seems evident that some form of life-long intervention may be needed in order to maintain weight loss in obese subjects. On the other hand, the long-term benefit of this strategy is not as obvious as it might look. Thus, there is an ongoing debate regarding the evidence suggesting that weight reduction, although having beneficial effects on risk factors for cardio-vascular diseases and type II diabetes, may be associated with increased long-term mortality (Sorensen 2003a; Yang et al. 2003). Evidently, improved primary prevention of weight gain in obesity-prone subjects, combined with improved strategies for management of obesity are the main tasks for the future. 10.5.1 Current strategies for the management of obesity Even a weight reduction of 5±10% of the initial weight can markedly improve insulin sensitivity and other features of the metabolic syndrome (Bosello et al. 1997; Pasanisi et al. 2001; Van Gaal et al. 1997). The US National Institute of Health accordingly recommends an initial weight loss of approximately 10% in obese subjects (National Institute of Health 1998). It has become evident that abdominal obesity is associated with greater health risk compared to increased fat accumulation on the hips and legs (Bigaard et al. 2003; Larsson et al. 1984). Therefore, subjects with abdominal obesity may need more intensive treatment, especially if they also suffer from obesity-related comorbidities. Surgical treatment of obesity as well as some of the pharmacological treatments is restricted to subjects who are massively obese. The

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different strategies for treatment are all related to the severity of the obesity and not to the pathophysiological nature of the obesity. Surgical treatment is recommended only for morbidly obese subjects, or for severely obese subjects with co-morbidities or physical conditions conferring a high risk. Several surgical techniques are available. One is gastroplasty or gastric banding, which reduces the volume of the stomach, thereby increasing post-meal satiety and inhibits intake of large meals. Another technique is intestinal bypass surgery, which reduces intestinal absorption but which often is associated with complications or severe side effects that require the bypass to be reversed. This is then followed by rapid weight regain. In general, both the initial and the long-term weight loss are large after these treatments compared with other regimens (Colquitt et al. 2003). Most pharmacological treatments are approved only for short-term usage in obesity treatment but the newer drugs, sibutramine and olistat, may be used for long-term treatment including prevention of weight regain. Sibutramine inhibits the central re-uptake of noradrenaline and serotonine, which reduces hunger and produces a minor increase in energy expenditure (Leung et al. 2003; Luque and Rey 2002). Orlistat inhibits pancreatic lipase, which reduces intestinal fat absorption (Leung et al. 2003). This treatment also invites the patients to reduce fat intake in order to avoid steatorrhea. Both treatments should be combined with dietary intervention to achieve the optimal results. Most other pharmacological agents available reduce food intake, via modulation of the central regulatory centres in the hypothalamus, combined with a varying degree of thermogenic effect. None of the currently available drugs directly target the adipose tissue fat metabolism but the PPARs expressed in this tissue may be a potential target (Ram 2003). The cornerstone of any dietary treatment of obesity is reduction of energy intake, and sustainment of a lower energy intake to prevent weight regain. The US National Institute of Health recommends an initial weight loss of approximately 10%, achieved over a six-month period with a daily energy deficit of 300±1000 kcal, depending on the initial weight (National Institute of Health 1998). The dietary approach may be combined with increased physical activity, which may aid both weight loss and subsequent maintenance of the weight loss. When a great weight loss is aimed at, an initial treatment with a Very Low Calorie Diet (VLCD), providing 2.4±4.2 MJ per day may be useful. VLCD should always be followed by a more lifestyle-orientated approach, providing the patient with new eating habits that will prevent regain. Although the initial weight loss rate is high for VLCDs, the maintained weight loss after one year is not different between subjects who initially followed a VLCD and subjects who followed a conventional energy-restricted diet (Wadden et al. 1994). More moderate reductions in energy intake can either be obtained directly by limiting the energy intake to a certain amount of calories per day (energy restriction), or by introducing a low-fat, low-energy density diet, which indirectly will reduce energy intake (low fat ad libitum diet). Previous studies

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have indicated that the two procedures may produce similar weight loss, but that the ad libitum approach may in general be perceived as more pleasant (Jeffery et al. 1995; Shah et al. 1994). However, other studies indicate that the passive underconsumption on the ad libitum diet may be a temporary phenomenon (Kendall et al. 1991; Stubbs et al. 1998), and that continued weight reduction over a longer time period may be achieved only by some degree of deliberate energy restriction (Astrup et al. 2000b). In controlled dietary intervention studies, medium- and low-fat diets may produce similar degrees of weight reduction, as long as they are subjected to the same degree of energy restriction (Petersen et al. 2003; Powell et al. 1994). However, the patients' ability and motivation actively to control calorie intake may be more important on a medium-fat diet than on a low-fat diet. Although there is little knowledge about interaction between dietary fat and genetic factors involved in the regulation of weight loss, it is plausible that some individuals are more susceptible than others to a high-fat diet, and hence that nutrient-gene interactions may play an important role in weight reduction. Lifestyle modification has become one of the most commonly used strategies for prevention and treatment of obesity. The overall aim of lifestyle modification is to induce a behavioural modification of eating and exercise patterns. The intervention will usually focus on reduction of intake of high-fat or other energydense foods and reduction of portion size, on reinforcement of the awareness to internal clues of hunger and satiety, on reduction of eating in response to external clues, and finally, on an increase of physical activity. However, such lifestyle changes have not proved to be as effective as might be expected. There are several possible reasons for the failure. Advising at-risk individuals to change their lifestyle will not have the same effect as changing the environment that promotes the maintenance of inappropriate habits. Most people are probably aware of the healthy choices, but these choices are often not the easy choices in terms of costs, social norms, etc. Little is known about the between-subject differences in response to different types of lifestyle changes. Finally, it is still unknown if generally recommended low-fat, low-energy density diets will reduce the risk of weight gain in the population. Even if this is the case, other prevention strategies may be more effective in subgroups of obesity-prone subjects. 10.5.2 Integrating knowledge on the genetics of obesity and nutrientgene interaction in treatment and prevention of obesity It seems likely that a polygenic, multifactorial disease such as obesity may be more effectively managed by individual tailoring of the treatment and prevention strategies. Other fields of nutritional science have provided insight into genetic mechanisms determining between-subject differences in nutrient requirements. One example is the abovementioned phenylketonuria. It is now known that genetic variations in apolipoproteins, in the vitamin D receptor, and in the thermo-labile variant of the 5,10-methylenetetrahydrofolate reductase (MTHER), may induce reduced sensitivity to dietary cholesterol and increased

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requirement for calcium and folate, respectively (Simopoulos 1999). Similar findings on genes or pathways involved in regulation of body fat accumulation, in sensitivity to physical inactivity and high-fat diet, and in response to antiobesity drugs and other forms of treatment or prevention of obesity may pave the way for development of a new obesity taxonomy defining different subtypes of obesity according sub-phenotypes as well as genetic aspects. Transforming this knowledge into new diagnostics and tailored treatment and prevention regimes may hold the key to combat the epidemic of obesity. The research on the genetics of obesity has mainly led to the discovery of rare monogenic forms of obesity, rather than improvement of our understanding of the genetic mechanisms behind the well-known genetic predisposition for the development of common obesity. The discovery of leptin offered an effective strategy for treating the rare cases of obesity caused by lack of leptin production (Farooqi et al. 1999). Current knowledge about nutrient-gene interaction in human obesity is very preliminary, however, studies have indicated that the long-term effect of dietary weight loss intervention may differ in subjects with different PPAR genotypes (Lindi et al. 2002; Nicklas et al. 2001), and an interaction between dietary fat composition and PPAR genotypes has been suggested (Luan et al. 2001; Nieters et al. 2002). Moreover, in obese subjects treated by gastric banding surgery, those carrying different types of mutation in the MC4 receptor may have more complications and less weight loss (Potoczna et al. 2003). Current and future large-scale research projects aim to identify predictors of weight gain and weight loss, and susceptibility to environmental obesitypromoting factors. Within the next decades it may therefore be possible to identify at-risk individuals before they develop obesity and to prevent the development of obesity using tailored intervention strategies. Further, tailoring the treatment and secondary prevention for obese subjects may increase the degree of success in initial weight loss and in prevention of subsequent regain. Prevention and treatment of obesity clearly demands testing changes in the environmental factors to which the individual is exposed (Hill and Peters 1998). In addition to more refined and targeted regimes for dietary intervention, advanced insight into the biological mechanisms regulating adipose tissue fat accumulation, energy expenditure and appetite regulation will offer new drug targets for the treatment of obesity. Also, in the pharmacological treatment of obesity, a sub-classification of the obese phenotype based on the affected genes or pathways may lead to specifically targeted treatment regimes, hopefully implying improved treatment and reduced side effects.

10.6

Future trends

During the past years, research in the field of nutrient-sensitive genes and nutrient-gene interaction with implications on body weight regulation has developed rapidly, from a research strategy focusing on the overall role of

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genetic factors in determining the between-subjects variation in response to habitual diet or dietary interventions such as overfeeding and calorie restriction, to research protocols aimed at addressing the specific interaction between genetic factors and dietary components. Studies have addressed the effect of calorie restriction on adipose tissue gene expression in humans. Future studies addressing the association between change in adipose tissue gene expression in response to dieting and subsequent weight loss, the effect of diet composition on change in gene expression, and the interaction between gene polymorphisms and change in gene expression in response to dietary intervention are under way. They are expected to increase our understanding of the systems biology of adipose tissue in response to weight loss, as well as the between-subject differences in response to dietary weight loss interventions. An improved understanding of the interaction between diet composition and specific gene variants, or pathways involved in the regulation of body fat accumulation, may pave the way for a new obesity taxonomy, and more targeted strategies for treatment. Future large-scale multi-disciplinary projects should aim at optimising the use of new research methods in combination with existing research methods. Metabolomics and proteomics, combined with gene expression profiling and genotype screening may provide new important insights into the system biology of obesity. This may further allow the identification of candidate pathways involved in the development of obesity, and thereby the identification of candidate drug targets for treatment and prevention of obesity, as well as biomarkers indicating the efficiency of the pathways on the individual subject level. It may be speculated that although a large number of genes and a complex interplay of environmental and genetic factors determines body fat accumulation, the number of pathways mediating the effect of these factors on body fat accumulation may be limited. Identifying the pathways could serve as a platform for a new classification, diagnostic and treatment of the common complex forms of obesity. Further, identifying biomarkers reflecting susceptibility to particular nutrients in pre-obese subjects would be a crucial step in the primary prevention of obesity.

10.7

Sources of further information

Section 10.2 Maes et al. 1997. Review of the literature on the genetic and environmental factors in human obesity. This review includes family studies, twin studies, adoption studies, and advantages and disadvantages of the study designs are discussed. Stunkard et al. 1986. An adoption study of the genetic and familial environmental effects on adult obesity. The study was based on 540 thin, medium weight, overweight and obese adoptees, their biological and adoptive

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parents, and their biological full and half siblings. The adoptees were selected among 3,580 Danish adoptees who had reported their height and weight. The average BMI of the relatives of the selected adoptees was analysed and supported a genetic influence and no sustained influence of the rearing family environment. Sorensen et al. 1989. An adoption study of genetic effects on obesity in adulthood. The study is based on 341 thin, medium weight, overweight and obese adoptees selected among 4,000 adoptees from the region of Copenhagen, Denmark. The association between BMI of the adoptees and their biological full and half siblings was examined. Comuzzie et al. 2001; Comuzzie and Allison 1998. Review papers addressing the strategies for identifying candidate genes for obesity. The papers discuss the strategies for identifying candidate genes involved in the pathogenesis of complex phenotypes, expected to be determined by the action and interaction of multiple genes and environmental factors. Clement et al. 2002a. Review of genetics in Obesity. The review presents examples of monogenetic forms of obesity, and the insight gained from these rare cases of obesity in terms of understanding the complex pathways involved in the regulation of food intake and identifying targets for future drug development. The candidate gene and the genome-wide scan approach are presented and discussed as strategies for identifying genes involved in the common forms of obesity. Updated results from genome-wide scan studies are presented. Finally the strategies for medical application of this knowledge in terms of drug development and optimal integration of knowledge about the genetic mechanisms in the pharmacotherapy of obesity is discussed. Chagnon et al. 2003. The ninth update of the human obesity gene map, incorporating published results through October 2002. Evidence from singlegene mutation obesity cases, Mendelian disorders exhibiting obesity as a clinical feature, quantitative trait loci (QTLs) from human genome-wide scans and various animal cross-breeding experiments, and association and linkage studies with candidate genes and other markers are reviewed. In addition, transgenic and knockout murine models exhibiting obesity as a phenotype are incorporated. Barsh et al. 2000. Review addressing the search for human obesity genes, rodents as a model system for human obesity, and gene-environment interaction. West et al. 1995. Study addressing the effect of varying dietary macronutrient content on body composition and the possible interaction between genetic background and macronutrient content of the diet on body composition of different strains of mice.

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Lissner and Heitmann 1995. Review paper addressing the role of dietary fat in human obesity. The evidence for a role of dietary fat in human obesity is addressed based on the epidemiological evidence from various epidemiological methods and the consistency of these findings with experimental data is discussed. Section 10.3 Copland et al. 2003. Review paper describing gene array technology. This technology is expected to pave the way for identifying candidate genes and pathways for specific diseases, a way of subclassifying diseases according to patterns of gene expression in various tissues, and a means of identifying new molecular targets for the development of drug therapeutics. Clement et al. 2002b; Rome et al. 2003. Studies applying microarray technology addressing the effects of thyroid homeone and insulin on gene expression in human skeletal muscle. The studies also address the interpretation of microarray data. Section 10.4 Lindi et al. 2002, 2003; Luan et al. 2001; Nicklas et al. 2001; Nieters et al. 2002. Studies addressing nutrient-gene interaction between fat intake or calorie restriction and the Pro12Ala variant of the PPAR gamma gene. Masson et al. 2003. Review addressing the effect of genetic variation on the lipid response to dietary intervention. Schadt et al. 2003. Study combining gene expression studies and genomewide scan in standard inbred mice strains. The study describes comprehensive genetic screens of mouse, plant and human transcriptomes by considering gene expression values as quantitative traits. The study identified a gene expression pattern strongly associated with obesity in a murine cross, and observed two distinct obesity subtypes that appeared to be under the control of different loci. Section 10.5 Simopoulos 1999. Review paper addressing the concept of nutrient±gene interaction and implications for dietary recommendations. Useful websites in relation to Chapter 10 www.nugenob.com: the website for the NUGENOB project, funded by the European Commission within the 5th Framework Programme. http://www.adipositas-online.com: the website for the Diet and Obesity project, funded by the European Commission within the 5th Framework Programme.

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http://www.nugo.org/everyone/: the website for the European NutriGenomics Organisation. A Network of Excellence on Nutrition and Genomics funded by the European Commission within the 6th Framework Programme. http://www.flair-flow.com/: FLAIR-FLOW EUROPE disseminates information from food R&D and nutrition projects funded by the European Union. http://obesitygene.pbrc.edu/: electronic version of the Human Obesity Gene Map (Chagnon et al. 2003). Claude Bouchard, Pennington Biomedical.

10.8

References

and TOUBRO, S. 1994, `Failure to increase lipid oxidation in response to increasing dietary fat content in formerly obese women', Am. J. Physiol, vol. 266, no. 4 Pt 1, p. E592±E599. ASTRUP, A., GRUNWALD, G. K., MELANSON, E. L., SARIS, W. H. and HILL, J. O. 2000a, `The role of low-fat diets in body weight control: a meta-analysis of ad libitum dietary intervention studies', Int. J. Obes. Relat Metab Disord., vol. 24, no. 12, pp. 1545± 1552. ASTRUP, A., RYAN, L., GRUNWALD, G. K., STORGAARD, M., SARIS, W., MELANSON, E. and HILL, J. O. 2000b, `The role of dietary fat in body fatness: evidence from a preliminary meta-analysis of ad libitum low-fat dietary intervention studies', Br. J. Nutr., vol. 83 Suppl 1, p. S25±S32. BABA, N. H., SAWAYA, S., TORBAY, N., HABBAL, Z., AZAR, S. and HASHIM, S. A. 1999, `High protein vs high carbohydrate hypoenergetic diet for the treatment of obese hyperinsulinemic subjects', Int. J. Obes. Relat Metab Disord., vol. 23, no. 11, pp. 1202±1206. BARSH, G. S., FAROOQI, I. S. and O'RAHILLY, S. 2000, `Genetics of body-weight regulation', Nature, vol. 404, no. 6778, pp. 644±651. ASTRUP, A., BUEMANN, B., CHRISTENSEN, N. J.

BASTARD, J. P., HAINQUE, B., DUSSERRE, E., BRUCKERT, E., ROBIN, D., VALLIER, P., PERCHE, S.,

and VIDAL, H. 1999, `Peroxisome proliferator activated receptor-gamma, leptin and tumor necrosis factor-alpha mRNA expression during very low calorie diet in subcutaneous adipose tissue in obese women', Diabetes Metab Res. Rev., vol. 15, no. 2, pp. 92± 98. BENECKE, H., TOPAK, H., VON ZUR, M. A. and SCHUPPERT, F. 2000, `A study on the genetics of obesity: influence of polymorphisms of the beta-3±adrenergic receptor and insulin receptor substrate 1 in relation to weight loss, waist to hip ratio and frequencies of common cardiovascular risk factors', Exp. Clin. Endocrinol. Diabetes, vol. 108, no. 2, pp. 86±92. BIGAARD, J., TJONNELAND, A., THOMSEN, B. L., OVERVAD, K., HEITMANN, B. L. and SORENSEN, T. I. 2003, `Waist Circumference, BMI, Smoking, and Mortality in Middle-Aged Men and Women', Obes. Res., vol. 11, no. 7, pp. 895±903. BOLTON-SMITH, C. and WOODWARD, M. 1994, `Dietary composition and fat to sugar ratios in relation to obesity', Int. J. Obes. Relat Metab Disord., vol. 18, no. 12, pp. 820± 828. ROBIN, P., TURPIN, G., JARDEL, C., LAVILLE, M., FOREST, C.

252

Functional foods, ageing and degenerative disease and FITCHET, M. 1997, `The benefits of modest weight loss in type II diabetes', Int. J. Obes. Relat Metab Disord., vol. 21 Suppl. 1, pp. S10±S13.

BOSELLO, O., ARMELLINI, F., ZAMBONI, M.

BOUCHARD, C., TREMBLAY, A., NADEAU, A., DESPRES, J. P., THERIAULT, G., BOULAY, M. R.,

and FOURNIER, G. 1989, `Genetic effect in resting and exercise metabolic rates', Metabolism, vol. 38, no. 4, pp. 364±370.

LORTIE, G., LEBLANC, C.

BOUCHARD, C., TREMBLAY, A., DESPRES, J. P., NADEAU, A., LUPIEN, P. J., THERIAULT, G.,

and FOURNIER, G. 1990, `The response to long-term overfeeding in identical twins', N. Engl. J. Med., vol. 322, no. 21, pp. 1477±1482. BOUCHARD, C. and TREMBLAY, A. 1997, `Genetic influences on the response of body fat and fat distribution to positive and negative energy balances in human identical twins', J. Nutr., vol. 127, no. 5 Suppl, pp. 943S±947S. BOWEN, J., NOAKES, M., FOSET, P. and CLIFTON. P. 2003, `Influence of dairy protein and calcium in energy restricted weight loss diets' Int. J. Obes. Relat Metab Disord., vol. 27, Suppl. 1, pp. S25. BRANSON, R., POTOCZNA, N., KRAL, J. G., LENTES, K. U., HOEHE, M. R. and HORBER, F. F. 2003, `Binge eating as a major phenotype of melanocortin 4 receptor gene mutations', N. Engl. J. Med., vol. 348, no. 12, pp. 1096±1103. CHAGNON, Y. C., RANKINEN, T., SNYDER, E. E., WEISNAGEL, S. J., PERUSSE, L. and BOUCHARD, C. 2003, `The human obesity gene map: the 2002 update', Obes. Res., vol. 11, no. 3, pp. 313±367. CLEMENT, K. and FERRE, P. 2003, `Genetics and the pathophysiology of obesity', Pediatr. Res., vol. 53, no. 5, pp. 721±725. DUSSAULT, J., MOORJANI, S., PINAULT, S.

CLEMENT, K., VAISSE, C., LAHLOU, N., CABROL, S., PELLOUX, V., CASSUTO, D., GOURMELEN, M., DINA, C., CHAMBAZ, J., LACORTE, J. M., BASDEVANT, A., BOUGNERES, P., LEBOUC, Y.,

FROGUEL, P. and GUY-GRAND, B. 1998, `A mutation in the human leptin receptor gene causes obesity and pituitary dysfunction', Nature, vol. 392, no. 6674, pp. 398±401. CLEMENT, K., BOUTIN, P. and FROGUEL, P. 2002a, `Genetics of obesity', Am. J. Pharmacogenomics., vol. 2, no. 3, pp. 177±187.

CLEMENT, K., VIGUERIE, N., DIEHN, M., ALIZADEH, A., BARBE, P., THALAMAS, C., STOREY, J. D.,

and LANGIN, D. 2002b, `In vivo regulation of human skeletal muscle gene expression by thyroid hormone', Genome Res., vol. 12, no. 2, pp. 281±291. COLQUITT, J., CLEGG, A., SIDHU, M. and ROYLE, P. 2003, `Surgery for morbid obesity', Cochrane. Database. Syst. Rev., no. 2, p. CD003641. COMUZZIE, A. G. and ALLISON, D. B. 1998, `The search for human obesity genes', Science, vol. 280, no. 5368, pp. 1374±1377. COMUZZIE, A. G., WILLIAMS, J. T., MARTIN, L. J. and BLANGERO, J. 2001, `Searching for genes underlying normal variation in human adiposity', J. Mol. Med., vol. 79, no. 1, pp. 57±70. COPLAND, J. A., DAVIES, P. J., SHIPLEY, G. L., WOOD, C. G., LUXON, B. A. and URBAN, R. J. 2003, `The use of DNA microarrays to assess clinical samples: the transition from bedside to bench to bedside', Recent Prog. Horm. Res., vol. 58, pp. 25±53. CURB, J. D. and MARCUS, E. B. 1991, `Body fat and obesity in Japanese Americans', Am. J. Clin. Nutr., vol. 53, no. 6 Suppl, pp. 1552S±1555S. DAVIES, K. M., HEANEY, R. P., RECKER, R. R., LAPPE, J. M., BARGER-LUX, M. J., RAFFERTY, K. and HINDERS, S. 2000, `Calcium intake and body weight', J. Clin. Endocrinol. Metab, BROWN, P. O., BARSH, G. S.

Nutrient-gene interactions in the control of obesity

253

vol. 85, no. 12, pp. 4635±4638. 1993, `Independence of genetic influences on body size, daily intake, and meal patterns of humans', Physiol Behav., vol. 54, no. 4, pp. 633±639. DIMEGLIO, D. P. and MATTES, R. D. 2000, `Liquid versus solid carbohydrate: effects on food intake and body weight', Int. J. Obes. Relat Metab Disord., vol. 24, no. 6, pp. 794± 800. DE CASTRO, J. M.

ENGERT, J. C., VOHL, M. C., WILLIAMS, S. M., LEPAGE, P., LOREDO-OSTI, J. C., FAITH, J., DORE, C., RENAUD, Y., BURTT, N. P., VILLENEUVE, A., HIRSCHHORN, J. N., ALTSHULER, D., GROOP, L.

and HUDSON, T. J. 2002, `5' flanking variants of resistin are associated with obesity', Diabetes, vol. 51, no. 5, pp. 1629±1634.

C., DESPRES, J. P., GAUDET, D.

ESTERBAUER, H., SCHNEITLER, C., OBERKOFLER, H., EBENBICHLER, C., PAULWEBER, B., SANDHOFER, F., LADURNER, G., HELL, E., STROSBERG, A. D., PATSCH, J. R., KREMPLER,

F. and PATSCH, W. 2001, `A common polymorphism in the promoter of UCP2 is associated with decreased risk of obesity in middle-aged humans', Nat. Genet., vol. 28, no. 2, pp. 178±183. FAROOQI, I. S., JEBB, S. A., LANGMACK, G., LAWRENCE, E., CHEETHAM, C. H., PRENTICE, A. M.,

HUGHES, I. A., MCCAMISH, M. A. and O'RAHILLY, S. 1999, `Effects of recombinant leptin therapy in a child with congenital leptin deficiency', N. Engl. J. Med., vol. 341, no. 12, pp. 879±884.

FOSTER, G. D., WYATT, H. R., HILL, J. O., MCGUCKIN, B. G., BRILL, C., MOHAMMED, B. S., SZAPARY,

P. O., RADER, D. J., EDMAN, J. S. and KLEIN, S. 2003, `A randomized trial of a lowcarbohydrate diet for obesity', N. Engl. J. Med., vol. 348, no. 21, pp. 2082±2090.

FUMERON, F., DURACK-BOWN, I., BETOULLE, D., CASSARD-DOULCIER, A. M., TUZET, S., BOUILLAUD, F., MELCHIOR, J. C., RICQUIER, D. and APFELBAUM, M. 1996, `Polymorphisms of uncoupling protein (UCP) and beta 3 adrenoreceptor genes in obese people submitted to a low calorie diet', Int. J. Obes. Relat Metab Disord., vol. 20, no. 12, pp. 1051±1054. HAINER, V., STUNKARD, A. J., KUNESOVA, M., PARIZKOVA, J., STICH, V. and ALLISON, D. B. 2000, `Intrapair resemblance in very low calorie diet-induced weight loss in female obese identical twins', Int. J. Obes. Relat Metab Disord., vol. 24, no. 8, pp. 1051±1057. HALSALL, D. J., LUAN, J., SAKER, P., HUXTABLE, S., FAROOQI, I. S., KEOGH, J., WAREHAM, N. J. and O'RAHILLY, S. 2001, `Uncoupling protein 3 genetic variants in human obesity: the c55t promoter polymorphism is negatively correlated with body mass index in a UK Caucasian population', Int. J. Obes. Relat Metab Disord., vol. 25, no. 4, pp. 472± 477. HEANEY, R. P. 2003, `Normalizing calcium intake: projected population effects for body weight', J. Nutr., vol. 133, no. 1, pp. 268S±270S. HEITMANN, B. L., LISSNER, L., SORENSEN, T. I. and BENGTSSON, C. 1995, `Dietary fat intake and weight gain in women genetically predisposed for obesity', Am. J. Clin. Nutr., vol. 61, no. 6, pp. 1213±1217. HEITMANN, B. L., LISSNER, L. and OSLER, M. 2000, `Do we eat less fat, or just report so?', Int. J. Obes. Relat Metab Disord., vol. 24, no. 4, pp. 435±442. HILL, J. O. and PETERS, J. C. 1998, `Environmental contributions to the obesity epidemic', Science, vol. 280, no. 5368, pp. 1371±1374. HODGE, A. M., DOWSE, G. K., TOELUPE, P., COLLINS, V. R., IMO, T. and ZIMMET, P. Z. 1994, `Dramatic increase in the prevalence of obesity in western Samoa over the 13 year period 1978±1991', Int. J. Obes. Relat Metab Disord., vol. 18, no. 6, pp. 419±428. HODGE, A. M., DOWSE, G. K., GAREEBOO, H., TUOMILEHTO, J., ALBERTI, K. G. and ZIMMET, P. Z. 1996, `Incidence, increasing prevalence, and predictors of change in obesity and

254

Functional foods, ageing and degenerative disease

fat distribution over 5 years in the rapidly developing population of Mauritius', Int. J. Obes. Relat Metab Disord., vol. 20, no. 2, pp. 137±146. HOFFSTEDT, J., ERIKSSON, P., HELLSTROM, L., ROSSNER, S., RYDEN, M. and ARNER, P. 2000, `Excessive fat accumulation is associated with the TNF alpha-308 G/A promoter polymorphism in women but not in men', Diabetologia, vol. 43, no. 1, pp. 117± 120. JEFFERY, R. W., HELLERSTEDT, W. L., FRENCH, S. A. and BAXTER, J. E. 1995, `A randomized trial of counseling for fat restriction versus calorie restriction in the treatment of obesity', Int. J. Obes. Relat Metab Disord., vol. 19, no. 2, pp. 132±137. KENDALL, A., LEVITSKY, D. A., STRUPP, B. J. and LISSNER, L. 1991, `Weight loss on a low-fat diet: consequence of the imprecision of the control of food intake in humans', Am. J. Clin. Nutr., vol. 53, no. 5, pp. 1124±1129. KNOWLER, W. C., PETTITT, D. J., SAAD, M. F., CHARLES, M. A., NELSON, R. G., HOWARD, B. V.,

BOGARDUS, C. and BENNETT, P. H. 1991, `Obesity in the Pima Indians: its magnitude and relationship with diabetes', Am. J. Clin. Nutr., vol. 53, no. 6 Suppl, pp. 1543S± 1551S. KOLEHMAINEN, M., VIDAL, H., OHISALO, J. J., PIRINEN, E., ALHAVA, E. and UUSITUPA, M. I. 2002, `Hormone sensitive lipase expression and adipose tissue metabolism show gender difference in obese subjects after weight loss', Int. J. Obes. Relat Metab Disord., vol. 26, no. 1, pp. 6±16. KROSNICK, A. 2000, `The diabetes and obesity epidemic among the Pima Indians', N. J. Med., vol. 97, no. 8, pp. 31±37. LARSSON, B., SVARDSUDD, K., WELIN, L., WILHELMSEN, L., BJORNTORP, P. and TIBBLIN, G. 1984, `Abdominal adipose tissue distribution, obesity, and risk of cardiovascular disease and death: 13 year follow up of participants in the study of men born in 1913', Br. Med. J. (Clin. Res. Ed), vol. 288, no. 6428, pp. 1401±1404. LEE, G. H., PROENCA, R., MONTEZ, J. M., CARROLL, K. M., DARVISHZADEH, J. G., LEE, J. I. and FRIEDMAN, J. M. 1996, `Abnormal splicing of the leptin receptor in diabetic mice', Nature, vol. 379, no. 6566, pp. 632±635. LEIBEL, R. L. 1997, `Single gene obesities in rodents: possible relevance to human obesity', J. Nutr., vol. 127, no. 9, p. 1908S. LEUNG, W. Y., NEIL, T. G., CHAN, J. C. and TOMLINSON, B. 2003, `Weight management and current options in pharmacotherapy: orlistat and sibutramine', Clin. Ther., vol. 25, no. 1, pp. 58±80. LINDI, V. I., UUSITUPA, M. I., LINDSTROM, J., LOUHERANTA, A., ERIKSSON, J. G., VALLE, T. T.,

and 2002, `Association of the Pro12Ala polymorphism in the PPARgamma2 gene with 3±year incidence of type 2 diabetes and body weight change in the Finnish Diabetes Prevention Study', Diabetes, vol. 51, no. 8, pp. 2581±2586.

HAMALAINEN, H., ILANNE-PARIKKA, P., KEINANEN-KIUKAANNIEMI, S., LAAKSO, M. TUOMILEHTO, J.

LINDI, V., SCHWAB, U., LOUHERANTA, A., LAAKSO, M., VESSBY, B., HERMANSEN, K., STORLIEN, L.,

and RIVELLESE, A. 2003, `Impact of the Pro12Ala polymorphism of the PPAR-gamma2 gene on serum triacylglycerol response to n-3 fatty acid supplementation', Mol. Genet. Metab, vol. 79, no. 1, pp. 52±60. LISSNER, L. and HEITMANN, B. L. 1995, `Dietary fat and obesity: evidence from epidemiology', Eur. J. Clin. Nutr., vol. 49, no. 2, pp. 79±90. LUAN, J., BROWNE, P. O., HARDING, A. H., HALSALL, D. J., O'RAHILLY, S., CHATTERJEE, V. K. and WAREHAM, N. J. 2001, `Evidence for gene-nutrient interaction at the PPARgamma locus', Diabetes, vol. 50, no. 3, pp. 686±689. LUDWIG, D. S., PETERSON, K. E. and GORTMAKER, S. L. 2001, `Relation between consumption RICCARDI, G.

Nutrient-gene interactions in the control of obesity

255

of sugar-sweetened drinks and childhood obesity: a prospective, observational analysis', Lancet, vol. 357, no. 9255, pp. 505±508. LUQUE, C. A. and REY, J. A. 2002, `The discovery and status of sibutramine as an antiobesity drug', Eur. J. Pharmacol., vol. 440, no. 2±3, pp. 119±128. MACDIARMID, J. I., CADE, J. E. and BLUNDELL, J. E. 1996, `High and low fat consumers, their macronutrient intake and body mass index: further analysis of the National Diet and Nutrition Survey of British Adults', Eur. J. Clin. Nutr., vol. 50, no. 8, pp. 505± 512. MAES, H. H., NEALE, M. C. and EAVES, L. J. 1997, `Genetic and environmental factors in relative body weight and human adiposity', Behav. Genet., vol. 27, no. 4, pp. 325± 351. MAMMES, O., BETOULLE, D., AUBERT, R., HERBETH, B., SIEST, G. and FUMERON, F. 2000, `Association of the G-2548A polymorphism in the 50 region of the LEP gene with overweight', Ann. Hum. Genet., vol. 64, no. Pt 5, pp. 391±394. MAMMES, O., AUBERT, R., BETOULLE, D., PEAN, F., HERBETH, B., VISVIKIS, S., SIEST, G. and FUMERON, F. 2001, `LEPR gene polymorphisms: associations with overweight, fat mass and response to diet in women', Eur. J. Clin. Invest, vol. 31, no. 5, pp. 398± 404. MASSON, L. F., MCNEILL, G. and AVENELL, A. 2003, `Genetic variation and the lipid response to dietary intervention: a systematic review', Am. J. Clin. Nutr., vol. 77, no. 5, pp. 1098±1111. MERCER, J. G. 2001, `Dietary and genetic influences on susceptibility or resistance to weight gain on a high fat diet', Nutr. Metab Cardiovasc. Dis., vol. 11, no. 4 Suppl, pp. 114±117. MONTAGUE, C. T., FAROOQI, I. S., WHITEHEAD, J. P., SOOS, M. A., RAU, H., WAREHAM, N. J., SEWTER, C. P., DIGBY, J. E., MOHAMMED, S. N., HURST, J. A., CHEETHAM, C. H., EARLEY, A.

and O'RAHILLY, S. 1997, `Congenital leptin deficiency is associated with severe early-onset obesity in humans', Nature, vol. 387, no. 6636, pp. 903±908. NATIONAL INSTITUTES OF HEALTH 1998, Clinical guidelines on the identification, evaluation, and treatment of overweight and obesity in adults, NIH Publication No. 98±4083. NEEL, J. V. 1999, `The ``thrifty genotype'' in 1998', Nutr. Rev., vol. 57, no. 5 Pt 2, p. S2±S9. NICKLAS, B. J., VAN ROSSUM, E. F., BERMAN, D. M., RYAN, A. S., DENNIS, K. E. and SHULDINER, A. R. 2001, `Genetic variation in the peroxisome proliferator-activated receptorgamma2 gene (Pro12Ala) affects metabolic responses to weight loss and subsequent weight regain', Diabetes, vol. 50, no. 9, pp. 2172±2176. NIETERS, A., BECKER, N. and LINSEISEN, J. 2002, `Polymorphisms in candidate obesity genes and their interaction with dietary intake of n-6 polyunsaturated fatty acids affect obesity risk in a sub-sample of the EPIC-Heidelberg cohort', Eur. J. Nutr., vol. 41, no. 5, pp. 210±221. OZATA, M., OZDEMIR, I. C. and LICINIO, J. 1999, `Human leptin deficiency caused by a missense mutation: multiple endocrine defects, decreased sympathetic tone, and immune system dysfunction indicate new targets for leptin action, greater central than peripheral resistance to the effects of leptin, and spontaneous correction of leptin-mediated defects', J. Clin. Endocrinol. Metab, vol. 84, no. 10, pp. 3686±3695. PASANISI, F., CONTALDO, F., DE SIMONE, G. and MANCINI, M. 2001, `Benefits of sustained moderate weight loss in obesity', Nutr. Metab Cardiovasc. Dis., vol. 11, no. 6, pp. 401±406. R., BARNETT, A. H., PRINS, J. B.

256

Functional foods, ageing and degenerative disease

and VERDICH, C., et al. www.nugenob.com 2003, `Effect of hypo-caloric low-fat versus medium fat diet on body weight ± the NUGENOB EU multicentre, randomised trail' Int. J. Obes. Relat Metab Disord., vol. 27, Suppl. 1, pp. S25. POTOCZNA, N., BRANSON, R. PIEC, G. STEFFEN, R. RICKLIN, TH., HOEHE, M, R., LENTES, K. U. and HORBER, F. F. 2003, `Influence of dairy protein and calcium in energy restricted weight loss diets' Int. J. Obes. Relat Metab Disord., vol. 27, Suppl. 1, pp. S25. POWELL, J. J., TUCKER, L., FISHER, A. G. and WILCOX, K. 1994, `The effects of different percentages of dietary fat intake, exercise, and calorie restriction on body composition and body weight in obese females', Am. J. Health Promot., vol. 8, no. 6, pp. 442±448. RABEN, A., ANDERSEN, H. B., CHRISTENSEN, N. J., MADSEN, J., HOLST, J. J. and ASTRUP, A. 1994, `Evidence for an abnormal postprandial response to a high-fat meal in women predisposed to obesity', Am. J. Physiol, vol. 267, no. 4 Pt 1, pp. E549±E559. RABEN, A., VASILARAS, T. H., MOLLER, A. C. and ASTRUP, A. 2002, `Sucrose compared with artificial sweeteners: different effects on ad libitum food intake and body weight after 10 wk of supplementation in overweight subjects', Am. J. Clin. Nutr., vol. 76, no. 4, pp. 721±729. RAM, V. J. 2003, `Therapeutic role of peroxisome proliferator-activated receptors in obesity, diabetes and inflammation', Prog. Drug Res., vol. 60, pp. 93±132. REDONNET, A., BONILLA, S., NOEL-SUBERVILLE, C., PALLET, V., DABADIE, H., GIN, H. and HIGUERET, P. 2002, `Relationship between peroxisome proliferator-activated receptor gamma and retinoic acid receptor alpha gene expression in obese human adipose tissue', Int. J. Obes. Relat Metab Disord., vol. 26, no. 7, pp. 920±927. REILLY, J. J. and DOROSTY, A. R. 1999, `Epidemic of obesity in UK children', Lancet, vol. 354, no. 9193, pp. 1874±1875. PETERSEN, M., TAYLOR, M., SARIS, W.

RICHELSEN, B., PEDERSEN, S. B., KRISTENSEN, K., BORGLUM, J. D., NORRELUND, H., CHRISTIANSEN, J. S. and JORGENSEN, J. O. 2000, `Regulation of lipoprotein lipase and hormone-sensitive lipase activity and gene expression in adipose and muscle tissue by growth hormone treatment during weight loss in obese patients', Metabolism, vol. 49, no. 7, pp. 906±911.

ROME, S., CLEMENT, K., RABASA-LHORET, R., LOIZON, E., POITOU, C., BARSH, G. S., RIOU, J. P.,

LAVILLE, M. and VIDAL, H. 2003, `Microarray profiling of human skeletal muscle reveals that insulin regulates approximately 800 genes during a hyperinsulinemic clamp', J. Biol. Chem., vol. 278, no. 20, pp. 18063±18068. ROSMOND, R., CHAGNON, Y. C., CHAGNON, M., PERUSSE, L., BOUCHARD, C. and BJORNTORP, P. 2000, `A polymorphism of the 50 -flanking region of the glucocorticoid receptor gene locus is associated with basal cortisol secretion in men', Metabolism, vol. 49, no. 9, pp. 1197±1199. SAKANE, N., YOSHIDA, T., UMEKAWA, T., KOGURE, A., TAKAKURA, Y. and KONDO, M. 1997, `Effects of Trp64Arg mutation in the beta 3±adrenergic receptor gene on weight loss, body fat distribution, glycemic control, and insulin resistance in obese type 2 diabetic patients', Diabetes Care, vol. 20, no. 12, pp. 1887±1890. SALMON, D. M. and FLATT, J. P. 1985, `Effect of dietary fat content on the incidence of obesity among ad libitum fed mice', Int. J. Obes., vol. 9, no. 6, pp. 443±449. SAMAHA, F. F., IQBAL, N., SESHADRI, P., CHICANO, K. L., DAILY, D. A., MCGRORY, J., WILLIAMS, T., WILLIAMS, M., GRACELY, E. J. and STERN, L. 2003, `A low-carbohydrate as compared with a low-fat diet in severe obesity', N. Engl. J. Med., vol. 348, no. 21, pp. 2074± 2081.

Nutrient-gene interactions in the control of obesity

257

SARIS, W. H., ASTRUP, A., PRENTICE, A. M., ZUNFT, H. J., FORMIGUERA, X., VERBOEKET-VAN DE

and 2000, `Randomized controlled trial of changes in dietary carbohydrate/ fat ratio and simple vs complex carbohydrates on body weight and blood lipids: the CARMEN study. The Carbohydrate Ratio Management in European National diets', Int. J. Obes. Relat Metab Disord., vol. 24, no. 10, pp. 1310±1318. VENNE WP, RABEN, A., POPPITT, S. D., SEPPELT, B., JOHNSTON, S., VASILARAS, T. H.

KEOGH, G. F.

SCHADT, E. E., MONKS, S. A., DRAKE, T. A., LUSIS, A. J., CHE, N., COLINAYO, V., RUFF, T. G.,

and 2003, `Genetics of gene expression surveyed in maize, mouse and man', Nature, vol. 422, no. 6929, pp. 297±302. SEIDELL, J. C. 1998, `Societal and personal costs of obesity', Exp. Clin. Endocrinol. Diabetes, vol. 106 Suppl 2, pp. 7±9. SHAH, M., MCGOVERN, P., FRENCH, S. and BAXTER, J. 1994, `Comparison of a low-fat, ad libitum complex-carbohydrate diet with a low-energy diet in moderately obese women', Am. J. Clin. Nutr., vol. 59, no. 5, pp. 980±984. SHIWAKU, K., NOGI, A., ANUURAD, E., KITAJIMA, K., ENKHMAA, B., SHIMONO, K. and YAMANE, Y. 2003, `Difficulty in losing weight by behavioral intervention for women with Trp64Arg polymorphism of the beta(3)-adrenergic receptor gene', Int. J. Obes. Relat Metab Disord., vol. 27, no. 9, pp. 1028±1036. SIMOPOULOS, A. P. 1999, `Genetic variation and nutrition', Nutr. Rev., vol. 57, no. 5 Pt 2, pp. S10±S19. MILLIGAN, S. B., LAMB, J. R., CAVET, G., LINSLEY, P. S., MAO, M., STOUGHTON, R. B. FRIEND, S. H.

SINA, M., HINNEY, A., ZIEGLER, A., NEUPERT, T., MAYER, H., SIEGFRIED, W., BLUM, W. F.,

and HEBEBRAND, J. 1999, `Phenotypes in three pedigrees with autosomal dominant obesity caused by haploinsufficiency mutations in the melanocortin-4 receptor gene', Am. J. Hum. Genet., vol. 65, no. 6, pp. 1501±1507. SKOV, A. R., TOUBRO, S., RONN, B., HOLM, L. and ASTRUP, A. 1999, `Randomized trial on protein vs carbohydrate in ad libitum fat reduced diet for the treatment of obesity', Int. J. Obes. Relat Metab Disord., vol. 23, no. 5, pp. 528±536. SéRENSEN, T. I. A. 1996, `Adoption studies of Obesity', in Molecular and Genetic Aspects of Obesity, G. A. Bray and D. H. Ryan, eds, Louisiana State University Press, Baton Rouge and London, pp. 462±469. SéRENSEN, T. I. A. 2003a, `Weight loss causes increased mortality: pros', Obes. Rev., vol. 4, no. 1, pp. 3±7. SéRENSEN, T. I. A. 2003b, `Interpretation of positive energy balance', Int. J. Obes. Relat Metab Disord., vol. 27, Suppl. 1, pp. S32. SéRENSEN, T. I. A. and ECHWALD, S. M. 2001, `Obesity genes', BMJ, vol. 322, no. 7287, pp. 630±631. SéRENSEN, T. I. A., PRICE, R. A., STUNKARD, A. J. and SCHULSINGER, F. 1989, `Genetics of obesity in adult adoptees and their biological siblings', BMJ, vol. 298, no. 6666, pp. 87±90. SéRENSEN, T. I. A., HOLST, C. and STUNKARD, A. J. 1992, `Childhood body mass index± genetic and familial environmental influences assessed in a longitudinal adoption study', Int. J. Obes. Relat Metab Disord., vol. 16, no. 9, pp. 705±714. SéRENSEN, H. T., SABROE, S., GILLMAN, M., ROTHMAN, K. J., MADSEN, K. M., FISCHER, P. and SORENSEN, T. I. A. 1997, `Continued increase in prevalence of obesity in Danish young men.', Int. J. Obes., vol. 21, pp. 712±714. REMSCHMIDT, H.

STICH, V., HARANT, I., DE, G., I, CRAMPES, F., BERLAN, M., KUNESOVA, M., HAINER, V., DAUZATS,

and LANGIN, D. 1997, `Adipose tissue lipolysis and hormone-sensitive lipase expression during very-low-

M., RIVIERE, D., GARRIGUES, M., HOLM, C., LAFONTAN, M.

258

Functional foods, ageing and degenerative disease calorie diet in obese female identical twins', J. Clin. Endocrinol. Metab, vol. 82, no. 3, pp. 739±744.

STICH, V., MARION-LATARD, F., HEJNOVA, J., VIGUERIE, N., LEFORT, C., SULJKOVICOVA, H., LANGIN, D., LAFONTAN, M. and BERLAN, M. 2002, `Hypocaloric diet reduces exerciseinduced alpha 2±adrenergic antilipolytic effect and alpha 2±adrenergic receptor mRNA levels in adipose tissue of obese women', J. Clin. Endocrinol. Metab, vol. 87, no. 3, pp. 1274±1281. STRAUSS, R. S. and POLLACK, H. A. 2001, `Epidemic increase in childhood overweight, 1986±1998', JAMA, vol. 286, no. 22, pp. 2845±2848. STUBBS, R. J., JOHNSTONE, A. M., HARBRON, C. G. and REID, C. 1998, `Covert manipulation of energy density of high carbohydrate diets in ``pseudo free-living'' humans', Int. J. Obes. Relat Metab Disord., vol. 22, no. 9, pp. 885±892.

STUNKARD, A. J., SORENSEN, T. I., HANIS, C., TEASDALE, T. W., CHAKRABORTY, R., SCHULL, W. J.

and SCHULSINGER, F. 1986, `An adoption study of human obesity', N. Engl. J. Med., vol. 314, no. 4, pp. 193±198. STUNKARD, A. J., HARRIS, J. R., PEDERSEN, N. L. and MCCLEARN, G. E. 1990, `The body-mass index of twins who have been reared apart', N. Engl. J. Med., vol. 322, no. 21, pp. 1483±1487. SWARBRICK, M. M. and VAISSE, C. 2003, `Emerging trends in the search for genetic variants predisposing to human obesity', Curr. Opin. Clin. Nutr. Metab Care, vol. 6, no. 4, pp. 369±375. TEEGARDEN, D. 2003, `Calcium intake and reduction in weight or fat mass', J. Nutr., vol. 133, no. 1, pp. 249S±251S. THOMPSON, D. and WOLF, A. M. 2001, `The medical-care cost burden of obesity', Obes. Rev., vol. 2, no. 3, pp. 189±197. THOMSEN, B. L., EKSTROM, C. T. and SORENSEN, T. I. 1999, `Development of the obesity epidemic in Denmark: cohort, time and age effects among boys born 1930±1975', Int. J. Obes. Relat Metab Disord., vol. 23, no. 7, pp. 693±701. UKKOLA, O., ROSMOND, R., TREMBLAY, A. and BOUCHARD, C. 2001a, `Glucocorticoid receptor Bcl I variant is associated with an increased atherogenic profile in response to long-term overfeeding', Atherosclerosis, vol. 157, no. 1, pp. 221±224. UKKOLA, O., TREMBLAY, A., SUN, G., CHAGNON, Y. C. and BOUCHARD, C. 2001b, `Genetic variation at the uncoupling protein 1, 2 and 3 loci and the response to long-term overfeeding', Eur. J. Clin. Nutr., vol. 55, no. 11, pp. 1008±1015. UKKOLA, O., SUN, G. and BOUCHARD, C. 2001c, `Insulin-like growth factor 2 (IGF2) and IGF-binding protein 1 (IGFBP1) gene variants are associated with overfeedinginduced metabolic changes', Diabetologia, vol. 44, no. 12, pp. 2231±2236. UKKOLA, O., TREMBLAY, A. and BOUCHARD, C. 2001d, `Beta-2 adrenergic receptor variants are associated with subcutaneous fat accumulation in response to long-term overfeeding', Int. J. Obes. Relat Metab Disord., vol. 25, no. 11, pp. 1604±1608. UKKOLA, O., JOANISSE, D. R., TREMBLAY, A. and BOUCHARD, C. 2003, `Na+-K+-ATPase alpha 2±gene and skeletal muscle characteristics in response to long-term overfeeding', J. Appl. Physiol, vol. 94, no. 5, pp. 1870±1874. VAISSE, C., CLEMENT, K., GUY-GRAND, B. and FROGUEL, P. 1998, `A frameshift mutation in human MC4R is associated with a dominant form of obesity', Nat. Genet., vol. 20, no. 2, pp. 113±114. VAISSE, C., CLEMENT, K., DURAND, E., HERCBERG, S., GUY-GRAND, B. and FROGUEL, P. 2000, `Melanocortin-4 receptor mutations are a frequent and heterogeneous cause of morbid obesity', J. Clin. Invest, vol. 106, no. 2, pp. 253±262.

Nutrient-gene interactions in the control of obesity

259

and DE LEEUW, I. H. 1997, `The beneficial effects of modest weight loss on cardiovascular risk factors', Int. J. Obes. Relat Metab Disord., vol. 21 Suppl 1, p. S5±S9. VIGUERIE, N. and LANGIN, D. 2003, `Effect of thyroid hormone on gene expression', Curr. Opin. Clin. Nutr. Metab Care, vol. 6, no. 4, pp. 377±381. VAN GAAL, L. F., WAUTERS, M. A.

VINCENT, S., PLANELLS, R., DEFOORT, C., BERNARD, M. C., GERBER, M., PRUDHOMME, J., VAGUE,

and LAIRON, D. 2002, `Genetic polymorphisms and lipoprotein responses to diets', Proc. Nutr. Soc., vol. 61, no. 4, pp. 427±434. VOGLER, G. P., SORENSEN, T. I., STUNKARD, A. J., SRINIVASAN, M. R. and RAO, D. C. 1995, `Influences of genes and shared family environment on adult body mass index assessed in an adoption study by a comprehensive path model', Int. J. Obes. Relat Metab Disord., vol. 19, no. 1, pp. 40±45. WADDEN, T. A., FOSTER, G. D. and LETIZIA, K. A. 1994, `One-year behavioral treatment of obesity: comparison of moderate and severe caloric restriction and the effects of weight maintenance therapy', J. Consult Clin. Psychol., vol. 62, no. 1, pp. 165± 171. WEST, D. B., WAGUESPACK, J. and MCCOLLISTER, S. 1995, `Dietary obesity in the mouse: interaction of strain with diet composition', Am. J. Physiol, vol. 268, no. 3 Pt 2, pp. R658±R665. WILLIAMS, R. R. 1984, `The role of genetic analyses in characterising obesity', Int. J. Obes., vol. 8, no. 5, pp. 551±559. WORLD HEALTH ORGANISATION 1997, Obesity; Preventing and managing the global epidemic; Report of a WHO consultation on Obesity; Geneva, 3±5 June 1997, World Health Organisation, Geneva, Geneva. XINLI, W., XIAOMEI, T., MEIHUA, P. and SONG, L. 2001, `Association of a mutation in the beta3±adrenergic receptor gene with obesity and response to dietary intervention in Chinese children', Acta Paediatr., vol. 90, no. 11, pp. 1233±1237. YAMADA, K., YUAN, X., OTABE, S., KOYANAGI, A., KOYAMA, W. and MAKITA, Z. 2002, `Sequencing of the putative promoter region of the cocaine- and amphetamineregulated-transcript gene and identification of polymorphic sites associated with obesity', Int. J. Obes. Relat Metab Disord., vol. 26, no. 1, pp. 132±136. YANG, D., FONTAINE, K. R., WANG, C. and ALLISON, D. B. 2003, `Weight loss causes increased mortality: cons', Obes. Rev., vol. 4, no. 1, pp. 9±16. ZEMEL, M. B. 2003, `Mechanisms of dairy modulation of adiposity', J. Nutr., vol. 133, no. 1, pp. 252S±256S. ZHANG, Y., PROENCA, R., MAFFEI, M., BARONE, M., LEOPOLD, L. and FRIEDMAN, J. M. 1994, `Positional cloning of the mouse obese gene and its human homologue', Nature, vol. 372, no. 6505, pp. 425±432. P.

11 Nutrition, fat synthesis and obesity F. Foufelle and P. FerreÂ, INSERM, France

11.1

Introduction: fat synthesis and nutrition

11.1.1 The process of fat synthesis Storage of energy when food is available is essential for survival in periods of increased energy expenditure or decreased energy availability. Quantitatively, the main form of energy storage is represented by lipids in adipose tissue. The origin of the lipids stored can be either the diet or de novo synthesis from nonlipid substrates and this is called the lipogenic process. In mammals, nearly all tissues are able to synthesize fatty acids due to the absolute necessity of these compounds as components of cellular structures. However, a high and dietcontrolled rate of lipogenesis is found only in the liver and adipose tissue (and in the mammary gland of lactating females). The relative importance of these tissues depends upon the species considered. In rodents, lipogenesis is shared by both liver and adipose tissue whereas in man, the liver is predominant. In pigs, dogs, cats and ruminants, adipose tissue is the main lipogenic organ. Although carbohydrates are usually considered in mammals as the main lipogenic substrates, amino acids can also be used, especially in the case of low fat, high protein diets. In ruminants, since carbohydrates are fermented in the rumen, acetate is the main lipogenic precursor. The lipogenic pathway from glucose in the liver is summarized in Fig. 11.1. The main differences between liver and adipose tissue are that (i) in adipose tissue, glucose entry is mediated by a glucose transporter (GLUT 4), which in the absence of insulin is mainly intracellular and which translocates to the plasma membrane in the presence of insulin (Simpson and Cushman, 1986), whereas in the liver glucose is transported by a glucose transporter (GLUT 2), which is constitutively present in the plasma membrane (Thorens, 1996). (ii) in the adipocyte, glucose is

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Fig. 11.1

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The glycolytic and lipogenic pathways in the liver.

phosphorylated by a hexokinase (hexokinase II) whereas in the liver, glucose is phosphorylated by glucokinase (hexokinase IV) which requires insulin for its expression (Printz et al., 1993). When glucose is the main substrate used for fatty acid synthesis, the enzymes of the glycolytic pathway can be considered as an extended part of the lipogenic pathway, inasmuch as in the liver it has been proposed that the major function of glycolysis is not to provide ATP but to allow the transformation of carbohydrates into fat (Hers and Hue, 1983). Pyruvate originating from glycolysis enters into the mitochondria where it is transformed into acetylCoA by the pyruvate dehydrogenase complex. Acetyl-CoA condenses with oxaloacetate to form citrate in a reaction catalyzed by citrate synthase. Citrate is then transported outside the mitochondria into the cytoplasm where acetyl-CoA

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and oxaloacetate are produced in a reaction catalyzed by ATP citrate lyase. Oxaloacetate carbons can enter back into the mitochondria through the successive action of two malate dehydrogenases or they can be used to form pyruvate and NADPH through the action of Malic enzyme. This citrate shuttle is rendered necessary by the fact that inner mitochondrial membrane is not permeable to CoA-derivatives. In the cytoplasm, acetyl-CoA is then converted into malonyl-CoA in an ATP-dependent manner by acetyl-CoA carboxylase (ACC). Acetyl-CoA and malonyl-CoA are used in a seven-step reaction catalyzed by fatty acid synthase (FAS) in order to produce palmitate. Palmitate can then be further elongated and/or desaturated by specific enzymes. Then three fatty acyl-CoA will be esterified on an alpha-glycerophosphate backbone to form a triglyceride which can be stored in hepatocytes or exported as VLDL outside of the liver. NADPH, which is necessary for the reaction catalyzed by FAS, is provided first through the citrate shuttle (Fig. 11.1) then through the pentose phosphate pathway which begins with glucose-6-phosphate as a substrate with two dehydrogenation steps catalyzed successively by glucose-6phosphate and 6-phosphogluconate dehydrogenases. Since most of the carbohydrates in our diet are in the form of sucrose which contains equal amounts of glucose and fructose, the latter is also an important lipogenic substrate. Fructose metabolism is essentially hepatic due to the presence of an active fructokinase which yields fructose-1-phosphate (F1P). F1P is split by fructose bisphosphate aldolase yielding glyceraldehyde and dihydroxyacetone phosphate which will enter glycolysis after the reaction catalyzed by 6-phosphofructokinase (Fig. 11.1). 6-Phosphofructokinase is an important regulatory step for glycosis; fructose metabolism escapes this step and it might explain why fructose has been found as a better lipogenic substrate than glucose. 11.1.2 Lipogenesis in humans In laboratory rodents, the most studied model in terms of lipogenesis, the lipogenic rate is usually high due to the high-carbohydrate, low-fat content of the chow diet. In contrast, in humans hepatic lipogenesis has long been considered as negligible in the overall lipid economy since limited amounts of carbohydrate are available for lipogenesis with the typical western diet (35±40% of energy as fat). Recent quantitive analysis using isotopic methods in various nutritional and physiopathological conditions have shown that the fractional contribution of lipogenesis to VLDL triglycerides is 2±5% in normal subjects eating a typical western diet but that high-carbohydrate, low fat, or simple sugarenriched diets, obesity, alcohol consumption and infectious states can strongly increase this proportion up to 20±30% (Diraison et al., 1997; Hellerstein et al., 1993; Siler et al., 1999; Hudgins et al., 2000; Diraison et al., 2003). For instance, it was shown that increased hepatic lipogenesis in obese subjects represented an excess of 2±5g/day of triglycerides (0.7±1.8 kg on a yearly basis) (Diraison et al., 2002).

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11.1.3 Regulatory role of lipogenesis in energy metabolism In addition to quantitative considerations, a regulatory role of lipogenesis must be emphasized. Indeed, one of the intermediates of lipogenesis, malonyl-CoA, is a potent inhibitor of carnitine palmitoyl transferase I (CPT I) which catalyzes the entry of long chain fatty acyl-CoA into mitochondria and is a crucial regulatory step of mitochondrial -oxidation (McGarry and Brown, 1997). In other words, in the liver, there is an inverse relationship between lipogenic and fatty acid oxidation rates. Interestingly, in non-lipogenic tissues such as muscles for instance, although FAS activity is usually extremely low, a different isoform of ACC (called ACC2 or beta) is presently encoded by a different gene from the ACC (ACC1 or alpha) found in lipogenic tissues (Ruderman et al., 1999). ACC2 seems to be associated with the mitochondrial membrane, in contrast with ACC1 which is cytoplasmic (Abu-Elheiga et al., 2000). The role of ACC2 would then be to produce malonyl-CoA in the vicinity of CPT-I and would mainly be involved in the regulation of fatty acid oxidation rather than in lipogenesis per se (Ruderman et al., 1999). In animal models in which ACC2 activity is decreased by means of either genetic modifications (Abu-Elheiga et al., 2001), specific hormonal action (Yamauchi et al., 2002; Tomas et al., 2002) or changes in fuel supply (Ruderman et al., 1999), fatty acid oxidation is markedly increased and this is concomitant with an improvement of insulin sensitivity. This might be the consequence of a decreased intracellular concentration of fatty acyl-CoA which are considered as promoters of insulin resistance. In addition, in rodents, replacement of carbohydrate oxidation by fatty acid oxidation is concomitant with a resistance to diet-induced obesity (Abu-Elheiga et al., 2003). In a recent work, it was postulated that malonyl-CoA might be involved in the control of food intake in mice (Loftus et al., 2000). Indeed, it was shown that intra-cerebroventricular (ICV) administration of an inhibitor of FAS, which showed increased malonyl-CoA concentration by decreasing its removal, markedly inhibited feeding. Moreover, ICV administration of TOFA (tetrahydrofuroic acid), which is an inhibitor of ACC, opposed the anorectic effect of the FAS inhibitor. Although malonyl-CoA concentration could not be measured, these findings support the hypothesis that malonyl-CoA could be a regulator of food intake. Thus the lipogenic pathway of which activity signals carbohydrate availability (see below) could be an important regulator of energy intake and substrate partition. As such, it can be one of the components of obesity by providing substrates for adipose tissue development and by decreasing overall fatty acid oxidation and favoring weight gain. 11.1.4 Nutrition and lipogenesis It has been known for a long time that hepatic and adipose tissue lipogenic activities are dependent upon the nutritional environment (Saggerson and Greenbaum, 1970; Volpe and Vagelos, 1973). Lipogenic capacity is markedly increased by high-carbohydrate diets whereas fasting or high-fat diets are considerably reducing its activity (Volpe and Vagelos, 1973). An illustration of

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these regulations in both adipose tissue and liver is given by the sucklingweaning transition in the rat. Suckling rats are fed with milk which is a diet rich in fat (69% calories) and poor in carbohydrate (less than 10%) (Girard et al., 1992). At weaning the rat is fed with the laboratory chow which is a highcarbohydrate, low-fat diet. Lipogenesis activity is exceedingly low in the liver and adipose tissue of the suckling rat and strongly increases at weaning (Taylor et al., 1967; Tsujikawa and Kimura, 1980). This is due to an enhanced activity of the enzymes of the pathway, ACC, FAS, ATP citrate lyase and malic enzyme (Girard et al., 1992). Weaning onto a high-fat diet prevents these changes suggesting that they are the results from the change of diet rather than from a developmental stage (Coupe et al., 1990). Nutritional variations are concomitant with important hormonal changes. For instance, a high insulin-low glucagon ratio is characteristic of a highcarbohydrate-fed state whereas the converse is true for fasting or a high-fat diet. Thus the regulation of the lipogenic flux can be secondary to changes in nutrient but also hormone concentrations. Regulation of lipogenic flux is due to both short-term and long-term regulations of enzyme activities which are illustrated below by some examples.

11.2

Regulation of glycolytic/lipogenic enzymes

11.2.1 Short-term regulation of glycolytic/lipogenic enzymes Two enzymes, glucokinase and ACC have a high control strength on the lipogenic pathway from glucose in the liver. Their short-term regulation is detailed below. Glucokinase (hexokinase IV) is the major glucose phosphorylating enzyme in hepatocytes. The regulation of hepatic glucokinase involves binding to a glucokinase regulatory protein (GKRP) (Van Schaftingen et al., 1994). When glucokinase is bound to GKRP, its activity is totally inhibited. The interaction of these two proteins is stimulated by fructose 6-phosphate and inhibited by fructose 1-phosphate (Agius and Peak, 1993; Agius et al., 1995). GKRP is located mainly in the nucleus of hepatocytes. Glucokinase binds GKRP in the nucleus under basal glucose conditions (~5.5 mM) (Brown et al., 1997). However, in the presence of either high glucose (10±30 mM) or fructose (50 M to 1 mM), glucokinase is released from GKRP and translocates to the cytoplasm. The fact that a fructose metabolite is an activator of glucokinase could explain why fructose potentiates lipogenesis from glucose. Thus the shortterm regulation of glucokinase involves the availability of carbohydrates. The activity of ACC changes rapidly in response to dietary and hormonal factors. Its regulation is complex and involves both allosteric and reversible phosphorylation events which interact with each other (Kim, 1997; Munday, 2002). In the liver, ACC activity which represents mainly ACC1 (or ACC alpha) is increased by citrate (a lipogenic precursor) and inhibited by long chain fatty acyl-CoA (lipogenic products). Activation is concomitant of a polymeric state of the protein. Seven phosphorylation sites have been identified for rat ACC1.

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Among them, two are specially important; serine 1200 is phosphorylated by protein kinase A which is active in the liver in the presence of a low insulin/high glucagon ratio (e.g. state of fast). This phosphorylation seems to strongly increase the Ka for citrate thus decreasing the activity of ACC. Serine 79 is phosphorylated by AMP-activated protein kinase (AMPK) and it induces a large decrease in Vmax. AMPK is activated by a decrease in the energy charge (decrease in ATP and increase in AMP) and its role is to restore the energy homeostasis by inhibiting anabolic pathways, including lipogenesis and activating catabolic pathway such as fatty acid oxidation. It is very likely that these latter consequences of AMPK activation have a common origin, i.e., inhibition of ACC, thus alleviating the inhibition of CPT-1 and allowing fatty acyl-CoA to be oxidized. Recent studies indicate that some hormones produced by adipose tissue, leptin and adiponectin activate fatty acid oxidation in liver and muscle and increase insulin sensitivity by stimulating AMPK with ACC as the ultimate target. It can be pointed out that the ACC2 isoform is also phosphorylated and inhibited by AMPK (the equivalent serine in ACC2 is serine 218). AMPK and ACC are thus extremely interesting targets for drugs aimed at improving insulin sensitivity in syndromes such as type-2 diabetes or obesity. Insulin stimulates ACC activity but its effects on the protein are not clear. Insulin increases the phosphorylation of ACC but there is no evidence that this is concomitant with ACC activation. In fact, insulin could promote the association of ACC with a specific phosphatase suggesting that insulin acts by dephosphorylation to activate the enzyme. In conclusion, hormones and substrates which are linked to nutrient availability are able to modulate on a short time-basis the activity of the lipogenic pathway. 11.2.2 Long-term regulation of glycolytic/lipogenic enzymes The expression of several key glycolytic and lipogenic enzymes is induced by a high-carbohydrate diet in the liver: glucokinase (Iynedjian et al., 1987), 6phosphofructo-1-kinase (Rongnoparut et al., 1991), 6-phosphofructo-2-kinase/ fructose-2-6-bisphosphatase (Colosia et al., 1988), aldolase B (Weber et al., 1984) and L-pyruvate kinase (L-PK) (Vaulont et al., 1986) for glycolysis, ATPcitrate lyase (Elshourbagy et al., 1990), ACC (Pape et al., 1988), FAS (Katsurada et al., 1990; Paulauskis and Sul, 1989), stearoyl-CoA desaturase (Ntambi, 1992) for lipogenesis, glucose-6-phosphate dehydrogenase (Kletzien et al., 1985; Katsurada et al., 1989), 6-phosphogluconate dehydrogenase (Miksicek and Towle, 1983) for the pentose-phosphate pathway. We have to mention also here the induction of the S14 gene which encodes for a small acidic polypeptide which seems to be related to lipogenesis. It is expressed in lipogenic tissues (white and brown adipose tissue, liver, lactating mammary gland) and shares a similar regulation with genes involved in the lipogenic pathway (Mariash et al., 1986; Kinlaw et al., 1987; Clarke et al., 1990b). The specific function of the S14 protein remains unclear, although some studies have suggested that S14 could be

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involved itself in the machinery which regulates lipogenic enzyme expression (Kinlaw et al., 1995; Zhu et al., 2001). For most of these genes involved in glucose carbon utilisation, the induction of their mRNA expression by a carbohydrate-rich diet is powerful (from 4 to 25 fold), rapid (in the one-two hours range) and involves a transcriptional mechanism. Absorption of a carbohydrate diet is concomitant with increases in the concentration of substrates such as glucose and lactate but also with changes in the concentration of pancreatic hormones, insulin and glucagon. Differentiating between the respective roles of hormones and substrates on the regulation of gene expression has then strongly benefited from in vitro studies either on primary cultured cells or cell lines. From these studies, different kinds of gene regulation have emerged for genes involved in the lipogenic pathway. Purely insulin sensitive genes, such as hepatic glucokinase which can be induced by a high insulin concentration independently from the presence of glucose (Iynedjian et al., 1989), and genes which require both an increased insulin and glucose concentration in order to be induced, such as L-pyruvate kinase (L-PK), FAS, ACC, S14 and stearoyl-CoA desaturase (Decaux et al., 1989; Prip- Buus et al., 1995; O'Callaghan et al., 2001; Koo et al., 2001; Waters and Ntambi, 1994). One potential reason why in contrast with glucokinase, these genes require both a high insulin and a high glucose concentration could be linked to the fact that after a meal the metabolic priority is to replenish glycogen stores, and it is only if glucose is particularly abundant that the glucose carbons are orientated towards lipid synthesis. Interestingly, glucagon is able to antagonize the effect of insulin and glucose on the expression of glycolytic/lipogenic genes.

11.3 Molecular mechanisms involved in controlling glycolytic/lipogenic genes 11.3.1 Mechanisms involved in insulin action: the SREBP-1c transcription factor Recent studies have shown that insulin action on this family of gene is mediated by a transcription factor called SREBP-1c (Sterol Regulatory Element Binding Protein). SREBP-1c belongs to a family of transcription factors originally involved in the regulation of genes by the cellular availability in cholesterol (Wang et al., 1994). Three members of the SREBP family have been described in several mammalian species (mice, rat, hamster and human). SREBP-1a and SREBP-1c are encoded by a single gene through the use of alternative transcription start sites and differ by their first exon (Hua et al., 1995). The third member of the family, SREBP-2 is derived from a different gene and presents 50% homology with the SREBP-1 amino acid sequence. Another major difference between the 1a and 1c isoform is their tissue distribution. SREBP-1c is mainly expressed in most of the tissues of mice and humans with specially high levels in the liver, white adipose tissue, adrenal gland and brain (Shimomura et al., 1997). SREBP-1c is also expressed in

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various muscles in adult rats and humans at appreciable levels (Ducluzeau et al., 2001, Guillet-Deniau et al., 2002). By contrast, SREBP-1a is mainly expressed in cell lines and in tissues with a high capacity of cell proliferation such as spleen and intestine (Shimomura et al., 1997). The three isoforms have a common structure: (i) an amino-terminal fragment of 480 amino acids which is in fact a transcription factor of the basic domain-helix loop helix, leucine zipper family, (ii) a region of 80 amino acids containing two transmembrane domains separated by 31 amino acids which are in the lumen of the endoplasmic reticulum and a regulatory C-terminal domain of 590 amino acids. Brown and Goldstein have elegantly unraveled the mechanisms by which the transcriptionally active fragment of SREBP-2 and 1a is liberated (Brown and Goldstein, 1997, 1999; Yang et al., 2002). When the concentration of cholesterol decreases in the membranes, the precursor form of SREBP-2 and SREBP-1a is increased through an enhanced gene transcription. Then this precursor form is cleaved by a complex mechanism involving two proteolytic cleavages catalyzed by two distinct proteases (S1P and S2P), a protein `sensor' for the cholesterol concentration (SCAP) and an anchoring protein (INSIG). The mature form migrates inside the nucleus where it activates the promoter of genes involved in cholesterol uptake or in cholesterol synthesis. In contrast to SREBP-2 and SREBP-1a, SREBP-1c expression and nuclear abundance is not increased in case of low cholesterol availability (Sheng et al., 1995) but is related to carbohydrate and lipid metabolism. In mouse and rat liver, the expression and the presence of the mature form of SREBP-1c in nuclei are increased when starved animals are fed with a high carbohydrate diet (Horton et al., 1998). In contrast, this experimental protocol has only a modest effect on SREBP-2 expression (Horton et al., 1998). Several studies have shown that overexpression of SREBP-1c is concomitant with the induction of most of the lipogenesis-related genes such as FAS, ACC, ATP-citrate lyase, glucose-6phosphate dehydrogenase (Horton et al., 2002). Insulin is able to strongly activate SREBP-1c expression (Foretz et al., 1999b). This effect is transcriptional as shown by run-on assays. Glucagon opposes the effects of insulin on SREBP-1c expression via its second messenger cAMP (Foretz et al., 1999b). At the protein level, the induction of SREBP-1c expression by insulin is followed by an increase in the precursor form of SREBP-1c and a concomitant increase in the nuclear mature form (Azzout-Marniche et al., 2000). These effects of insulin on the SREBP-1c proteins are antagonized by glucagon. The effect of insulin on SREBP-1c is corroborated by in vivo studies showing that SREBP-1c expression and nuclear abundance are low in the liver of diabetic rats and increase markedly after an insulin treatment (Shimomura et al., 1999). Studies using inhibitors of various branches of the insulin signalling pathway have shown that the effect of insulin on SREBP-1c expression and synthesis involves mainly the PI-3±kinase pathway (Azzout-Marniche et al., 2000). At present, the mechanism by which glucagon reduces the expression of SREBP-1c is totally unknown except that it occurs through the usual cAMP system (Foretz et al., 1999b).

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A number of in vivo and in vitro experiments, reviewed in Foufelle and Ferre (2002) involving dominant positive and negative forms of this transcription factor have demonstrated that SREBP-1c is indeed the mediator of insulin action on glucokinase and is also able to account for the insulin part on lipogenic gene expression which requires both insulin and glucose as transcriptional inducers. 11.3.2 Mechanisms involved in glucose action: involvment of the CHREBP transcription factor As stated above, a subset of glycolytic/lipogenic genes in the liver requires both an increased insulin and glucose concentration to be fully expressed (Girard et al., 1997; Towle et al., 1997; Vaulont et al., 2000). This regulation involves the stimulation of their transcription rate. In the absence of glucose, insulin by itself is unable to induce their expression. In the absence of insulin, the effect of glucose is greatly reduced in adipose tissue and nearly absent in cultured hepatocytes. Glucose must be metabolized in order to activate gene expression. Glucose-6phosphate and xylulose-5-phosphate, an intermediate of the non-oxidative branch of the metabolic pathway, have both been proposed as the metabolic signal. Glucose or carbohydrate response elements (ChoRE) have been characterized on the promoter of most glucose-responsive genes, L-PK, FAS, S14, ACC (Thompson and Towle, 1991; Bergot et al., 1992; Shih and Towle, 1992; Rufo et al., 2001; O'Callaghan et al., 2001). The ChoRE is described as two E-box or E-box like sequences of the form CANNTG, separated by a defined distance. The first 4 bp of each E-box are critical for the glucose response as well as the length of the spacing sequence (Shih and Towle, 1994; Shih et al., 1995). The group of Uyeda has purified a transcription factor from rat liver, based on its capacity to bind to the ChoRE of the L-PK promoter and named it ChREBP (Carbohydrate Response Element Binding Protein) (Yamashita et al., 2001). This factor fulfils a number of criteria for a bona fide ChoRE binding protein. It is a basic domain helix-loop-helix leucine zipper protein with a bipartite nuclear localization signal and its binding capacity to mutated ChoRE parallels the capacity of these ChoRE to respond to a high glucose concentration in cultured hepatocytes. When transfected in hepatocytes ChREBP is able to stimulate the L-PK promoter which contains the ChoRE and this effect is dramatically increased in the presence of glucose. Finally, although the mRNA of this factor is found in tissues such as the cerebellum, intestine, kidney and liver, the DNA binding to ChoRE is found only in liver (Yamashita et al., 2001). The mechanism suggested in order to explain the glucose effect on target gene transcription is a translocation of ChREBP between the cytoplasm and the nucleus. ChREBP is located in the cytoplasm when the glucose concentration is low and enters into the nucleus at high glucose levels (Kawaguchi et al., 2001). This would be secondary to a dephosphorylation of a specific serine (Ser196) otherwise phosphorylated by protein kinase A (PKA) in conditions of high cAMP concentrations such as the fasting state for instance. A second PKA

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phosphorylation near the DNA binding domain and which precludes ChREBP binding would also be dephosphorylated in the presence of high glucose (Kawaguchi et al., 2001). Thus, the main effect of glucose would be to activate a phosphatase counteracting the effect of cAMP, inducing the translocation of ChREBP in the nucleus and stimulating its DNA binding activity. This would be consistent with the antagonistic effect of glucagon and insulin/glucose on L-PK and lipogenic genes. Recently, phosphatases activated by xylulose-5±phosphates have been identified (Kabashima et al., 2003). They are able to dephosphorylate Ser 196 of ChREBP, thus favoring its translocation into the nucleus. 11.3.3 Integration of insulin/glucose regulation of glycolytic and lipogenic genes When a carbohydrate-rich meal is absorbed, plasma glucose concentration is increased and it induces a secretion of insulin by the pancreatic beta-cell. High concentrations of both glucose and insulin then reach the liver through the portal vein. Insulin will induce SREBP-1c transcription by a PI3±kinase dependent mechanism and hence the synthesis of the precursor form in the endoplasmic reticulum (Fig. 11.2). This precursor form will then be cleaved and the mature form of SREBP-1c will translocate into the nucleus and activate glucokinase. Synthesis of glucokinase increases glucose phosphorylation and glycogen

Fig. 11.2

Integration of insulin/glucose regulation of glycolytic and lipogenic genes.

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repletion. A signal metabolite downstream of the glucokinase step (like xylulose-5-phosphate) activates a transcription factor (ChREBP) by a dephosphorylation-mediated mechanism (Fig. 11.2). ChREBP then translocates into the nucleus where together with SREBP-1c, it activates genes of the glycolytic/lipogenic pathway such as L-PK, ACC, FAS and S14. This will finally lead to the synthesis of fatty acyl-CoA from glucose carbons.

11.4

Improving lipogenesis using functional foods

11.4.1 Regulation of lipogenic gene expression by fatty acids It is now clearly established that fatty acids can modulate the transcription of many genes involved in lipid transport and metabolism. Most of these genes, such as those coding for aP2 (Amri et al., 1991), phosphoenolpyruvate carboxykinase (Antras-Ferry et al., 1994), carnitine palmitoyltransferase I (Chatelain et al., 1996) and liver fatty-acid-binding protein (Meunier-Durmort et al., 1996), are activated by both mono-unsaturated and polyunsaturated fatty acids (PUFA). It has been shown that fatty acids are ligands of peroxisomeproliferator-activated receptors (PPAR), which are members of the nuclearhormone-receptor superfamily (Forman et al., 1997; Kliewer et al., 1997). PPAR regulates the transcription of target genes by binding to DNA sequence elements named the `PPAR response element' (PPAR-RE). A number of PPARREs have been identified in various gene promoters, such as aP2 (Tontonoz et al., 1994) and phosphoenolpyruvate carboxykinase (Tontonoz et al., 1995). Three PPAR isoforms have been described. PPAR alpha is present mainly in liver, heart, and to a lesser extent in skeletal muscles. When activated it promotes fatty acid oxidation, ketone body synthesis and glucose sparing. PPAR delta is ubiquitous and could also favor fatty acid oxidation in tissues in which PPAR alpha is less, or not at all, expressed, such as skeletal muscles. PPAR gamma is expressed in adipose tissues, lower intestine and in cells involved in immunity. Activation of PPAR gamma induces the differentiation of preadipocytes into adipocytes and favors lipid storage pathways. Over the past 25 years, numerous studies have demonstrated that high-fat low-carbohydrate diets inhibit the hepatic lipogenic pathway (Clarke, 2001). It was clearly shown that this was due to an inhibition of the expression of the enzymes such as FAS, ACC and S14 (Clarke et al., 1990a; Blake and Clarke, 1990; Girard et al., 1994; Liimatta et al., 1994). This inhibition is induced by the presence of small amounts of PUFA in the diet (Clarke et al., 1990a; Girard et al., 1994; Katsurada et al., 1990). The inhibitory effect of PUFA appears to be a liver-specific response, since they are not effective in other lipogenic tissues, e.g., adipose tissue and lung. In vitro studies in primary cultures of hepatocytes have confirmed the inhibition of glucokinase, L-PK, glucose-6-phosphate dehydrogenase, ATP citrate lyase FAS and S14 gene expression by PUFA, whereas saturated and mono-unsaturated fatty acids have no effect (Liimatta et al., 1994; Jump et al., 1993, 1994). Transfection analysis of the L-PK and S14

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promoter in primary cultures of hepatocytes have led to the identification of PUFA response elements (Liimatta et al., 1994; Jump et al., 1993). It should be underlined that these PUFA-sensitive elements are located in key activatory transcriptional regions. Finally, it is noteworthy that the four genes (coding for L-PK, S14, ACC and FAS) which are specifically inhibited by PUFA are also the genes which are activated by glucose and insulin. PPARs do not appear to interact with PUFA response sequences of lipogenic genes (Clarke, 2000; Jump and Clarke, 1999; Kakuma et al., 2000). Moreover, PUFA continue to suppress the transcription of hepatic lipogenic genes in PPARalpha KO mice (Jump and Clarke, 1999). The PUFA response region of the FAS gene is the one which is responsible for insulin responsiveness and contains DNA binding sites for SREBP-1. It bears similarities with the PUFA response region of the S14 gene (Jump and Clarke, 1999). It has been shown that diets rich in 18:2(n-6) or 20:5 and 22:6(n-3) reduce the hepatic nuclear and precursor content of mature SREBP-1 (Xu et al., 1999) whereas saturated and monounsaturated fats had no effect on the nuclear and precursor forms of SREBP-1 or on lipogenic gene expression (Xu et al., 1999, 2001; Hannah et al., 2001). The PUFA-dependent decrease in hepatic content of SREBP-1 is consistent with the inhibition of glycolytic/lipogenic genes which depend upon this transcription factor for their insulin-mediated induction. The mechanisms which are involved in the decrease in SREBP-1c nuclear abundance could involve a decrease in the proteolytic process of the precursor form as well as a decreased SREBP-1c mRNA abundance (Clarke, 2001). However the mechanisms by which PUFA interfere with these mechanisms is not yet clear. It has been suggested that PUFA effect on gene expression is somehow linked to peroxidative mechanisms (Foretz et al., 1999a). 11.4.2 Targeting foods to improve lipogenesis When dealing with the regulation of a specific metabolic pathway in a given direction by functional foods, it is obviously necessary that the specific nutrients chosen have no adverse consequences that will alter the global health status. The other necessity is that the functional food can be used at a reasonable dose compatible with an otherwise normal nutrition. Is it advantageous to modulate the lipogenic rate? Hyperilipidemia with high concentrations of VLDL-triglycerides is associated with insulin resistance, obesity and cardiovascular diseases. Plasma triglyceride concentration is dependent upon the rates of hepatic VLDL production and of clearance from the plasma. As discussed above, hepatic lipogenesis from carbohydrate can contribute signficantly to the rate of triglycerides production by the liver in conditions such as obesity or when ingesting simple-sugar enriched diets. It is thus reasonable to try to decrease de novo fatty acid synthesis. In addition, this could favor hepatic fatty acid oxidation by reducing malonyl-CoA concentration thus alleviating the inhibition of CPT I, and ultimately improve hepatic insulin sensitivity (Yamauchi et al., 2002). At present, two directions can be explored.

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The first one involves the use of PUFA of the n-3 and n-6 families. As seen above, small amounts of PUFA in the diet are able to reduce very efficiently the hepatic rate of lipogenesis at least in rodents (Clarke et al., 1990a). In addition, this effect is specific of hepatic lipogenesis. Moreover, PUFA have been described as potentially beneficial for atherosclerosis (Jump and Clarke, 1999). Thus the use of PUFA is an interesting clue as a beneficial modulator of hepatic lipogenesis. The second possibility involves the use of non-digestible oligosaccharides belonging to the fructan class such as inulin or one of its fraction oligofructose. They are usually obtained from chicory roots or Jerusalem artichokes. These oligosaccharides are not digested in the upper intestine since the anomeric carbon 2 in their fructose monomer is in a beta configuration precluding hydrolysis by human digestive enzymes which are specific for alpha-glycosidic bonds (Kaur and Gupta, 2002). Inulin and oligofructose are thus fermented in the caeco-colon yielding short-chain fatty acids such as acetate, butyrate and propionate. It has been repeatedly shown in rodent models that inulin and oligofructose given in a 10% range in the diet are able to modulate triglyceride metabolism at the hepatic level (Delzenne et al., 2002). When fed with oligofructose-enriched diets, a lower triglyceridemia in non-obese animals and a decreased hepatic steatosis in Zucker fa/fa obese rats have been observed (Delzenne et al., 2002). A lower rate of lipogenesis due to a reduction in the expression and activity of lipogenic enzymes seems to be a key explanation for the effects of these oligosaccharides (Delzenne et al., 2002). In humans, conflicting results have been reported concerning the effect of inulin and oligofructose on lipid metabolism (Kaur and Gupta, 2002). Some studies have reported a beneficial effect on serum triglycerides where others have not. It must be underlined that the doses used in humans are much lower than in rats due to the unpleasant gastro-intestinal effects when consuming doses in excess of 30g per day. In addition, if lipogenesis is one of the key target of their effects, then the diet fed during the study is of importance (Kaur and Gupta, 2002). It can be predicted that the anti-lipogenic effects of fructans would increase with the sugar content of the diet. A recent study has addressed the question of the effects of fructans on hepatic lipogenesis in humans (Letexier et al., 2003). Inulin (10g per day) was added to a moderately high-carbohydrate diet (55% of total energy). Plasma triglycerides and hepatic lipogenesis were indeed lower in the group consuming the inulin-enriched diet. Finally, it can be pointed out that fructans which are already widely used in the food industry (texture modifier, sweetener) have no obvious adverse effects and are even usually considered as having health-promoting properties (Kaur and Gupta, 2002).

11.5

Future trends

One of the most important future issues in the field of lipogenesis is probably the confirmation of its regulatory role in food intake and in fatty acid oxidation and

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hence resistance to obesity. Decreased lipogenesis in liver and muscle should favor fatty acid oxidation. This is turn would favor weight loss by a yet unknown mechanism but which could be linked to an uncoupling effect of fatty acids on the mitochondrial respiratory chain. However, an inhibition of lipogenesis in specific hypothalamic nuclei would mimic starvation and thus should increase food intake and favor weight gain. Activation of the lipogenic pathway would thus have an opposite effect on weight. Part of the problem would then be to find a way of reaching only one of the two regulatory systems. PUFA might be one possibility. Indeed, they reduce hepatic lipogenesis in animal models and do not enter readily into the brain. However, the reality of this effect needs to be studied convincingly in man. Concerning fructans, a number of problems have to be solved. The links between fructan fermentation and effects on gene expression are not entirely clear and this kind of experiment must be repeated and further analyzed in different models. The effects of fructans on human lipid metabolism must be addressed in a more systematic way. The effect of fructans on hepatic steatosis in obese rats is particularly interesting since in humans, Non-Alcoholic SteatoHepatitis (NASH) is diagnosed in a large percentage of obese people and has been associated with the metabolic syndrome (Luyckx et al., 2000). Whether such an effect of non-digestible oligosaccharides can be observed on the steatosis of patients is obviously also an interesting issue that needs to be documented.

11.6

Sources of further information

Among the references listed here, the reader can find extensive information in reviews on the regulation of the lipogenic pathways by carbohydrates (Girard et al., 1997; Foufelle and Ferre, 2002) and fatty acids (Jump and Clarke, 1999), the regulatory role of lipogenesis on fatty acid oxidation (Ruderman et al., 1999) and the effects of fructans and their potential applications to health (Roberfroid and Delzenne, 1998; Delzenne et al., 2002; Kaur and Gupta, 2002).

11.7

References

ABU-ELHEIGA, L., BRINKLEY, W. R., ZHONG, L., CHIRALA, S. S., WOLDEGIORGIS, G.

and WAKIL, S. (2000) Proc Natl Acad Sci USA, 97, 1444±9. ABU-ELHEIGA, L., MATZUK, M. M., ABO-HASHEMA, K. A. and WAKIL, S. J. (2001) Science, 291, 2613±6. ABU-ELHEIGA, L., OH, W., KORDARI, P. and WAKIL, S. J. (2003) Proc Natl Acad Sci USA, 14, 14. AGIUS, L. and PEAK, M. (1993) Biochem J, 296, 785±96. AGIUS, L., PEAK, M. and VAN SCHAFTINGEN, E. (1995) Biochem J, 309, 711±13. AMRI, E. Z., BERTRAND, B., AILHAUD, G. and GRIMALDI, P. (1991) J Lipid Res, 32, 1449±56. ANTRAS-FERRY, J., LE BIGOT, G., ROBIN, P., ROBIN, D. and FOREST, C. (1994) Biochem. J.

274

Functional foods, ageing and degenerative disease Biophys. Res, Comm., 203, 385±91.

AZZOUT-MARNICHE, D., BECARD, D., GUICHARD, C., FORETZ, M., FERRE, P.

(2000) Biochem J, 350, 389±93.

and

FOUFELLE, F.

BERGOT, M. O., DIAZ-GUERRA, M. J. H., PUIZENAT, N., RAYMONDJEAN, M.

and KAHN, A. (1992) Nucleic Acids Res., 20, 1871±8. BLAKE, W. L. and CLARKE, S. D. (1990) J. Nutr., 120, 1727±9. BROWN, K. S., KALINOWSKI, S. S., MEGILL, J. R., DURHAM, S. K. and MOOKHTIAR, K. A. (1997) Diabetes, 46, 179±86. BROWN, M. S. and GOLDSTEIN, J. L. (1997) Cell, 89, 331±40. BROWN, M. S. and GOLDSTEIN, J. L. (1999) Proc Natl Acad Sci USA, 96, 11041±8. CHATELAIN, F., KOHL, C., ESSER, V., MC GARRY, J. D., GIRARD, J. and PEÂGORIER, J. P. (1996) Eur. J. Biochem., 235, 789±98. CLARKE, S. D. (2000) Br J Nutr, 83, S59±66. CLARKE, S. D. (2001) Am J Physiol Gastrointest Liver Physiol, 281, G865±9. CLARKE, S. D., ARMSTRONG, M. K. and JUMP, D. B. (1990a) J. Nutr., 120, 225±31. CLARKE, S. D., ARMSTRONG, M. K. and JUMP, D. B. (1990b) J. Nutr., 120, 218±24. COLOSIA, A. D., MARKER, A. J., LANGE, A. J., EL-MAGHRABI, M. R., GRANNER, D. K., TAULER, A., PILKIS, J. and PILKIS, S. J. (1988) J Biol Chem, 263, 18669±77. COUPEÂ, C., PERDEREAU, D., FERREÂ, P., HITIER, Y., NARKEWICZ, M. and GIRARD, J.

(1990) Am. J. Physiol., 258, E126±E133. DECAUX, J. F., ANTOINE, B. and KAHN, A. (1989) J Biol Chem, 264, 11584±90. DELZENNE, N. M., DAUBIOUL, C., NEYRINCK, A., LASA, M. and TAPER, H. S. (2002) Br J Nutr, 87, S255±9. DIRAISON, F., PACHIAUDI, C. and BEYLOT, M. (1997) J Mass Spectrom, 32, 81±6. DIRAISON, F., DUSSERRE, E., VIDAL, H., SOTHIER, M. and BEYLOT, M. (2002) Am J Physiol Endocrinol Metab, 282, E46±51. DIRAISON, F., YANKAH, V., LETEXIER, D., DUSSERRE, E., JONES, P. and BEYLOT, M. (2003) J Lipid Res, 44, 846±53. DUCLUZEAU, P. H., PERRETTI, N., LAVILLE, M., ANDREELLI, F., VEGA, N., RIOU, J. P. and VIDAL, H. (2001) Diabetes, 50, 1134±42. ELSHOURBAGY, N. A., NEAR, J. C., KMETZ, P. J., SATHE, G. M., SOUTHAN, C., STRICKLER, J. E., GROSS, M., YOUNG, J. F., WELLS, T. N.

5.

FORETZ, M., FOUFELLE, F.

and

FERRE, P.

and GROOT, P. H. (1990) J Biol Chem, 265, 1430±

(1999a) Biochem J, 341, 371±6.

FORETZ, M., PACOT, C., DUGAIL, I., LEMARCHAND, P., GUICHARD, C., LE LIEPVRE, X., BERTHELIER-LUBRANO, C., SPIEGELMAN, B., KIM, J. B., FERRE, P. and FOUFELLE, F. (1999b) Mol Cell Biol, 19, 3760±8. FORMAN, B. M., CHEN, J. and EVANS, R. M. (1997) Proc. Natl. Acad. Sci. USA, 94, 4312±7. FOUFELLE, F. and FERRE, P. (2002) Biochem J, 366, 377±91. GIRARD, J., FERRE, P., PEGORIER, J. P. and DUEE, P. H. (1992) Physiol Rev, 72, 507±62. GIRARD, J., PERDEREAU, D., FOUFELLE, F., PRIP-BUUS, K. and FERREÂ, P. (1994) FASEB J., 8, 36±42. GIRARD, J., FERREÂ, P. and FOUFELLE, F. (1997) Annu. Rev Nutr., 17, 325±52. GUILLET-DENIAU, I., MIEULET, V., LE LAY, S., ACHOURI, Y., CARRE, D., GIRARD, J., FOUFELLE, F. and FERREÂ, P. (2002) Diabetes, 51, 1722±8. HANNAH, V. C., OU, J., LUONG, A., GOLDSTEIN, J. L. and BROWN, M. S. (2001) J Biol Chem, 276, 4365±72. HELLERSTEIN, M. K., GRUNFELD, C., WU, K., CHRISTIANSEN, M., KAEMPFER, S., KLETKE, C. and SHACKLETON, C. H. (1993) J Clin Endocrinol Metab, 76, 559±65.

Nutrition, fat synthesis and obesity HERS, H. G.

and

275

(1983) Annu Rev Biochem, 52, 617±53. and SHIMANO, H. (1998) Proc. Natl. Acad. Sci., 95, 5987±92. HORTON, J. D., GOLDSTEIN, J. L. and BROWN, M. S. (2002) J. Clin. Invest., 109, 1125±31. HUA, X., WU, J., GOLDSTEIN, J. L., BROWN, M. S. and HOBBS, H. H. (1995) Genomics, 25, 667± 73. HUDGINS, L. C., HELLERSTEIN, M. K., SEIDMAN, C. E., NEESE, R. A., TREMAROLI, J. D. and HIRSCH, J. (2000) J Lipid Res, 41, 595±604. IYNEDJIAN, P. B., UCLA, C. and MACH, B. (1987) J Biol Chem, 262, 6032±8. IYNEDJIAN, P. B., JOTTERAND, D., NOUSPIKEL, T., ASFARI, M. and PILOT, P. R. (1989) J Biol Chem, 264, 21824±9. JUMP, D. B. and CLARKE, S. D. (1999) Annu Rev Nutr, 19, 63±90. JUMP, D. B., CLARKE, S. D., MAC DOUGALD, O. and THELEN, A. (1993) Proc. Natl. Acad. Sci. USA, 90, 8454±8. JUMP, D. B., CLARKE, S. D., THELEN, A. and LIIMATTA, M. (1994) J. Lipid Res., 35, 1076±84. KABASHIMA, T., KAWAGUCHI, T., WADZINSKI, B. E. and UYEDA, K. (2003) Proc Natl Acad Sci USA, 100, 5107±12. KAKUMA, T., LEE, Y., HIGA, M., WANG, Z., PAN, W., SHIMOMURA, I. and UNGER, R. H. (2000) Proc Natl Acad Sci USA, 97, 8536±41. KATSURADA, A., IRITANI, N., FUKUDA, H., MATSUMURA, Y., NOGUCHI, T. and TANAKA, T. (1989) Biochim Biophys Acta, 1006, 104±10. KATSURADA, A., IRITANI, N., FUKUDA, H., MATSUMARA, Y., NISHIMOTO, N., NOGUCHI, T. and TANAKA, T. (1990) Eur. J. Biochem., 190, 427±35. KAUR, N. and GUPTA, A. K. (2002) J Biosci, 27, 703±14. KAWAGUCHI, T., TAKENOSHITA, M., KABASHIMA, T. and UYEDA, K. (2001) Proc Natl Acad Sci USA, 98, 13710±5. KIM, K. H. (1997) Annu Rev Nutr, 17, 77±99. KINLAW, W. B., PEREZ-CASTILLO, A. M., FISH, L. H., MARIASH, C. N., SCHWARTZ, H. L. and OPPENHEIMER, J. H. (1987) Mol Endocrinol, 1, 609±13. KINLAW, W. B., CHURCH, J. L., HARMON, J. and MARIASH, C. N. (1995) J Biol Chem, 270, 16615±18. KLETZIEN, R. F., PROSTKO, C. R., STUMPO, D. J., MCCLUNG, J. K. and DREHER, K. L. (1985) J Biol Chem, 260, 5621±4. HUE, L.

HORTON, J. D., BASHMAKOV, Y., SHIMOMURA, I.

KLIEWER, S. A., SUNDSETH, S. S., JONES, S. A., BROWN, P. J., WISELY, G. B., KOBLE, C. S., DEVCHAND, P., WAHLI, W., WILLSON, T. M., LENHARD, J. M.

and LEHMANN, J. M. (1997) Proc. Natl. Acad. Sci. USA, 94, 4318±23. KOO, S. H., DUTCHER, A. K. and TOWLE, H. C. (2001) J Biol Chem, 276, 9437±45. LETEXIER, D., DIRAISON, F. and BEYLOT, M. (2003) Am J Clin Nutr, 77, 559±64. LIIMATTA, M., TOWLE, H. C., CLARKE, S. and JUMP, D. B. (1994) Mol. Endocrinol., 8, 1147±53. LOFTUS, T. M., JAWORSKY, D. E., FREHYWOT, G. L., TOWNSEND, C. A., RONNETT, G. V., LANE, M. D. and KUHAJDA, F. P. (2000) Science, 288, 2379±81. LUYCKX, F. H., LEFEBVRE, P. J. and SCHEEN, A. J. (2000) Diabetes Metab, 26, 98±106. MARIASH, C. N., SEELIG, S., SCHWARTZ, H. L. and OPPENHEIMER, J. H. (1986) J Biol Chem, 261, 9583±6. MCGARRY, J. D. and BROWN, N. F. (1997) Eur J Biochem, 244, 1±14. MEUNIER-DURMORT, C., POIRIER, H., NIOT, I., FOREST, C. and BESNARD, P. (1996) Biochem. J., 319, 483±7. MIKSICEK, R. J. and TOWLE, H. C. (1983) J Biol Chem, 258, 9575±9. MUNDAY, M. R. (2002) Biochem Soc Trans, 30, 1059±64.

276

Functional foods, ageing and degenerative disease

NTAMBI, J. M.

(1992) J Biol Chem, 267, 10925±30.

O'CALLAGHAN, B. L., KOO, S. H., WU, Y., FREAKE, H. C.

276, 16033±9.

PAPE, M. E., LOPEZ-CASILLAS, F.

and

TOWLE, H. C.

(2001) J Biol Chem,

and KIM, K. H. (1988) Arch Biochem Biophys, 267, 104±9. (1989) J. B iol. Chem., 264, 574±7. PRINTZ, R. L., MAGNUSON, M. A. and GRANNER, D. K. (1993) Ann. Rev. Nutr., 13, 463±96. PRIP-BUUS, K., PERDEREAU, D., FOUFELLE, F., MAURY, J., FERReÂ, P. and GIRARD, J. (1995) Eur. J. Biochem., 230, 309±15. ROBERFROID, M. P. and DELZENNE N. M. (1998) Annu. Rev Nutr., 18, 117±43. RONGNOPARUT, P., VERDON, C. P., GEHNRICH, S. C. and SUL, H. S. (1991) J Biol Chem, 266, 8086±91. RUDERMAN, N. B., SAHA, A. K., VAVVAS, D. and WITTERS, L. A. (1999) Am J Physiol, 276, E1± E18. RUFO, C., TERAN-GARCIA, M., NAKAMURA, M. T., KOO, S. H., TOWLE, H. C. and CLARKE, S. D. (2001) J Biol Chem, 276, 21969±75. SAGGERSON, E. D. and GREENBAUM, A. L. (1970) Biochem J, 119, 221±42. SHENG, Z., OTANI, H., BROWN, M. S. and GOLDSTEIN, J. L. (1995) Proc. Natl. Acad. Sci. USA, 92, 935±8. SHIH, H. M. and TOWLE, H. C. (1992) J Biol Chem, 267, 13222±8. SHIH, H. M. and TOWLE, H. C. (1994) J Biol Chem, 269, 9380±7. SHIH, H. M., LIU, Z. and TOWLE, H. C. (1995) J Biol Chem, 270, 21991±7. SHIMOMURA, I., SHIMANO, H., HORTON, J. D., GOLDSTEIN, J. L. and BROWN, M. (1997) J. Clin. Invest., 99, 838±45. SHIMOMURA, I., BASHMAKOV, Y., IKEMOTO, S., HORTON, J. D., BROWN, M. S. and GOLDSTEIN, J. L. (1999) Proc Natl Acad Sci USA, 96, 13656±61. SILER, S. Q., NEESE, R. A. and HELLERSTEIN, M. K. (1999) Am J Clin Nutr, 70, 928±36. SIMPSON, I. A. and CUSHMAN, S. W. (1986) Annu Rev Biochem, 55, 1059±89. TAYLOR, C. B., BAILEY, E. and BARTLEY, W. (1967) Biochem J, 105, 717±22. THOMPSON, K. S. and TOWLE, H. C. (1991) J Biol Chem, 266, 8679±82. THORENS, B. (1996) Am J Physiol, 270, G451±3. TOMAS, E., TSAO, T.-S., SAHA, A. K., MURREY, H. E., ZHANG, C. C., ITANI, S. I., LODISH, H. F. and RUDERMAN, N. B. (2002) Proc Natl Acad Sci USA, 99, 16309±13. TONTONOZ, P., HU, E., GRAVES, R., BUDAVARI, A. and SPIEGELMAN, B. (1994) Genes Dev., 8, 1224±34. TONTONOZ, P., HU, E., DEVINE, J., BEALE, E. and SPIEGELMAN, B. (1995) Mol. Cell. Biol., 15, 351±7. TOWLE, H. C., KAYTOR, E. N. and SHIH, H. M. (1997) Annu. Rev. Nutr., 17, 405±33. TSUJIKAWA, M. and KIMURA, S. (1980) J. Nutr. Sci. Vitaminol., 26, 367±74. VAN SCHAFTINGEN, E., DETHEUX, M. and VEIGA DA CUNHA, M. (1994) Faseb J, 8, 414±9. VAULONT, S., MUNNICH, A., DECAUX, J. F. and KAHN, A. (1986) J Biol Chem, 261, 7621±5. VAULONT, S., VASSEUR-COGNET, M. and KAHN, A. (2000) J Biol Chem, 275, 31555±8. VOLPE, J. J. and VAGELOS, P. R. (1973) Annu Rev Biochem, 42, 21±60. WANG, X., SATO, R., BROWN, M. S., HUA, X. and GOLDSTEIN, J. L. (1994) Cell, 77, 53±62. WATERS, K. M. and NTAMBI, J. M. (1994) J Biol Chem, 269, 27773±7. WEBER, A., MARIE, J., COTTREAU, D., SIMON, M. P., BESMOND, C., DREYFUS, J. C. and KAHN, A. (1984) J Biol Chem, 259, 1798±802. XU, J., NAKAMURA, M. T., CHO, H. P. and CLARKE, S. D. (1999) J Biol Chem, 274, 23577±83. XU, J., TERAN-GARCIA, M., PARK, J. H., NAKAMURA, M. T. and CLARKE, S. D. (2001) J Biol Chem, 276, 9800±7. PAULAUSKIS, J. D.

and

SUL, H. S.

Nutrition, fat synthesis and obesity

277

YAMASHITA, H., TAKENOSHITA, M., SAKURAI, M., BRUICK, R. K., HENZEL, W. J., SHILLINGLAW, W., ARNOT, D.

and

UYEDA, K.

(2001) Proc Natl Acad Sci USA, 98, 9116±21.

YAMAUCHI, T., KAMON, J., MINOKOSHI, Y., ITO, Y., WAKI, H., UCHIDA, S., YAMASHITA, S., NODA, M., KITA, S., UEKI, K., ETO, K., AKANUMA, Y., FROGUEL, P., FOUFELLE, F., FERRE, P., CARLING, D., KIMURA, S., NAGAI, R., KAHN, B. B.

1288±95.

and KADOWAKI, T. (2002) Nat Med, 8,

YANG, T., ESPENSHADE, P. J., WRIGHT, M. E., YABE, D., GONG, Y., AEBERSOLD, R., GOLDSTEIN, J. L.

and

BROWN, M. S.

(2002) Cell, 110, 489±500.

ZHU, Q., MARIASH, A., MARGOSIAN, M. R., GOPINATH, S., FAREED, M. T., ANDERSON, G. W. MARIASH, C. N.

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(2001) Endocrinology, 142, 4363±70.

Abbreviations

ACC: acetyl-CoA carboxylase AMPK: AMP-activated protein kinase ATP-CL: ATP citrate lyase ChoRE: carbohydrate response element ChREBP: carbohydrate response element binding protein CPTI: carnitine palmitoyl transferase I FAS: fatty acid synthase F1P: fructose-1-phosphate GK: glucokinase GKRP: glucokinase regulatory protein GLUT2: glucose transporter 2 GLUT4: glucose transporter 4 G-6-PDH: glucose-6-P dehydrogenase ICV: intracerebroventricular L-PK: liver pyruvate kinase MDH: malate dehydrogenase NASH: non-alcoholic steatohepatitis OAA: oxaloacetate PDH: pyruvate dehydrogenase 6-PG-DH: 6-phosphogluconate dehydrogenase PEP: phosphoenolpyruvate P: phosphate 6-PFK: 6-phosphofructo-1-kinase PKA: protein kinase A PPAR: peroxisome proliferator activated receptor PPAR-RE: peroxisome proliferator activated receptor response element PUFA: polyunsaturated fatty acids SREBP: sterol regulatory element binding protein VLDL: very low density lipoprotein.

and

12 Satiety and the control of obesity W. A. M. Blom, A. Stafleu and C. de Graaf, TNO Nutrition and Food Research, The Netherlands

12.1

Introduction: satiety and obesity

Overweight and obesity are growing health problems worldwide. Currently, 30± 50% of Europeans are overweight and 15±30% suffer from obesity. For the USA, these figures are even higher. The principal causes of the epidemic of overweight and obesity are sedentary lifestyles and high-fat, energy-dense diets, resulting in increased energy intake and decreased energy expenditure. Increasing physical activity helps to increase energy expenditure. Lowering food intake will lead to a decrease in energy intake. Energy intake is predominately determined by two processes: satiety and satiation. We start eating when we get hungry (= absence of satiety) and stop when we feel full (satiation). Therefore there is an urgent need for food products that help to lower or maintain body weight. Substances that speed up the process of satiation (feeling full) and/or induce longer-term feelings of satiety (absence of hunger) may help to control weight. Development of suitable biomarkers is very important for efficacy and safety studies of newly developed foods or ingredients aimed at weight management.

12.2

Factors influencing satiety and satiation

Although there has been much research, the mechanism of the regulation of food intake in humans has not been fully elucidated yet. One reason for this is that an important part of this regulation takes place at the level of the central nervous system. Animal research has unravelled part of this central regulation which takes place mainly in the hypothalamus. However, the regulation of food intake in humans can be different from animals. The problem is, however, that these

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central mechanisms are hard if not almost impossible to investigate in humans. With the rapid development of brain imaging techniques during the past decade non-invasive methods of measuring brain function in response to various stimuli can be investigated. The two most important techniques used to study appetite are Positron Emission Tomography (PET) and functional Magnetic Resonance Imaging (fMRI) (Berns, 1999; Raichle, 1998). The applicability of these techniques is limited, they require special expensive technology and can measure activity only of specific brain areas. Another reason why the mechanisms of the regulation of food intake have not been completely unravelled is that many factors important for the regulation of food intake are still unknown. Though the regulation of food intake has been studied quite intensively, new factors involved in this regulation are still being found (e.g. leptin and ghrelin). The reason for this is that a lot of those important regulators are present in very low concentrations. The development of new, more powerful techniques has led and will lead to the discovery of many of those unknown factors. Lastly, humans not only eat in response to a metabolic need for nutrients, but also in response to non-physiological factors, which are hard to control in a research setting. 12.2.1 Non-physiological factors influencing food intake Although food intake in humans is regulated by physiological (internal) signals, external factors modulate physiologically derived hunger and satiety signals. Non-physiological factors such as hedonic (palatability, taste, texture, odour), social (culture, religion), psychological (preferences, aversions, emotions, dieting behaviour), environmental (temperature, time of day, other people), economical (cost, availability) and pharmacological (anorectants) factors influence food intake. The extent to which different individuals respond to these various factors may vary markedly. This may explain some discrepancies among human food intake studies, and some reports of high between-subject variability. 12.2.2 Physiological factors influencing food intake The regulation of food intake is a complex interaction between numerous signals acting both peripherally and centrally. Three phases can be distinguished; a preprandial, prandial and a postprandial (pre-absorptive and post-absorptive) phase. However, the regulation of food intake can also be divided into two different phases; satiation (meal termination) and satiety (absence of satiety leads to meal initiation). Roughly speaking, factors important during the prandial phase are involved in satiation, and factors important during the postprandial phase are involved in meal initiation though in practice this distinction is less clear. Satiation (meal termination) During the pre-prandial phase, visual, olfactory, gustatory and tactile input stimulate processes at multiple sites (i.e. salivary glands, gastro-intestinal tract,

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pancreas, cardiovascular and renal systems) resulting in a cascade of physiological processes termed cephalic phase response, occurring within seconds to minutes after exposure to foods (Halford and Blundell, 2000; Mattes, 1997). The taste and smell of foods stimulate, for example, gastrin and gastric acid release (Mattes, 1997). The cephalic phase responses improve or optimize the efficiency of the digestion, absorption and use of nutrients (Halford and Blundell, 2000; Mattes, 1997). During the prandial phase the central nervous system (CNS) receives sensory afferent input reflecting the amount of food eaten and initial estimations of its nutrient content (Halford and Blundell, 2000). Mechano-receptors in the stomach detect the distension of the stomach caused by the presence of food. This helps to estimate the volume of food consumed (Halford and Blundell, 2000). Chemo-receptors in the gastro-intestinal tract detect the chemical presence of nutrients, and provide information on the composition of the foods consumed (Halford and Blundell, 2000). Peripheral satiety factors like cholecystokinine (CCK) and glucagon-like peptide 1 (GLP-1) are released in response to the physical and chemical presence of food in the gastro-intestinal tract. CCK, is a hormone released in the duodenum in response to the consumption of fat (i.e. long chain fatty acids) or protein (i.e. amino acids). GLP-1 is a hormone released in the blood by mucosal cells of the gut in response to the presence of carbohydrates and fat (MacIntosh et al., 2001). CCK and GLP-1 suppress appetite by decreasing gastric emptying through affecting the pyloric pressure, stomach motility and stomach muscle relaxation. By decreasing stomach emptying, the stomach distension increases, leading to sensations of fullness (Cecil et al., 1998; Geliebter et al., 1988; Melton et al., 1992; Rolls et al., 1998). GLP-1 stimulates the islet B-cells in the pancreas to secrete insulin, thereby lowering blood glucose levels in response to carbohydrate consumption. The effect of nutrients on satiety and satiation depends on the position of the nutrients in the digestive tract. The presence of physiological amounts of nutrients in the intestine provides a weak stimulus for the regulation of appetite. The same physiological amount of nutrients in the stomach leads to an increased suppression of appetite. Fullness is directly correlated with gastric content, and hunger and desire to eat are inversely correlated (Cecil et al., 1998). Oral ingestion of a physiological amount of nutrients leads to the greatest suppression of appetite. Orosensory stimulation (taste and smell perception) enhances the appetite suppressing effects produced by gastric distension, probably partly caused by slower gastric emptying (Cecil et al., 1998). Satiety (meal initiation) Because of its central role in the regulation of energy metabolism, the role of glucose in meal initiation has been extensively investigated. Though absolute concentrations of glucose do not seem to be very important in the regulation of food intake (Chapman, 1998; Gielkens et al., 1998), transient and dynamic declines in blood glucose concentration seem to be strongly related with meal

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initiation (Campfield and Smith, 1990; Kovacs et al., 2002; Melanson et al., 1999a,b,c). Also intraduodenal glucose influences appetite, possibly through glucoreceptors or osmoreceptors in the intestine, which may induce satiety through direct vagal stimulation or via the release of insulin and/or incretin hormones like GLP-1 (Lavin et al., 1996). Unlike glucose, studies examining exogenous as well as studies investigating endogenous insulin give mixed results concerning the role of insulin in the regulation of food intake (Campfield et al., 1996; Chapman, 1998; Holt et al., 1992; Speechly and Buffenstein, 2000; Verdich et al., 2001). Ghrelin is a recently discovered hormone which is abundantly synthesized in the fundus of the human stomach (Ariyasu et al., 2001), and is suggested to be involved in meal initiation. Before each meal, ghrelin concentrations rise whereas plasma ghrelin decreases to baseline levels in between meals (Cummings et al., 2001). Moreover, an intravenous infusion of ghrelin in humans has been shown potently to increase food intake and enhance appetite by approximately 28% (Wren et al., 2001). In response to oral and intravenous administration of glucose, plasma ghrelin concentrations decrease. Intake of an equivalent volume of water does not influence ghrelin concentrations (Shiiya et al., 2002), suggesting that secretion of ghrelin is not affected by stomach expansion. The data on ghrelin so far are very exciting, however, it having been discovered only a few years ago; it is not yet known which other factors influence ghrelin secretion. PYY, which is also a gut hormone, is postprandially released in response to medium and long chains fatty acids but not after sucrose polyester (Maas et al., 1998). PYY suppresses 24-hour food intake in humans (Batterham et al., 2002) and is correlated with measures of appetite (MacIntosh et al., 1999). Long-term regulation of food intake The hormone leptin is synthesized mainly by adipose tissue. Plasma leptin concentrations correlate positively with total body fat stores (Saad et al., 1998; Sinha et al., 1996). An energy deficit of more than 24 hours leads to decreases of plasma leptin concentration (Boden et al., 1996; Chin-Chance et al., 2000; Heini et al., 1998; Keim et al., 1998; Weigle et al., 1997; Wisse et al., 1999), whereas an energy surplus of more than 24 hours results in increased leptin concentrations (Chin-Chance et al., 2000; Kolaczynski et al., 1996). Plasma leptin is negatively correlated with appetite and food intake when the energy balance is distorted (Chin-Chance et al., 2000; Heini et al., 1998; Keim et al., 1998). When subjects are in energy balance, the relation between leptin concentrations and food intake and appetite is less clear (Joannic et al., 1998; Karhunen et al., 1997a; Romon et al., 1999; Cummings et al., 2001; Dallongeville et al., 1998; English et al., 2002; Sinha et al., 1996). Therefore, leptin seems to have a role in the regulation of food intake when energy stores are depleted or increased, rather then during energy balance.

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12.3

The impact of different food components on satiety

In the past twenty years numerous studies have been carried out to investigate the impact of different food components on satiety. Research has been done on three levels: 1. 2. 3.

Effects of food components on subjective ratings of hunger and satiety. Effects of food components on energy intake. Effects of food components on body weight and long-term energy balance.

Much work has been done in short-term preload ÿ! test meal studies, following a paradigm developed twenty years ago by Kissileff (Kissileff et al., 1984). In this paradigm subjects ingest preloads that differ in one particular property of food, whereas other properties are held constant, e.g., the fat content of a certain food is varied, while an attempt is made to hold the other properties (e.g., weight, volume, taste) constant. After the preloads, subjects record their feelings of hunger and satiety, and/or get a test meal from which they can eat ad libitum. The degree to which a particular property suppresses subsequent energy intake, and/or ratings of hunger and satiety is then a measure of the satiating efficiency (Kissileff et al., 1984). Many of the longer-term studies use a somewhat different approach to investigate the impact of properties of food on satiety. In many of these studies, subjects are offered diets that differ in one or more properties, from which they eat ad libitum for a certain amount of time. For example, in one study on the longer-term effect of the fat content of the diet on energy intake and energy balance, one group of subjects was offered a diet with foods with a regular fat content, and another group of subjects was provided with the same foods but then with a reduced fat content (de Graaf et al., 1997). The time span of these studies varies from a few days to a few years, with many more short-term studies than longer-term studies. The effects of food components on satiety work through their effects on gastrointestinal hormones and neural activity in the central nervous system. Most of the identified hormones are anorexins (CCK, GLP-1, leptin, PYY), whereas recently the orixen ghrelin has been identified. The effects of the various food components on satiety are described below. 12.3.1 Macronutrient content The results of a number of studies suggest that the order of satiating efficiency of macronutrients is protein > carbohydrate > fat > alcohol, although this conclusion was not reinforced in one very recent study (Raben et al., 2003). The weak effect of fat on satiety is well documented. In many preload ÿ! test meal studies that covertly manipulated the fat content of foods, subjects do not respond to higher fat levels in preloads with subsequent lower hunger ratings and/or lower food and energy intakes. This is a consistent finding across studies with various foods (e.g. foods with fat replacers, regular foods with high/low fat levels) (de Graaf et al., 1996), and with different groups of subjects (Rolls et al., 1994).

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The low satiating efficiency of fat is confirmed in short- and long-term studies on the ad libitum energy intake and energy balance from diets with various levels of fat. These studies show that ad libitum energy intake is lower on low-fat diets than with high-fat diets (e.g. Kendall et al., 1991; Lissner et al., 1987). In a recent review paper, Astrup et al. (2002) summarized the data from 13 clinical trials on low-fat diets and concluded that `the evidence strongly supports the low-fat diets as the optimal choice for the prevention of weight gain and obesity'. The results of a number of studies suggest that per calorie protein is the most satiating macronutrient (Raben et al., 2003). This idea was confirmed with a long-term weight trial of Haulrik et al. (2002), in which it was shown that a high-protein diet led to a lower body weight. The results of some studies suggest that carbohydrates are more satiating than fats (Rolls and Hammer, 1995). In general, high carbohydrate/low-fat diets lead to a lower energy intake than high-fat/low-carbohydrate diets. However, it is not clear whether this effect is related to the lower energy density of carbohydrates compared to fat. The effect of alcohol on satiety is difficult to investigate because of its strong behavioural effects. Results of studies by de Castro indicated that the energy intakes from meals with alcohol are on average higher than the energy intakes from meals without alcohol (de Castro and Orozco, 1990). 12.3.2 Fibre Fibre contributes to post-ingestive satiety. This result is clear from many studies (Delargy et al., 1997; Holt et al., 1995; Marlett et al., 2002). The mechanisms through which it operates are less clear. This could be through slower gastric emptying, increasing bulk, and/or increased transient time and nutrient exposure in the gut. The fibre content of foods also contributes to a lower glycaemic index, which may facilitate the control of food intake (Jenkins et al., 2002; Rizkalla et al., 2002). 12.3.3 Weight and energy density The weight and energy density of foods play a crucial role with respect to the impact of food components on satiety. From a large number of short-term studies it is clear that humans primarily regulate their food intake on the basis of the weight of foods, and not the energy content (Poppitt and Prentice, 1996). For example, when one gives subjects ad libitum access to foods (e.g. yoghurts) with varying energy densities (e.g. by manipulating the fat content), subjects will generally ingest equal weights of the different foods. The energy intake is then positively related to the energy density. The constant weight intake can be conceived as a learned response, based on the association between the sensory properties of foods and their post-ingestive hunger and satiety consequences. For example, after a number of exposures we learn that we need to eat a certain amount of certain foods for breakfast (e.g. two sandwiches with cheese) in order

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to stay satiated until lunch. This learning must be based on the association between the sensory properties of bread and cheese, and the associated postingestive consequences. The idea that we gradually learn this association between sensory properties and post-ingestive consequences, explains why in many short-term studies, subjects do not respond very sensitively to covert manipulations of the energy content of foods (Stubbs et al., 2000). These learned associations enable us to know how much to eat from various foods, e.g., for breakfast or other meals. Regulation on the basis of weight may also explain the weak satiating efficiency of fat that is found in many studies. Foods/diets with a high fat content generally have a high energy density, and consequently a low satiating efficiency. 12.3.4 Physical state/solid ! liquid Solid foods have a larger effect on satiety than liquid foods with an equivalent composition (DiMeglio and Mattes, 2000; Hulshof et al., 1993; Mattes and Rothacker, 2001). Some studies found that sugar-containing drinks have little impact on satiety, implying that the energy content of these drinks adds to energy intake from other foods (Raben et al., 2002; Tordoff and Alleva, 1990).

12.4

Developing biomarkers of satiety

12.4.1 The need for biomarkers of satiety At present, there are no validated biomarkers of satiety and satiation. Information on satiation and satiety can be assessed only by means of subjective introspection, by measuring the intervals between spontaneous requests for meals (satiety) or by measuring the energy intake from the meal (satiation). There is a need for more objective measures (biomarkers) of satiety and satiation, for example, for efficacy testing of bioactive functional food ingredients. Biomarkers give more insight into the processes and mechanisms involved in satiety than subjective reports therefore biomarkers are more suitable for supporting claims, for which a plausible mechanism is needed. Another advantage of objective measures of satiety is that the number of subjects needed in an efficacy study could be reduced because in general there is less variation in blood parameters compared to subjective reports. 12.4.2 Developing new biomarkers Definition of biomarkers Biomarkers of satiety and satiation could be defined as physiological measures that relate to subjectively rated appetite and/or actual food intake. Markers can be either indicators of appetite, or they can be proved to be causal factors of appetite (Diplock et al., 1999).

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What makes a parameter a good biomarker? Biomarkers should be (Diplock et al., 1999): · relatively immediate outcomes to enable interventions on a reasonable timescale · validated and of high quality · clearly linked to the phenomenon (in this case satiety or satiation) rather than accurately measured · sensitive and specific and reproduced by many centres · measurable in easily accessible biological materials (like urine and blood), according to ethical standards and minimal invasive procedures · dynamic responses or static measurements. Biomarkers in blood Recently developed techniques and acquired knowledge on the regulation of blood parameters known to be involved in signalling satiety and satiation, such as cholecystokinin, glucose, insulin, leptin, GLP-1 and others, enable the measurement of physiological correlates of satiation and satiety. In addition to these `classic' parameters, new techniques can be used to find biomarkers of satiety. Nuclear Magnetic Resonance spectroscopy (NMR) combined with pattern recognition is a promising technique to identify potential biomarkers in blood and urine. With NMR techniques, a broad range of compounds with different physico-chemical properties can be detected simultaneously. Sophisticated statistical software is needed to explore patterns in NMR data. From these patterns it is possible to nominate potential biomarkers. Fractionation of the samples and subsequent NMR liquid chromatographic and mass-spectrometric analysis on the fractions will elucidate the structure of the biomarkers. Also proteomics, metabolomics and transcriptomics are promising techniques that can be used for identifying biomarkers of satiety (Werf et al., 2001). Transcriptomics and proteomics can be employed to determine changes in gene expression and proteome relevant to the state of hunger or satiety. Central biomarkers There is limited knowledge of how the brain contributes to the regulation of food intake in humans. After eating, the human brain senses a biochemical change and then signals satiation, but when this occurs precisely is unknown. With respect to CNS biomarkers of satiety and satiation, there have been a number of recent studies in literature using PET (Positron Emission Tomography)-scan techniques and f-MRI (functional-Magnetic Resonance Imaging). These studies have identified various regions in the brain that can distinguish between fasted state and after food ingestion, for example, Liu et al. (2000). Some studies suggest that the responses of the brain to a meal differ between obese and lean persons (Gautier et al., 2000; Karhunen et al., 1997b; Matsuda et al., 1999).

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12.5 Future trends: using biomarkers to assess weightcontrol foods Many components of foods have an effect on satiety and satiation, as they have an effect on energy metabolism and on hormones related to hunger and satiety. For example, protein-rich and solid foods have a higher satiating effect, whereas high-fat and liquid foods have a low satiating efficiency. This knowledge can be used to develop new foods with a higher satiating efficiency. Another issue is the identification of new components/substances that have an effect on satiety and satiation. New developments in molecular biology, pharmacology and nutrigenomics enhance our insight in the complex pathways involved in energy balance. From these insights new substances will become available that will affect mechanisms involved in hunger and satiety. New substances with a plausible mechanism should first be tested in animal studies with respect to toxicological aspects, and their potential to influence short- and long-term energy balance. This may be followed by short-term safety and efficacy tests in humans. In the end substances may be used in more longterm trials with humans. Both short- and long-term studies would benefit from the inclusion of relevant biomarkers of satiety. Several reviews (Allison et al., 2001; Egger et al., 1999) have been published on the effect of supplements on weight loss, like chrome, conjugated linoleic acid (CLA), hydroxycitric acid (HCA), chitosan and Ma Huang (ephedra). In general there is no good evidence for the effectiveness of the substances reviewed. Apart from influence on feelings of hunger and satiety, two other potential mechanisms are involved in products aimed at weight loss or weight maintenance: 1. 2.

Reduction of energy intake. Examples are substances purported to block the absorption of fat; fat replacers, like Olestra (sucrosepolyester); lowcalorie products, like light products and meal replacers. Increasing energy expenditure. Substances with an effect on fat burning, changes in basal metabolism and thermogenesis increase energy expenditure.

For the design of new products that influence feelings of hunger and satiety it is necessary to identify food components with a satiating effect. Products could either speed up satiation (so that one stops eating sooner) or induce long-term satiety (so that one does not feel hungry for a long time after eating the product). These could be products or substances with an effect on noradrenalin and serotonin, like St John's Wort and capsaicin or products or substances that influence stomach filling, like fibre, resistant starch, pectin. To evaluate whether new or existing weight management products are effective in influencing satiety and satiation, biomarkers of both satiety and satiation should be used in controlled human intervention studies. Upon intervention, the marker should change in a statistically significant as well as a physiologically relevant way.

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Sources of further information and advice

Halford J, Blundell J (2000), `Pharmacology of appetite suppression', Prog Drug Res, 5425±58. When new to the subject, this review article is recommended as starting literature, because it describes the different phases and domains of the expression of appetite in a clear, structured manner. The most important peripheral and central satiety factors are also described and discussed. Westerterp-Plantenga MS, Steffens A, Tremblay (1999), `Regulation of food intake and energy expenditure', Milan, EDRA Medical Publishing & New Media. This reviews the variability in food intake and energy expenditure in humans, as well as in animals. A series of integrated topics like the complex integration of hormonal, neuronal, physiological, and metabolic controls, including the way the reward value of taste is determined in the brain are discussed. Also the social, cultural and psychological factors and the integration of food intake regulation with energy expenditure are dealt with.

12.7

References and HEYMSFIELD SB (2001), `Alternative treatments for weight loss: a critical review', Crit Rev Food Sci Nutr , 41 (1), 1±28.

ALLISON DB, FONTAINE KR, HESHKA S, MENTORE JL

ARIYASU H, TAKAYA K, TAGAMI T, OGAWA Y, HOSODA K, AKAMIZU T, SUDA M, KOH T, NATSUI K, TOYOOKA S, SHIRAKAMI G, USUI T, SHIMATSU A, DOI K, HOSODA H, KOJIMA M,

KANGAWA K and NAKAO K (2001), `Stomach is a major source of circulating ghrelin, and feeding state determines plasma ghrelin-like immunoreactivity levels in humans', J Clin Endocrinol Metab, 86 (10), 4753±4758. ASTRUP A, ASTRUP A, BUEMANN B, FLINT A and RABEN A (2002), `Low-fat diets and energy balance: how does the evidence stand in 2002?', Proceedings of the Nutrition Society, 61 (2), 299±309.

BATTERHAM RL, COWLEY MA, SMALL CJ, HERZOG H, COHEN MA, DAKIN CL, WREN AM, BRYNES

AE, LOW MJ, GHATEI MA, CONE RD and BLOOM SR (2002), `Gut hormone PYY(3±36) physiologically inhibits food intake', Nature, 418 (6898), 650±654. BERNS GS (1999), `Functional neuroimaging', Life Sciences, 65 (24), 2531±2540. BODEN G, CHEN X, MOZZOLI M and RYAN I (1996), `Effect of fasting on serum leptin in normal human subjects', J Clin Endocrinol Metab, 81 (9), 3419±3423. CAMPFIELD LA and SMITH FJ (1990), `Transient declines in blood glucose signal meal initiation', International Journal of Obesity, 14 Suppl 3 15±31. CAMPFIELD LA, SMITH FJ, ROSENBAUM M and HIRSCH J (1996), `Human eating: evidence for a physiological basis using a modified paradigm', Neurosci-Biobehav-Rev, 20 (1), 133±137. CECIL J, FRANCIS J and READ N (1998), `Relative contributions of intestinal, gastric, orosensory influences and information to changes in appetite induced by the same liquid meal', Appetite, 31 (3), 377±390. CHAPMAN IM (1998), `Effect of intravenous glucose and euglycemic insulin infusions on short-term appetite and food intake', American Journal of Physiology, 274 (43), R596±R603. CHIN-CHANCE C, POLONSKY KS and SCHOELLER DA (2000), `Twenty-four-hour leptin levels

288

Functional foods, ageing and degenerative disease

respond to cumulative short-term energy imbalance and predict subsequent intake', J Clin Endocrinol Metab, 85 (8), 2685±2691. CUMMINGS DE, PURNELL JQ, FRAYO RS, SCHMIDOVA K, WISSE BE and WEIGLE DS (2001), `A preprandial rise in plasma ghrelin levels suggests a role in meal initiation in humans', Diabetes, 50 (8), 1714±1719. DALLONGEVILLE J, HECQUET B, LEBEL P, EDME JL, LE FUR C, FRUCHART JC, AUWERX J and ROMON M (1998), `Short term response of circulating leptin to feeding and fasting in man: influence of circadian cycle', Int J Obes Relat Metab Disord, 22 (8), 728±733. DE CASTRO JM and OROZCO S (1990), `Moderate alcohol intake and spontaneous eating patterns of humans: evidence of unregulated supplementation', American Journal of Clinical Nutrition, 52 (2), 246±253. DE GRAAF C, HULSHOF T, WESTSTRATE JA and HAUTVAST JG (1996), `Nonabsorbable fat (sucrose polyester) and the regulation of energy intake and body weight', Am J Physiol, 270 (6 Pt 2), R1386±R1393. DE GRAAF C, DRIJVERS JJ, ZIMMERMANNS NJ, VAN HET HK, WESTSTRATE JA, VAN DEN BH,

and (1997), `Energy and fat compensation during longterm consumption of reduced fat products', Appetite, 29 (3), 305±323. DELARGY HJ, O'SULLIVAN KR, FLETCHER RJ and BLUNDELL JE (1997), `Effects of amount and type of dietary fibre (soluble and insoluble) on short-term control of appetite', International Journal of Food Sciences and Nutrition, 48 (1), 67±77. DIMEGLIO DP and MATTES RD (2000), `Liquid versus solid carbohydrate: effects on food intake and body weight', Int J Obes Relat Metab Disord, 24 (6), 794±800. DIPLOCK AT, AGGETT PJ, ASHWELL M, BORNET F, FERN EB and ROBERTFROID MB (1999), `Scientific concepts of functional foods in Europe: Consensus Document', Br. J. Nutr, 81 S1±S27. EGGER G, CAMERON-SMITH D and STANTON R (1999), `The effectiveness of popular, nonprescription weight loss supplements', Med J Aust, 171 (11±12), 604±608. ENGLISH PJ, GHATEI MA, MALIK IA, BLOOM SR and WILDING JP (2002), `Food fails to suppress ghrelin levels in obese humans', J Clin Endocrinol Metab, 87 (6), 2984. VELTHUIS-TE WIERIK EJ, WESTERTERP KR, VERBOEKET-VAN DE VENNE WP

WESTERTERP-PLANTENGA MS

GAUTIER JF, CHEN K, SALBE AD, BANDY D, PRATLEY RE, HEIMAN M, RAVUSSIN E, REIMAN EM

and TATARANNI PA (2000), `Differential brain responses to satiation in obese and lean men', Diabetes, 49 (5), 838±846. GELIEBTER A, WESTREICH S and GAGE D (1988), ` Gastric distention by balloon and testmeal intake in obese and lean subjects', American Journal of Clinical Nutrition, 48 (3), 592±594. GIELKENS HA, VERKIJK M, LAM WF, LAMERS CB and MASCLEE AA (1998), `Effects of hyperglycemia and hyperinsulinemia on satiety in humans', Metabolism, 47 (3), 321±324. HALFORD J and BLUNDELL J (2000), `Pharmacology of appetite suppression', Prog. Drug Res 5425±5458. HAULRIK N, TOUBRO S, DYERBERG J, STENDER S, SKOV AR and ASTRUP A (2002), `Effect of protein and methionine intakes on plasma homocysteine concentrations: a 6±mo randomized controlled trial in overweight subjects', American Journal of Clinical Nutrition, 76 (6), 1202±1206. HEINI AF, LARA-CASTRO C, KIRK KA, CONSIDINE RV, CARO JF and WEINSIER RL (1998), `Association of leptin and hunger-satiety ratings in obese women', Int J Obes Relat Metab Disord, 22 (11), 1084±1087.

Satiety and the control of obesity

289

and HANSKY J (1992), `Relationship of satiety to postprandial glycaemic, insulin and cholecystokinin responses', Appetite, 18 (2), 129±141. HOLT SH, MILLER JC, PETOCZ P and FARMAKALIDIS E (1995), `A satiety index of common foods', European Journal of Clinical Nutrition, 49 (9), 675±690. HULSHOF T, DE GRAAF C and WESTSTRATE JA (1993), `The effects of preloads varying in physical state and fat content on satiety and energy intake', Appetite, 21 (3), 273± 286. HOLT S, BRAND J, SOVENY C

JENKINS DJ, KENDALL CW, AUGUSTIN LS, FRANCESCHI S, HAMIDI M, MARCHIE A, JENKINS AL

and AXELSEN M (2002), `Glycemic index: overview of implications in health and disease', American Journal of Clinical Nutrition, 76 (1), 266S±273S. JOANNIC JL, OPPERT JM, LAHLOU N, BASDEVANT A, AUBOIRON S, RAISON J, BORNET F and GUYGRAND B (1998), `Plasma leptin and hunger ratings in healthy humans', Appetite, 30 (2), 129±138. KARHUNEN L, HAFFNER S, LAPPALAINEN R, TURPEINEN A, MIETTINEN H and UUSITUPA M (1997a), `Serum leptin and short-term regulation of eating in obese women', Clin Sci (Lond), 92 (6), 573±578. KARHUNEN LJ, LAPPALAINEN RI, VANNINEN EJ, KUIKKA JT and UUSITUPA MI (1997b), `Regional cerebral blood flow during food exposure in obese and normal-weight women', Brain, 120 (Pt 9) 1675±1684. KEIM N, STERN J and HAVEL P (1998), `Relation between circulating leptin concentrations and appetite during a prolonged, moderate energy deficit in women', Am. J. Clin. Nutr., 68 (4), 794±801. KENDALL A, LEVITSKY DA, STRUPP BJ and LISSNER L (1991), `Weight loss on a low-fat diet: consequence of the imprecision of the control of food intake in humans', American Journal of Clinical Nutrition, 53 (5), 1124±1129. KISSILEFF HR, GRUSS LP, THORNTON J and JORDAN HA (1984), `The satiating efficiency of foods', Physiol Behav, 32 (2), 319±332. KOLACZYNSKI JW, OHANNESIAN JP, CONSIDINE RV, MARCO CC and CARO JF (1996), `Response of leptin to short-term and prolonged overfeeding in humans', J Clin Endocrinol Metab, 81 (11), 4162±4165. KOVACS EM, WESTERTERP-PLANTENGA MS, SARIS WH, MELANSON KJ, GOOSSENS I, GEURTEN P

and BROUNS F (2002), `Associations between spontaneous meal initiations and blood glucose dynamics in overweight men in negative energy balance', British Journal of Nutrition, 87 (1), 39±45. LAVIN JH, WITTERT G, SUN WM, HOROWITZ M, MORLEY JE and READ NW (1996), `Appetite regulation by carbohydrate: role of blood glucose and gastrointestinal hormones', Am J Physiol, 271 (2 Pt 1), E209±E214. LISSNER L, LEVITSKY DA, STRUPP BJ, KALKWARF HJ and ROE DA (1987), `Dietary fat and the regulation of energy intake in human subjects', American Journal of Clinical Nutrition, 46 (6), 886±892. LIU Y, GAO J, LIU H and FOX P (2000), `The temporal response of the brain after eating revealed by functional MRI', Nature, 405 (6790), 1058±1062. MAAS MI, HOPMAN WP, KATAN MB and JANSEN JB (1998), `Release of peptide YY and inhibition of gastric acid secretion by long-chain and medium-chain triglycerides but not by sucrose polyester in men', Eur J Clin Invest, 28 (2), 123±130. MACINTOSH CG, ANDREWS JM, JONES KL, WISHART JM, MORRIS HA, JANSEN JB, MORLEY JE,

and CHAPMAN IM (1999), `Effects of age on concentrations of plasma cholecystokinin, glucagon-like peptide 1, and peptide YY and their relation to appetite and pyloric motility', American Journal of Clinical Nutrition, 69 (5), 999±1006.

HOROWITZ M

290

Functional foods, ageing and degenerative disease

MACINTOSH CG, HOROWITZ M, VERHAGEN MA, SMOUT AJ, WISHART J, MORRIS H, GOBLE E, MORLEY JE and CHAPMAN IM (2001), `Effect of small intestinal nutrient infusion on appetite, gastrointestinal hormone release, and gastric myoelectrical activity in young and older men', American Journal of Gastroenterology, 96 (4), 997±1007. MARLETT JA, MCBURNEY MI and SLAVIN JL (2002), `Position of the American Dietetic Association: health implications of dietary fiber', Journal of the American Dietetic Association, 102 (7), 993±1000. MATSUDA M, LIU Y, MAHANKALI S, PU Y, MAHANKALI A, WANG J, DEFRONZO RA, FOX PT and GAO JH (1999), `Altered hypothalamic function in response to glucose ingestion in obese humans', Diabetes, 48 (9), 1801±1806. MATTES RD (1997), `Physiologic responses to sensory stimulation by food: nutritional implications', Journal of the American Dietetic Association, 97 (4), 406±413. MATTES RD and ROTHACKER D (2001), `Beverage viscosity is inversely related to postprandial hunger in humans', Physiol Behav, 74 (4±5), 551±557. MELANSON K, WESTERTERP PM, SARIS W, SMITH F and CAMPFIELD L (1999a), `Blood glucose patterns and appetite in time-blinded humans: carbohydrate versus fat', Am. J. Physiol, 277 (2 Pt 2), R337±R345. MELANSON KJ, WESTERTERP-PLANTENGA MS, CAMPFIELD LA and SARIS WH (1999b), `Appetite and blood glucose profiles in humans after glycogen-depleting exercise', J Appl Physiol, 87 (3), 947±954. MELANSON KJ, WESTERTERP-PLANTENGA MS, CAMPFIELD LA and SARIS WH (1999c), `Blood glucose and meal patterns in time-blinded males, after aspartame, carbohydrate, and fat consumption, in relation to sweetness perception', British Journal of Nutrition, 82 (6), 437±446. MELTON PM, KISSILEFF HR and PI-SUNYER FX (1992), `Cholecystokinin (CCK-8) affects gastric pressure and ratings of hunger and fullness in women', Am J Physiol, 263 (2 Pt 2), R452±R456. POPPITT SD and PRENTICE AM (1996), `Energy density and its role in the control of food intake: evidence from metabolic and community studies', Appetite, 26 (2), 153±174. RABEN A, VASILARAS TH, MOLLER AC and ASTRUP A (2002), `Sucrose compared with artificial sweeteners: different effects on ad libitum food intake and body weight after 10 wk of supplementation in overweight subjects', American Journal of Clinical Nutrition, 76 (4), 721±729. RABEN A, AGERHOLM-LARSEN L, FLINT A, HOLST JJ and ASTRUP A (2003), `Meals with similar energy densities but rich in protein, fat, carbohydrate, or alcohol have different effects on energy expenditure and substrate metabolism but not on appetite and energy intake', American Journal of Clinical Nutrition, 77 (1), 91±100. RAICHLE ME (1998), `Behind the scenes of functional brain imaging: a historical and physiological perspective', Proc Natl Acad Sci USA, 95 (3), 765±772. RIZKALLA SW, BELLISLE F and SLAMA G (2002), ` Health benefits of low glycaemic index foods, such as pulses, in diabetic patients and healthy individuals', British Journal of Nutrition, 88 Suppl 3 S255±S262. ROLLS BJ and HAMMER VA (1995), `Fat, carbohydrate and the regulation of energy intake', The-American-journal-of-clinical-nutrition (USA), 62 (5S), 1086±1095. ROLLS BJ, KIM-HARRIS S, FISCHMAN MW, FOLTIN RW, MORAN TH and STONER SA (1994), `Satiety after preloads with different amounts of fat and carbohydrate: implications for obesity', American Journal of Clinical Nutrition, 60 (4), 476±487. ROLLS B, CASTELLANOS V, HALFORD J, KILARA A, PANYAM D, PELKMAN C, SMITH G and THORWART M (1998), `Volume of food consumed affects satiety in men', Am. J.

Satiety and the control of obesity

291

Clin. Nutr, 67 (6), 1170±1177.

and DALLONGEVILLE J (1999), `Leptin response to carbohydrate or fat meal and association with subsequent satiety and energy intake', Am J Physiol, 277 (5 Pt 1), E855±E861.

ROMON M, LEBEL P, VELLY C, MARECAUX N, FRUCHART JC

SAAD MF, RIAD-GABRIEL MG, KHAN A, SHARMA A, MICHAEL R, JINAGOUDA SD, BOYADJIAN R

and STEIL GM (1998), `Diurnal and ultradian rhythmicity of plasma leptin: effects of gender and adiposity', J Clin Endocrinol Metab, 83 (2), 453±459. SHIIYA T, NAKAZATO M, MIZUTA M, DATE Y, MONDAL MS, TANAKA M, NOZOE S, HOSODA H,

and MATSUKURA S (2002), `Plasma ghrelin levels in lean and obese humans and the effect of glucose on ghrelin secretion', J Clin Endocrinol Metab, 87 (1), 240±244. SINHA M, OHANNESIAN J, HEIMAN M, KRIAUCIUNAS A, STEPHENS T, MAGOSIN S, MARCO C and CARO J (1996), `Nocturnal rise of leptin in lean, obese, and non-insulin-dependent diabetes mellitus subjects', J-Clin-Invest, 97 (5), 1344±1347. SPEECHLY DP and BUFFENSTEIN R (2000), `Appetite dysfunction in obese males: evidence for role of hyperinsulinaemia in passive overconsumption with a high fat diet', European Journal of Clinical Nutrition, 54 (3), 225±233. STUBBS J, FERRES S and HORGAN G (2000), `Energy density of foods: effects on energy intake', Crit Rev Food Sci Nutr, 40 (6), 481±515. TORDOFF MG and ALLEVA AM (1990), `Effect of drinking soda sweetened with aspartame or high-fructose corn syrup on food intake and body weight', American Journal of Clinical Nutrition, 51 (6), 963±969. VERDICH C, TOUBRO S, BUEMANN B, LYSGARD MJ, JUUL HJ and ASTRUP A (2001), `The role of postprandial releases of insulin and incretin hormones in meal-induced satiety ± effect of obesity and weight reduction', Int J Obes Relat Metab Disord, 25 (8), 1206±1214. WEIGLE DS, DUELL PB, CONNOR WE, STEINER RA, SOULES MR and KUIJPER JL (1997), `Effect of fasting, refeeding, and dietary fat restriction on plasma leptin levels', J Clin Endocrinol Metab, 82 (2), 561±565. WERF MJ, SCHUREN FHJ, BIJLSMA S, TAS AC and OMMEN BV (2001), `Nutrigenomics: Application of Genomics Technologies in Nutritional Sciences and Food Technology', Journal of Food Science, 66 (6), 772±780. WISSE BE, CAMPFIELD LA, MARLISS EB, MORAIS JA, TENENBAUM R and GOUGEON R (1999), `Effect of prolonged moderate and severe energy restriction and refeeding on plasma leptin concentrations in obese women', American Journal of Clinical Nutrition, 70 (3), 321±330. KANGAWA K

WREN AM, SEAL LJ, COHEN MA, BRYNES AE, FROST GS, MURPHY KG, DHILLO WS, GHATEI MA

and BLOOM SR (2001), `Ghrelin enhances appetite and increases food intake in humans', J Clin Endocrinol Metab, 86 (12), 5992.

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Part III Gut health and immune function

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13 Functional foods for gut health: an overview R. Tahvonen and S. Salminen, University of Turku, Finland

13.1

Introduction: the human gut

The human gut is defined either as the lower part of the alimentary canal ± the intestinal tract, or the entire gastrointestinal tract: mouth, oesophagus, stomach, small intestine (duodenum, jejunum, ileum), large intestine and rectum (Fig. 13.1). The gastrointestinal tract (GI) is designed to comminute and digest food, to absorb and secrete nutrients and other compounds, including toxic compounds, and to excrete wastes. There is a persistent chemical and immunological challenge and transient mechanical challenge. To ensure normal function the gut with its microbiota forms the largest sensory, endocrine and immunological organ of the body. Epithelial cells and cells of the enteric nervous system (ENS) `taste' or `sense' the contents of the lumen to regulate digestion and absorption. The ENS is also connected to the central nervous system. Communication works in both directions and is partly mediated via gut hormones. Epithelial cells secrete hormones in varying compositions and concentrations that are specific to each meal. Despite regulation of digestion and absorption, gut hormones also regulate many other physiological processes in the body. The gut immune system consists of enterocytes, specialised immune cells in the epithelium, cells aggregated in follicles and the Peyer's paths and diffuse lymphosytes populations existing in mucosa lamina propria. The immune system secretes immunoglobulins as a first-line defence and evolves specific immune protection. Oral tolerance, hyporesponsiveness to dietary antigens and gut microbiota result from purposeful immune regulation. Several hundred microbial species form the gut microbiota, most of which are strict anaerobes. The gut microbiota provide energy from carbohydrates not digested in the upper

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Fig. 13.1

The human gut.

gut, produces vitamins and regulates the immune system. The total gut area, the gut wall, the ENS, gut hormone secreting cells, the gut immune system and the gut microbiota, interact through crosstalk influencing each other and the health of the host.

13.2

The structure of the gut and its immune system

13.2.1 Gut wall, epithelium and mucus The basic structure of the gut wall is, from inside to outside: mucosa, submucosa, muscularis propria and serosa (Fig. 13.2). The epithelium is lined with a protective mucus layer. The neurons form clusters (ganglia) which lie in the plexus of muscularis propria, and in submucous plexus. Mucosa has its own muscle layer able to move the villi, and the longitudinal and circular muscles of muscularis propria participate in comminution and peristaltis. The gut epithelium consists of enterocytes, sensory cells of neural system and specialised cells of the immune system. Enterocytes line the intestines as a monolayer. They secrete digestive enzymes and absorb nutrients in controlled

Functional foods for gut health: an overview

Fig. 13.2

297

The basic structure of the gut wall.

and selective ways. As enterocytes join each other with tight junctions they form a physical barrier between the luminal contents of the intestine and the human body. Enterocytes are renewed about once a week and factors contributing to their regeneration include growth factors and local hormones (Pitman and Blumberg, 2000; Ray et al., 2002). The intestinal epithelium area is about 200 square metres due to crypt and villi structure. The structure of the epithelium is highly specialised in different gut areas. The epithelial cells protect themselves by secreting mucins, viscous glykoprotein polymers which form a protecting and lubricating gel between the luminal contents and the epithelium. As all nutrients must be transported through this gel prior to absorption, the mucus also acts as a modulating barrier (Deplancke and Gaskins, 2001). In the stomach, the mucus layer maintains a pH gradient and prevents the effect of acid and pepsin on the epithelium by chanelling the secretes to the lumen (Atuma et al., 2001). The composition of mucins varies depending on site in the gut, age, diet, gut microbiota, and the genetic background of the subject. The mucus gel layer is thickest in the stomach and thinner in the small intestine to ease absorption processes and thickens again gradually from the ascending colon. The layer is continuous in the stomach and colon, but in the small intestine for example Peyer's patches are not covered thereby enabling their normal antigen sampling function. Goblet cells secrete mucins by a baseline model (simple exocytosis) and a compound exocytosis (accelerated release of centrally stored mucin granules). Compound exocytosis is induced by several factors like hormones, neuropeptides, and inflammatory mediators (Deplancke and Gaskins, 2001). Atuma et al. (2001) have found two layers of mucus in rat intestines: a loosely adherent layer, which can be removed by suction, and a layer that is

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firmly attached to the mucosa. They suggest that the sloppy gel layer might be a more lubricative layer, and the firmly adherent gel acts as a relatively stable protective barrier. Two different gels have also been found in the human stomach (Ota and Katsuyama, 1992) and colon. The ability of mucus to facilitate Bifidobacterium adhesion seems to be significantly reduced during ageing (Ouwehand et al., 1999). 13.2.2 Gut hormones Gut hormones are secreted after chemical, mechanical or neural signals throughout the whole digestive tract beginning from the mouth. Several peptide hormones induce secretion both of hydrochloric acid in the stomach, and digestive enzymes in the stomach, intestine and pancreas, secretion of bicarbonate and contraction of the gallbladder thus ensuring effective digestion and absorption. Gut hormones also stimulate the secretion of mucus, modulate gastric emptying rate and bowel movements. More than 30 peptide hormone genes are known to be expressed throughout the digestive tract and these genes produce more than 100 different hormonally active peptides. Five important characteristics of gut hormones are: 1. 2.

3. 4. 5.

The structural homology groups the hormones into families (a common ancestral gene). The individual hormone gene is often expressed in multiple bioactive peptides due to tandem genes encoding different hormonal peptides, alternative splicing of the primary transcript, or differentiated processing of the primary translation product. Similar peptide hormone genes are widely expressed also outside the gut. The different cell types often express different products of the same gene, `cell-specific expression'. Gastrointestinal hormone producing cells release the peptides in different ways, classic endocrine, neurocrine, paracrine, autocrine and spermiocrine. Thus the same peptide may act as an acute circulating hormone, as a local growth factor, and as a fertility factor.

As many gut hormones and their receptors are specifically expressed in several tissues, they may secondarily also have effects elsewhere in the body (Rehfeld, 1998). An interesting example of gut hormones is the new group of gut peptides expressed also in the central nervous system, orexins. These increase food consumption, wakefulness, locomotory activity (Willie et al., 2001), excite secretomotor neurons in the gut area and affect immunoreactive enteric neurons (Kirchgessner, 2002). Many gut hormones have trophic effect on epithelium growth and renewation (NaÈslund et al., 2001; Burrin et al., 2001; Drucker, 2002; Lovshin and Drucker, 2000; Alavi et al., 2000; Attele et al., 2002; Barrenetxe et al., 2002; Liddle, 2000). Glucagon-like-peptide-2 (GLP-2) may have an especially important role in gut barrier function (Burrin et al., 2001).

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Several gut hormonal activities still require identification of new hormones. Some of the structurally unidentified hormonal activities can be partly explained by activity of later identified hormones; for instance, incretin activity is partly due to gastric inhibitory polypeptide (GIP) and glucagons-like peptide I (GLP-I) or entero- vago-, and bulbogastrone effects may be explained by various combinations of somatostatin, GIP, EGF, and TGF- (Rehfeld, 1998). The potency or acting time of a hormone effect can also be varied by producing different peptides (chain length, other processing) having a similar active sequence that acts through a specific reseptor. 13.2.3 The enteric neuronal system The gut area has an impressive neuronal system consisting of 100 million neurons. The three main classes of neurons found on the gut area are: 1. 2. 3.

intrinsic primary afferent neurons (IPANs), with cell bodies and connections in the gut wall extrinsic primary afferent neurons (EPANs), cell bodies located in vagal and dorsal root intestinofugal neurons, projecting to neurons outside the gut wall but with cell bodies in the gut.

IPANs generate reflex responses to intestinal contents: mixing and propulsive movements of the muscles, local changes in blood flow, and secretion of water and electrolytes. They are involved with reflexes between gastrointestinal organs. IPANs detect chemical changes in the intestinal lumen, distension of the intestine, and mechanical distortion of the mucosa via specific receptors. They convey the information to other neurons of the enteric nervous system. These then integrate the information and result in appropriate changes in the gut functions. The overlapping populations of IPANs may contribute to motility, secretomotor and vasomotor reflexes, since similar stimuli evoke the responses. The stimuli may be direct or indirect via the release of gut hormones and peptides (Furness et al., 1999). Extrinsic primary afferent neurons (EPANs) mediate information about the status of the gastrointestinal tract to the central nervous system. The information may be directly perceived, like sensations of gastric or intestinal fulness, pain sensations or a sensation of warmth. The feeling of satiety is indirect, not specific to a particular organ. The perceptions are modulated psychosensorily, and most of the information is not consciously perceived. Low- and highthreshold mechanosensitive neurons have been defined, the latter ones being responsible for extreme or pathological stimuli. Again the stimuli may be direct or mediated by hormones and peptides. The information carried by EPANs can be interpreted consciously as satiety, pain, hunger, and nausea. Additionally, the information is used to automatically regulate the functions of organs via sympathetic motor pathways (Furness et al., 1999). The axons of intestinofugal neurons make excitatory, cholinergic synapses with the cell bodies of sympathetic neurons that project back to the gut. They

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mediate the reflexes between proximal and distal parts of the gut, feed-back responses to nutrients and reflexes passed from the small intestine through the central nervous system and returning via vagal motoneurons (Furness et al., 1999). 13.2.4 The specific features of the gut immune system The antigens of the gut are derived from the diet and the resident (commensal) microbiota. The gut immune system must be able to avoid unnecessary and potentially harmful reactions to dietary proteins and enteric microbiota (oral tolerance), but at the same time rapidly respond to episodic threats from pathogens. The antigens must be sampled continuously, innocuous and danger signals discriminated effectively, and effector responses regulated tightly (Shanahan, 2000; Mowat, 2003). The gut immune system consists of enterocytes, which have been shown to have several immunological functions discussed shortly below, and specialised immune cells of the gut-associated lymphatic tissue, GALT, and immune cells in lamina propria. Innate immunity, the first line of defence, is a rapid response to fight against infection before the slower development of adaptive, specific, memoryexhibiting immunity. Innate immunity inludes macrophages, natural killer cells, defensins, and the complement cascade, which all exist within the gut mucosa. They recognise micro-organisms by pattern-recognition molecules or receptors. Specially activated macrophages express multiple immunomodulatory genes including cytokines and costimulatory molecules required for antigen presentation and generation of adaptive immunity by T and B cells. Thus, in addition to early defence, the innate immune response also intitiates the adaptive immune response. Specific intestinal epithelial lymphocytes, T cells, seem to coordinate interplay between innate and adaptive immunity (Shanahan, 2000). Enterocytes as a part of the immune system Enterocytes form a barrier that offers only a very limited passage of material in both directions. Cell-cell adhesion is ensured by tight junctions, desmosomes and adherens junctions. Epithelial goblet cells secrete mucins, which act as another physical barrier. Epithelial cells also secrete several microbicidal or antiviral agents and transfer immunoglobulins secreted from mucosal plasma cells to the lumen. Enterocytes can, upon certain stimuli, secrete large quatities of Clÿ -rich fluid that flushes the surface. Many intermediate and end products of the digestion process are also microbicidal (fatty acids, lysophospholipids, etc.), and digestive enzymes can digest, at least partly, microbe cell walls (Pitman and Blumberg, 2000). Enterocytes communicate with regional immune cell populations and influence their growth, migration, and state of responsiveness to antigenic stimuli. They express surface factors, secrete chemokines, cytokines and prostanoids. By expressing cytokine receptors, enterocytes are also able to alter their own innate capacity for defence and their ability to interact with and

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influence local immune cell populations (Pitman and Blumberg, 2000). Enterocytes sample bacterial and other foreign antigens within the lumen and present them to cells of the mucosal immune system thus directing immune responses to potentially harmful antigens. Enterocytes have been shown to possess maybe several processing and presentation pathways for antigens. The solubility of antigens seems to be a key factor. Enterocytes preferentially take up soluble antigens, but specialised epithelial immune cells, M-cells, take up most particulate antigens (Shao et al., 2001). Byrne et al. (2002) suggest that healthy human duodenal epithelial cells process and present antigens, but they may induce anergy, rather than activation, of local T-cells. Enterocytes may also enhance the level of antigen presentation by other cells and maintain the intestinal mucosa immune responsiveness (Pitman and Blumberg, 2000). Apparently, under normal circumstances, the presentation of antigens by intestinal epithelial cells leads to inactivation or suppression of the normal immune response (oral tolerance). The intestinal epithelial cell may regulate the mucosal immune system by controlling the transit of antigens (Shao et al., 2001). Other gut lymphoid and myeloid cells The organised GALT consists of isolated or aggregated lymphoid follicles (Peyer's patches), smaller isolated lymphatic nodules and mesenteric lymph nodes. A more diffusively arranged GALT is distributed throughout the lamina propria and epithelium, consisting of two distinct effector cell populations above and below the basement membrane: the intraepithelial lymphocytes (IEL) and the lamina propria mononuclear cells including lamina propria lymphocytes (Shanahan, 2000; Mowat, 2003). The lamina propria also contains macrophages, dendritic cells, neutrophils, and mast cells (Wittig and Zeitz, 2003). Collectively, the gut immune cells form the largest lymphoid mass in the body. Isolated lymphoid follicles/lymphatic nodules occur throughout the intestine. Their amount is highest in the distal ileum and colon, where the microbiota are abundant and diverse, and in humans the greatest frequency of follicles and follicle-associated epithelium (FAE) occurs in the rectum, cecum and appendix. In the distal ileum, lymphoid follicles form large Peyer's patches (Neutra et al., 2001). The FAE differs from the villus epithelium, containing only a few goblet and enteroendocrine cells. Most cells in FAE are specialised M-cells that sample foreign material in the lumen by active transepithelial vesicular transport directly to intraepithelial lymphoid cells and to subepithelial organised lymphoid tissue. M-cells are typical polarised epithelial cells with tight junction, but their hallmark is the presence of a large intraepithelial `pocket' providing a docking site for a specific subpopulation of intraepithelial lymphocytes (IELs: B and T lymphocytes, and occasionally dendritic cells). M-cells and the underlying immune cells are crucial for the induction of protective mucosal immune responses. FAE may also play a role in down-regulation of immune responses (oral tolerance). The Peyer's patches can thus function as efficient immune inductive sites when pathogens enter the gut but undesirable T cell responses for

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antigens from food and commensal microbes are dampened (Neutra et al., 2001). The lamina propria contains a high proportion of activated and memory Tcells that allow rapid immune response against pathogens (Wittig and Zeitz, 2003). B-cells in mucosa and in the lamina propria secrete immunoglobulins for the first line of defence. Their role, activation, and class-switch recombination process has been reviewed recently (Fagarasan and Honjo, 2003). Different sets of dendritic cells in the gut wall have several functions in innate and adaptive immunity. They are important antigen-presenting cells, they may also be sample antigens from the gut lumen, shape the intestinal immune response, and activate immunoglobulin-class switching to IgA production (Mowat, 2003). The formation and location of organised GALT seem to be genetically determined. However, in response to the normal microbiota or to microbial challenge, mucosal lymphoid follicles can also form after birth and in adult life. Chemokines secreted by the epithelial cells may have a crucial role in the induction of new lymphoid follicles (Neutra et al., 2001). Oral tolerance Oral tolerance is a state of systemic specific immunologic hyporesponsiveness to the specific innocuous antigen (food, commensal microbiota) challenge. It is an actively regulated state that is maintained by multiple and probably nonmutually exclusive immunoregulatory mechanisms. The site of antigen sampling and especially the nature of the local antigen-presenting cells are critical to the development of oral tolerance or immunity to intestinal antigens. Oral tolerance does not prevent efficient immune functions against harmful antigens (Shanahan, 2000; Garside and Mowat, 2001). The gut prevents excessive inflammation and tissue injury by several mechanisms (Garside and Mowat, 2001; Monteleone et al., 2002). The mechanisms have been recently reviewed (Isolauri et al., 2002; Wittig and Zeitz, 2003). The role of dendritic cells in T cell tolerance seem to be important. Steinman et al. (2003) discuss the possibility that distinct developmental stages and subsets of dendritic cells and T cells can account for the different pathways to peripheral tolerance, such as deletion or suppression. 13.2.5 The gut microbiota and health (host-microbe crosstalk) The generation of immunophysiological regulation in the gut depends on the establishment of indigenous microbiota. The microbiota of a newborn baby develops rapidly after birth and it is initially strongly dependent on the mother's microbiota, mode of birth and birth environment, and subsequently influenced by feeding practices and the environment of the child. Most microbiota succession studies have been based on culture method studies. Recent molecular studies have indicated that the microbiota in infants develops rapidly during the first week and remains unstable for the first year of life. We know that lactic acid bacteria account for <1% of the total microbiota in infants but

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bifidobacteria can range from 60 to 90% of the total faecal microbiota in breastfed infants (Vaughan et al., 2002; Favier et al., 2002). Pioneer bacteria modulate expression of genes in host epithelial cells, create a favourable habitat for themselves, and can prevent growth of other bacteria introduced later in the ecosystem (Hooper et al., 2001). The initial microbiota may be relevant to the final microbiota in adults. The composition of bifidobacteria microbiota in infants was first clarified by Benno and Mitsuoka (1986). Usually bifidobacteria appear after birth and within a week they were reported as the dominant bacterial group with Bifidobacterium breve and Bifidobacterium bifidum as the most common species present in healthy infants. Comparing breast-fed and formula-fed infants the greatest differences appear to be in lactic acid bacteria colonisation and species of bifidobacteria present. In breast-fed infants Lactobacillus gasseri and Bifidobacterium breve were the most common species present in culture method studies (Matsuki et al., 1999). Similar results have been reported with more detailed information on distribution or bifidobacterial species using 16S rRNA primers and modern taxonomy (Satokari et al., 2001). The species composition is now known to stabilise in adulthood, but it has been reported to decrease and the numbers of several species decrease in the gastrointestinal tract of the elderly. Bifidobacterial species and numbers are especially reduced during ageing (Benno and Mitsuoka, 1986; He et al., 2001). A decrease in anaerobes and bifidobacteria population and an increase in enterobacteria, together with the reduced intestinal immunity of the aged may favour gastrointestinal infections (HeÂbuterne, 2003). The complex microbial community differs in composition both along the length and across the diameter of the gut. The amount of species and microbes increase from the stomach to the colon and consist both of rapidly transiting and relatively persistent components. The gastrointestinal tract provides distinct physiological environments varying from the acidic stomach to the alkaline pH in the small bowel and the microbiota is subjected to the influence of bile juices, pancreatic secretions and the active mucosal immune system (Dunne, 2001). Pancreatic enzymes, epithelial mucin secretion and the sloughing-off of epithelia further modify the microbiota (Van der Meer and Bovee-Oudenhoven, 1998). The adult human gut contains 1±2 kg of microbes, which are metabolically very active. Only part of these microbes have been identified, but more than 500 species are already known. The most common genera are Bacteroides, Eubacterium, Bifidobacterium, Peptostreptococcus and Fusobacterium (Neish, 2002). The species and their dominance vary greatly between individuals. The composition of the microbiota may fluctuate during acute diarrhoeal illnesses, antibiotic treatments and to a lesser extent during dietary interventions, but usually it remains constant (Moore and Moore, 1995). The key physiological roles of gut microbes are: 1.

establishment of a barrier to protect the organism from undesirable microbial invasion

304 2. 3. 4. 5.

Functional foods, ageing and degenerative disease help in the establishment of effective gut-associated lymphoid tissue help in the absorption of vitamins and minerals fermentation and recovery of energy from nutrients which are partly or totally resistant to digestion in the upper gastrointestinal tract production of acetate, probionate and butyrate, the short chain fatty acids that play key roles as modulators of cell turnover and cell differentation in the colonic mucosa and which also act as regulators of the metabolism in systemic tissues.

Gut microbes also synthesise amino acids, and some of these, especially the amino acids synthesised in the small intestine, may be absorbed. The nutritional role of this synthesis is so far unclear (Fuller and Reeds, 1998). Further, the human enteric microbiota provides for the metabolism of bile acids, bilirubin, and cholesterol (Neish, 2002). Gut microbes may also produce harmful compounds, NH3 from proteins and urea (Clinton, 1992) and phenols and cresols from amino acids (Macfarlane and Macfarlane, 1995), N-nitroso compounds (Rowland, 1996), diacylglycerols, and secondary bile acids. Many of these compounds are potentially harmful and even cancer promoting (Salminen et al., 1998). Also the microbiota composition in the colon may have cancer-promoting effects (Moore and Moore, 1995). Commensal gut microbes are essential to the normal development of the intestinal humoral and cellular immune system. They activate both the innate and adaptive functions especially by increasing circulating specific and natural antimicrobial antibodies and maintain the normal physiological inflammation state, oral tolerance, throughout life (Cebra, 1999). Processing of antigens by proteases and competitive exclusion of pathogens belongs to the protective mechanisms of commensal microbes. The strains bifidobacteria and lactobacilli especially seem to have beneficial health-promoting properties (Isolauri et al., 2002). Commensal microbes try to circumvent the immune response, for instance, by changing their surface antigenity and thus reshape and keep the immune system active (Guarner and Malagelada, 2003). Gut microbes are able to modulate the expression of several host genes. They affect intestinal functions like nutrient absorption, mucosal barrier fortification, xenobiotic metabolism, angiogenesis, and postnatal intestinal maturition. A commensal microbe, Bacteroides thetaiotaomicron has been shown to regulate production of ileal glycans for its own nutritional benefit. It also may affect the components of the enteric nervous system and motility, and angiogenesis. The interactions between resident micro-organisms and their hosts seem to be essential (Hooper et al., 2001; Guarner and Malagelada, 2003). A variety of potentially harmful micro-organisms are members of the normal gut microbiota. They may be autochthonous within the intestinal niche, held in check by the normal microbiota and become pathogenic only when the ecosystem undergoes abnormal changes. Alternatively, particular microbes may be autochthonous within a specific gut area, but allochthonous within another (Dunne, 2001). Many gut microbes are able to produce bacteriocins, which may

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be important in localised niches (Guarner and Malagelada, 2003). Stress can affect gut microbiota. With probiotics a transient change in balance may be achieved but long-term effects of probiotics can be expected only during infancy.

13.3

Nutrients and gut function

Gastrointestinal epithelial cells especially, but also other cells like M-cells, enteroendocrine cells, intraepithelial lymphocytes and the multiple cell types of lamina propria, are all affected by intra- and extraluminal nutrient intake. Many nutrients play an important role in the maintenance of normal mucosal function in the gut area (Duggan et al., 2002). There are also age-related changes which could influence nutrient utilisation and especially microbiota on the mucosa. Thus, the intestinal microbiota becomes less complex during ageing and the metabolic role of the intestinal microbiota is altered. This may result in poorer utilisation of nutrients and reduced detoxification of harmful components in the gut as well as increased risk for intestinal infections (Benno and Mitsuoka, 1986; Hopkins et al., 2001; HeÂbuterne, 2003). 13.3.1 Proteins and amino acids The gut utilises a large proportion of the total protein intake and is responsible for the main part of the first-pass metabolism of dietary amino acids. The gut uses also arterial essential amino acids, but so far very little is known about the nature of the amino acid transporters on the basolateral membrane of the enterocytes. Intestinal tissues, especially small intestinal mucosa synthesises proteins at a high rate. Most of these proteins are secretory proteins, which are at least partly recycled to the body. The gut utilises glutamine, aspartate, glycine and glutamate also for the biosynthesis of nucleotides, glutathione, polyamines, and non-essential amino acids and for energy. The gut regulates actively the flow of amino acids from the diet to the body, perhaps by systemic hormones or by gut regulatory peptides. The gut seems to remove the amino acids necessary for its own integrity and physiologic function (Reeds and Burrin, 2000). Glutamine is an important fuel source for cells rapidly turning over, like epithelial cells, lymphocytes, fibroblasts and reticulocytes. It is a non-essential amino acid under normal conditions. Several in vitro and in vivo studies show an important role for glutamine in the maintenance and repair of gut mucosa in critically ill patients and it may be a conditionally essential nutrient. More studies are, however, needed on its effects on intestinal permeability, bacterial translocation, and small-bowel histology (Duggan et al., 2002). Glutamate is another amino acid playing an important role as an enterocyte energy source but also elsewhere in intestinal physiology and metabolism. Ziegler et al. (2003) give a long list of the mechanisms of glutamate action in

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intestinal growth, repair, and function, including stimulation of cell proliferation and epithelial cell migration, enhanced growth factor signalling, upregulation of mucosal cell glutathione production and heat-shock proteins and effects on the immune system. Arginine is also considered to be a non-essential amino acid, but like glutamine it may be essential during catabolic states such as trauma and sepsis. Arginine is a precursor for nitric oxide NO). NO may have both anti- and proinflammatory effects and excessive NO production has been found in endotoxemia, septic shock, and increased intestinal vascular permeability. Arginine-containing formulas seem to reduce infectious complications in some surgical patients. The benefits to other patient groups need further studies (Duggan et al., 2002; Ziegler et al., 2003). Dietary or luminal glycine and histidine may have protective effects on gastrointestinal tissues (Ziegler et al., 2003) and studies on their usefulness in diarrhoeal illness are currently being undertaken. 13.3.2 Vitamins and minerals Vitamin A is important for epithelial cell integrity and immune function. In animal experiments vitamin A deficiency leads to reduced epithelial cell renewal and when paired to other inflammatory or infectious conditions results in significant histologic abnormalities. Infants and children with mild vitamin A deficiency are at increased risk of diarrhoea, and vitamin A supplementation seems to decrease mortality. However, it has not been proved whether vitamin A is protective also when its intake is adequate and in other population groups (Duggan et al., 2002). Zinc is needed in protein synthesis and transcription proteins and it is thus important to cells with a high rate of turnover. Zinc supplementation has been shown to be beneficial in the treatment of diarrhoea in children whose zinc intake may be marginal and/or phytate intake high. Clinical studies are going on to identify other patient populations whose dietary zinc intakes are higher than expected (Duggan et al., 2002). Deficiencies of several specific nutrients inhibit the growth and turnover of the intestinal mucosa (Ziegler et al., 2003). However, more clinical studies are needed to prove the usefulness of supplementation with doses higher than the present recommendations. 13.3.3 Dietary fibre Dietary fibres promote beneficial physiological effects, including laxation (faecal bulking and softening, increased frequency), and regulate bowel movements (The Dietary Fiber Definition Committee, 2001). Dietary fibre consists of several different groups of molecules with different effects. Insoluble fibre especially contributes to laxation and normal bowel function, but fermentable/ soluble fibre promotes faecal bulking through fermentation and microbial growth (Topping and Clifton, 2001).

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Gut microbes ferment part of the fibre to short chain fatty acids (SCFA). The concentration of SCFA produced vary depending on substrates, commensal microbes and site of fermentation. Assessing fermentation and SCFA production in humans is so far complicated. Anyway, SCFA seem to be beneficial to gut health via several actions. Colonocytes absorb and metabolise SCFA, which are major respiratory fuels and trophic to the small bowel and colon (Topping and Clifton, 2001). A part of starch also escapes digestion and thus belongs to dietary fibre. According to Topping and Clifton (2001) there is so far no physiologically relevant measure of resistant starch (RS) for foods as they are eaten. There are inhibitory components in food, and many physiological variables like chewing, transit, and microbiota. The more starch is eaten, the more enters the colon, and about 10% of total dietary starch seems to escape digestion in the human small intestine. Even RS types differ in fermentation speed and SCFA profiles. On the other hand, some data suggest that rates of SCFA production and SCFA profiles may even be characteristic to individuals and not influenced by diet. More information on the specific effects of dietary fibre may be found in Chapter 22. 13.3.4 Other bioactive compounds Plant phenolic compounds have been considered nutritionally important only recently. Two large groups, flavonoids and phytoestrogens especially, have raised interest. Flavonoids are effective antioxidants due to their phenolic hydroxyl groups but they have plenty of other beneficial effects like antiinflammatory and anticarcinogenic activities. Gut microbes metabolise phytoestrogens to biologically active mammalian compounds with weak estrogen activity. Like flavonoids, phytoestrogens are also antioxidants and have anticarcinogenic and antimicrobial properties (Puupponen-PimiaÈ et al., 2002). Thus the microbiota may have profound health effects, especially on the mucosa. The gastrointestinal glutathione system is an antitoxic barrier of the mucosa but appears also to be important for gut barrier function. Ziegler et al. (2003) suggest that administration of glutathione, specific dietary substrates for glutathione synthesis and certain growth factors may be a potential strategy to improve gut mucosal redox status. Several growth factors affect intestinal growth and repair (Howarth and Shoubridge, 2001; Playford et al., 2000) and enterocyte nutrient transport (Ray et al., 2002). Some of these growth factors are secreted in colostrum and milk (Playford et al., 2000), but also nutrients like glutamine, dipeptides, glucose, fructose and PUFA may act as growth factors by themselves or induce growth factor production in the gut (Ray et al., 2002). The importance of breast milk growth factors on epithelial growth has been recently reviewed by Cummins and Thompson (2002).

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13.3.5 Nutrition and gastrointestinal hormones and nerves GLP-2 is trophic for gut wall cells. Its secretion is stimulated by enteral nutrient intake, especially carbohydrates and lipids (Xiao et al., 1999; Thomsen et al., 1999). Endocrine stimulation by glucose-dependent-insulinotrophic peptide (GIP) and neural reflexes involving gastrin-releasing peptide (GRP) participate in nutrient stimulated secretion of GLP-2 (Rocca and Brubaker, 1999) demonstrating interactions between hormones and nerves. Since SCFA stimulate ileal proglucagon expression and GLP-2 secretion (Tappenden and McBurney, 1998; Tappenden et al., 1998; Thulesen et al., 1999), also fermentable dietary fibre and specific gut microbes may enhance GLP-2 secretion. SCFA themselves have trophic effects on colonocytes and seem to promote a normal phenotype in colonocytes (Topping and Clifton, 2001). Another trophic factor, peptide YY, is released by glutamine and free fatty acids (Ray et al., 2002). Since high-energy and high-fat diets suggest environmental risk factors of colon cancer and induce large release of peptide YY, the proliferative action of peptide YY might also be the link between diet and colon cancer risk (Mannon, 2002). CCK is also known to stimulate growth and motility in the gut in addition to its other effects on the gut area. Protein and fat are the primary macronutrients that stimulate the release of CCK. Increasing the amount of fat increases the height and the length of CCK response. Non-digestible polysaccharides prolong the time that CCK is significantly elevated above baseline (Schneeman, 2002). Caseinomacropeptide can also release CCK and other gut peptides (Blundell and NaÈslund, 1999).

13.4

Nutrients and the gut immune system

13.4.1 Long chain polyunsaturated fatty acids (LCPUFA) In their recent review, Anderson and Fritsche (2002) point out that in animal studies n-3 fatty acids have both improved and impaired host resistance to a number of pathogens. Kelley (2001) found discrepancies also in human studies, but points out that fish oils have been used successfully in the management of several inflammatory and autoimmune diseases. Donnet-Hughes et al. (2001) have reviewed the effects of both dietary n-6 and n-3 polyunsaturated fatty acids and concluded that the efficacy of dietary PUFA will depend on several factors, and maybe especially the antioxidant status of the host's gastrointestinal tract. Recently, Kew et al. (2003a) showed that the fatty acid composition of peripheral blood mononuclear cells significantly affects the variation of some immune cell functions, but they also found that modest doses of n-3 fatty acids do not impair innate or acquired immune response and early host defence (Kew et al., 2003b). According to Donnet-Hughes et al. (2001), eicosanoids derived from dietary PUFA affect intestinal secretion, mucus, phospholipid synthesis, and the density of surfactants in the mucus. Other possible mechanisms are the effects of tight junctions, interleukin production and antigen presentation. However, much research is needed to understand how dietary lipids affect the immune regulation

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of the gut area. Cunningham-Rundles (2003) states that membrane phospholipid composition has a major regulatory effect on immune response, which develops over time and is closely controlled by host factors rather than being a simple reflection of dietary intake. 13.4.2 Minerals and vitamins In a rat model, Ca was proved beneficial against experimental Salmonella enteritidis infection. The authors suggest that Ca can be a functional ingredient in foods designed to inhibit intestinal infections due to its cytoprotective effects in the gut and/or stimulation of the growth of protective intestinal bacteria, like lactobacilli (Van der Meer and Bovee-Oudenhoven, 1998). Several reviews report the importance of vitamins A, E and C for immune functions (Gill et al., 2001; Duggan et al., 2002; Calder and Kew, 2002), either especially in the gut area or all over the body. 13.4.3 Dietary fibre On the basis of animal studies it seems clear that dietary fibre content and type can modulate immune function. The mechanisms for the effects are not clear, but several interesting hypotheses have been proposed. Consumption of prebiotic fibres inulin and oligofructose and maybe other prebiotic fibres like lactulose and other olisaccharides, seems to increase the proportion of beneficial lactic acid bacteria in the human colon. Lactic acid bacteria further contact with the gut immune cells affecting their function, produce SCFA by fermenting the fibres and modulate mucin production (Schley and Field, 2002). The immunomodulatory effects of SCFA have been demonstrated in rat models (Schley and Field, 2002). Short chain fatty acids are energy fuel for epithelial cells thus sparing glutamine for other cells, like immune cells (Jenkins et al., 1999). As discussed earlier, glutamine is important for all rapidly increasing cells in the gut area. SCFA also seem to increase mucin production, which might contribute to the lower incidence of bacterial transport across the gut barrier (Schley and Field, 2002). SCFA at their physiological concentration stimulate mucus secretion in the rat colon via a cholinergic nerve mechanism, and thus improve the lubricating and barrier functions (Shimotoyodome et al., 2002).

13.5

Nutrition and gut health

13.5.1 Nutrition and commensal microbes The diet may affect the composition and activity of the gut microbiota. Breastfed infants have a microbiota consisting mainly of bifidobacteria, while formulafed infants have more complex microbiota similar to that of adults (Harmsen et al., 2000). During and after weaning the microbiota of both breast- and formulafed infants still develop and become more complex than during breast-feeding.

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Fermentable dietary fibre is an important source of energy for gut microbes. In the basic fermentation poly-, oligo-, and disaccharides are hydrolysed to the constituent sugars. Fermenting yields SCFA (mainly acetate, propionate and butyrate), gases (CO2, CH4 and H2) and increases biomass (Topping and Clifton, 2001). SCFA also lower pH in the intestines and is therefore believed to prevent the overgrowth of pH-sensitive pathogenic bacteria. Since PUFA are also antimicrobials, they may affect commensal microbiota. According to DonnetHughes et al. (2001) specific dietary PUFA might be used to antagonise potential pathogens and/or encourage health-promoting lactic acid bacteria. Plant phenolics (flavonoids and phytoestrogens) may selectively affect growth of intestinal microbes thus influencing the bacterial population dynamics. Plant phenolic from olives, tea, wine and berries have antimicrobial properties (Puupponen-PimiaÈ et al., 2002). The effects of phenolics on gastrointestinal microbiota are poorly understood. The gut microbes metabolise phenolic compounds, and the metabolites can affect the epithelium and the colonic microbiota. Absorbed metabolites are found in plasma and urine and may have systemic health effects (Puupponen-PimiaÈ et al., 2002). Elevated enterolactone levels in urine and in serum are thought to be related to the activity and composition of colonic microbiota. Increased use of fibre and plant foods raises serum enterolactone concentrations in animal studies and high levels have been reported in human subjects consuming large amounts of whole-grain cereals, fruit and vegetables. The use of antibiotics decreases enterolactone concentrations therefore it is likely that the intestinal microbiota has a role in enterolactone production (Kilkkinen et al., 2002). Microbiota composition in the upper intestine may have different actions on lignan precursors when compared to faecal bacteria and thus characterisation of the active composition during different phases of life is important. As constipation is positively associated with serum enterolactone levels, the longer intestinal transit time may expose the precursors to enhanced microbial metabolism. However, studies among the elderly are not available to assess this phenomenon in aged individuals. This is a matter to be clarified for assessing the health effects of both the microbiota and plant lignans among the elderly. The interest in probiotic bacteria as dietary adjuncts has focused work on their ability to convert lignans to enterolactone. The currently used commercial probiotic strains on their own appear to have little or no ability to produce enterolactone from its precursors (Lahtinen et al., 2002). The effects of other probiotics and members of the intestinal microbiota should be further clarified to find optimal compositions for enterolactone production also among the elderly. Probiotics have been defined as bacterial preparations which impart beneficial health effects on the host when consumed orally (Salminen et al., 1998). Current knowledge of probiotics shows that mechanisms of probiotic action are mutifaceted and each probiotic may have specific functions on the host. The criteria for effective probiotics were defined based on the general properties of probiotics. Today, it may be necessary to redefine these criteria and

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develop new selection criteria which would allow the selection and development of probiotics for specific functions and targets (Isolauri et al., 2002). 13.5.2 Nutrition and colon cancer Several dietary factors, like consumption of red meat, animal and saturated fat, refined carbohydrates, and alcohol, as well as total energy intake have been associated with increased risk of colorectal carcinogenesis. Possible protective factors are fibre, vegetables, fruits, antioxidant vitamins, calcium and folate (Kim, 2000). Other possible protective compounds include starch, carotenoids, the allium compounds, isoflavones, polyphenols, glucosinolates, plant sterols, etc. However, very few epidemiological and intervention studies are so far available (Van Poppel, 2002). Bresalier (1999) has reviewed the recent studies concerning the protective effects of increased calcium intake. Overall, it seems that the total composition of diet, not single components, may be protective (Negri et al., 2002). Although the results of different types of studies about the effects of dietary fibre are inconsistent, the American Gastroenterological Association (AGA) recommends total dietary fibre intake of at least 30±35 g/d, fibre from different sources. AGA stresses also the importance on lifestyle (physical activity and no smoking) and overall diet (Kim, 2000). Kim (2000) reviews the possible protective mechanisms: increased stool bulk, binding with potential carcinogens, binding with bile acids, lowering faecal pH, altering colonic microbiota, fermentation by faecal microbiota to SCFA and prevention of insulin resistance and hyperinsulinemia. Recent studies associate high fibre intake with decreased risk of distal colon adenoma (Peters et al., 2003) and low fibre intake with increased risk of colorectal cancer (Bingham et al., 2003). According to Ferguson and Harris (2003), one reason for discrepancies in earlier studies might be the relatively low fibre intake even in the highest intake groups. In epidemiological studies, intake of starch and resistant starch correlate negatively with colorectal cancer risk, maybe due to butyrate produced during fermentation (Topping and Clifton, 2001). Augenlicht et al. (2002) have shown several effects of SCFA in animal models on adenomas and adenocarcinomas in the small and large intestine and the rectum, and some fraction or type of dietary fibre and its microbial fermentation in the colon may prove significant in further studies. The colonic microbiota seem to be a major environmental factor modulating the risk of colonic cancer although conclusive evidence is still lacking (Guarner and Malagelada, 2003). In addition to different types of fibre, plant foods contain other possible protective components, like flavonoids, lignans, and anthocyanins, and maybe fibre is merely a marker for the intake of plant foods (Ferguson and Harris, 2003). Five or more servings of fruit and vegetables per day and physical activity are recommended for prevention of colon cancer (Byers et al., 2002).

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13.5.3 Nutrients and bowel diseases Resistant starch promotes colon function by alleviating infectious diarrhoea, maybe due to interaction with the microbiota (Topping and Clifton, 2001). The role of microbiota is also important therefore pro- and prebiotics may also be of use in future, when target, site and disease-specific species can be isolated. Dietary fibre alleviates the symptoms of several gut diseases like irritable bowel disease (IBD), diverticulosis and constipation (Salminen et al., 1998).

13.6

The role of functional foods in promoting gut health

According to Schneeman (2002) gut physiology and functions mediate the effects of functional foods in three different ways: 1. 2. 3.

Factors in food induce in the gut area responses, which may result in longterm adaptive changes (meal-induced responses). Foods or mixtures of food are able to alter the digestive and absorptive functions in manners influencing metabolism. The gut area, through its adaptation to diet, affects risk factors for disease. Functional foods can thus also affect overall health and well-being.

No health claims have been accepted so far for gut health in the US (Hasler, 2002). However, several applications are already used in clinical work and also marketed to consumers. Maybe the best-known group of functional foods for gut health are the probiotics (Table 13.1), which are considered to maintain or improve gut health by several mechanisms, for example: · · · ·

maintaining a barrier against colonisation by pathogenic bacteria inhibiting the growth of pathogens (maybe by producing bacteriocins) competing nutrients with the pathogens enhancing the gut immune response by contact and crosstalk with the host via the mucosa.

Different probiotics show different effects (Table 13.2). Probiotics colonise the gut temporarily and they must be consumed regularly (Calder and Key, 2002). The effects of probiotics can be enhanced by simultaneously providing prebiotics. Probiotics and prebiotics are reviewed in Chapter 17. Monteleone et al. (2002) suggest that the reason for inflammatory bowel diseases are genetic disorders that increase production of proinflammatory cytokines. These dysfunctions might be partly alleviated by suitable probiotics. Due to the heterogeneity of Crohn's disease and ulcerative colitis the patient may require subset-specific microbe strains/cocktails. Detailed knowledge about the composition and activities of the normal microbiota will help to optimise the probiotic therapy (Dunne, 2001; Shanahan, 2002). Probiotics are also suggested for treatment of gastrointestinal infections (Isolauri et al., 2002a), constipation,

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Table 13.1 The definition of probiotics ± development of the current definition (adapted from Isolauri et al., 2002) Definition

Source

Specific bacteria in yoghurt fermentation balance intestinal microbiota Substances excreted by one protozoan to stimulate the growth of another Substances that have a beneficial effect on animals by contributing to the balance of the intestinal microbiota Live microbial feed supplements which beneficially affect the host animal by improving the intestinal microbial balance Mono- or mixed cultures of live micro-organisms which when applied to man affect beneficially the host by improving the properties of the indigenous microbiota Live microbial food ingredients that are beneficial to health (efficacy and safety scientifically documented) Specific live or inactivated microbial cultures that have documented targets in reducing the risk of human disease or in their adjunct treatment

Metchnikoff (1907) Lilly and Stillwell (1965) Parker (1974) Fuller (1989) Huis in't Veld and Havenaar (1991) Salminen et al. (1998) Isolauri et al. (2002b)

irritable bowel syndrome, colorectal cancer (Salminen et al., 1998) and food allergy (Laiho et al., 2002). Another alleviating food ingredient for inflammatory bowel diseases is n-3 fatty acids. These can modulate the dysfunctions found in the immune system (Kelley, 2001; Donnet-Hughes et al., 2001; Anderson and Fritsche, 2002; Ziegler et al., 2003). For gut-related surgery and trauma patients, a mixture of arginine, glutamine, n-3 fatty acids and nucleotides has been tested and applied successfully (Alexander, 2002). However, optimising the treatment is still needed. According to Prosky (2000) dietary fibre can be considered a functional food when it imparts a special function to that food aside from the normal expected function and similarly when the dietary fibre is used as an additive to foods.

13.7

Future trends

13.7.1 Gut renewal and absorption Malabsortion of nutrients, impairment of the ability of the intestine to adapt, and immunosenescence has been described in the elderly. Enhancing absorption by dietary manipulation to prevent malnutrition may prove useful (Gill et al., 2001; Woudstra and Thomson, 2002). As the body of knowledge surrounding the molecular processes involved in the regulation of intestinal adaptation and transport grows, it should be possible to develop novel agents to improve the quality of life of patients suffering from inadequate nutrient absorption (Ray et

Table 13.2 Current probiotic bacteria and their important reported effects in human studies Strain

Reported effects in clinical studies

Selected key references and reviews

L. johnsonii LA1

Adherence to human intestinal cells, balances intestinal microbiota, immune enhancement, adjuvant in H. pylori treatment.

L. acidophilus NCFB 1748 L. casei Shirota

Lowering of faecal enzyme activity, decreasing faecal mutagenicity, prevention of radiotherapy-related diarrhoea. Prevention of intestinal disturbances, balancing intestinal bacteria, lowering faecal enzyme activities, positive effects on reducing the recurrence of superficial bladder cancer.

Salminen et al. (1998); MacFarland (2000); Fonden et al. (2000); Ouwehand et al. (2003) Fonden et al. (2000)

Ouwehand et al. (2003); Salminen et al. (1998); Aso and Akaza (1992); Aso et al. (1995); Ohashi et al. (2002) S. thermophilus; L. No effect on rotavirus diarrhoea, no immune enhancing effect during rotavirus Fonden et al. (2000); Ouwehand et bulgaricus al. (2003) diarrhoea, no effect on faecal enzymes, strain dependent improvement of lactose intolerance symptoms. L. acidophilus La-5 Balancing intestinal microbiota, protection against traveller's diarrhoea, Fonden et al. (2000); Ouwehand et al. (2003) immune enhancement. B. Lactis Bb-12 Treatment of viral diarrhoea including rotavirus diarrhoea, alleviation of Saavedra et al. (1994); Salminen et al. (1998); Isolauri et al. (2002b) symptoms of food allergy, balancing of intestinal microbiota. L. reuteri Shortening of rotavirus diarrhoea, colonising the intestinal tract. De Roos and Katan (2000); Fonden et al. (2000); Ouwehand et al. (2003) S. boulardii Prevention of antibiotic associated diarrhoea, treatment of C. difficile colitis. MacFarland (2000) Probiotic mix VSL 3 Positive effect in inflammatory bowel disease and irritable bowel syndrome; Delia et al. (2002a,b); Kim et al. (L. bulgaricus, L. plantarum, treatment and prevention of pouchitis, prevention and alleviation of (2002) S. thermophilus , B. longum, radiotherapy associated diarrhoea B. infantis, and B. breve)

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al., 2002). As gut health, digestion and absorption have an impact on metabolic effect in other organs (Verschuren, 2002), functional foods for this area might be appealing for each age group. Colostrum-derived preparations have already been tested in the clinical management of gastrointestinal diseases (Playford et al., 2000). They seem to be useful at least in infancy and childhood, and the possibilities for also developing products for adults and the elderly are also interesting. Since nutrients affect hormone secretion and release, they also affect mucosa, nerves and muscles. Specific nutrients might also be useful as enhancers of gut renewal. Even gut microbes might be employed ± they may stimulate nerves (Hooper et al., 2001). Hardman (2002) assumes that after the appropriate cancer therapy, consumption of n-3 fatty acids might slow or stop the growth of metastatic cancer cells, increase longevity of patients and improve their quality of life. N-3 fatty acids seem to alter the eicosanoid metabolism of cancer cells, slow cancer cell mitosis, increase cancer cell death, induce differentiation, suppress angiogenesis and alter estrogen metabolism. Animal studies also suggest that n-3 fatty acids might increase the efficacy of chemotherapy and reduce its side effects. One of the mechanisms in the treatment of colon cancer might be the suppression of the increased production of COX-2 typical to the disease. 13.7.2 Gut microbiota and allergy Our studies have indicated significant differences in the Bifidobacterium and Clostridium microbiota composition of children who later develop atopic diseases (KalliomaÈki et al., 2001). The result has been confirmed in Estonian and Swedish infants (BjoÈrksten et al., 2001). Taken together the results suggest that the focus on intestinal microbiota and the search for new probiotics for the newborn and infants should be centred on the lactic acid bacteria and Bifidobacteria composition and modulating the ratio of bifidobacteria to clostridia in the intestinal contents of infants. We have obtained similar results in studies among very old Finnish and Japanese subjects (He et al., 2000). 13.7.3 Gut microbiota and intestinal mucus or age-specific intestinal microbiota We have also shown that the mucus microbiota is not necessarily reflected by faecal microbiota. This finding offers a basis for further assessing the influence of mucosal microbiota on the immune system and mucosal cell gene expression. The role and crosstalk between specific bifidobacteria and the gut epithelium has been further clarified by understanding the genome sequences of specific bifidobacteria. Schnell and co-workers (2002) reported the genome sequence of B. longum. They identified polypeptides with homology to most major proteins needed for production of glycoprotein-binding fimbriae. Such fimbriae were also tentatively for the first time identified for a Bifidobacterium by electron microscopy. B. longum was also shown to have mucin utilising properties and it

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has a eukaryotic-type serine protease inhibitor possibly involved in earlier reported immunomodulatory activity of bifidobacteria. Interestingly, B. longum also has numerous high-affinity oligosaccharide transporters, including the use of over 80 oligosaccharides found in breast milk, which may enhance its presence and competition for nutrients in the newborn infant colon during breast feeding. In the same manner, B. adolescentis, the dominant bifidobacterium species among the adults and the elderly, may have adapted to changing nutrient sources during old age. This is an area of future research as the host physiology may have an influence on the gut health of the elderly and the microbiota may have a significant role in preserving the gut health of the elderly. High antioxidative activity may be useful for survival and defensive properties of probiotics (Kullisaar et al., 2002). Starch encapsulation of probiotics may in future enable utilisation of new probiotic strains (PuupponenPimiaÈ et al., 2002). 13.7.4 `Novel prebiotics' Chemical modification of starch and other cereal components (Topping and Clifton, 2001; Charalampopoulos et al., 2002) and inulin (Stevens et al., 2001) offers possibilities for modifying their digestibility and thus efficiency as prebiotics. According to Das (2002) the combination of LCPUFA and probiotics could offer significant protection against atopy. 13.7.5 Nutrigenomics Genetic polymorphism may be a contributing factor in the inconsistensies found between diet and risk of colorectal cancer (Cotton et al., 2000; Houlston and Tomlinson, 2001). Van Ommen and Stierum (2002) stress the importance of multiple minor changes in genomic responses to food. Co-operation of nutritional sciences with systems biology is needed for proving health claims of functional food in future. Weststrate et al. (2002) predict that within ten years there will be some functional foods on the market that take into account gene polymorphism relevant to the development of chronic diseases. However, Van Ommen and Stierum (2002) state that prevention or intervention of a disease may be fundamentally different from maintaining or improving good health. 13.7.6 Biotechnology Genetic engineering has been applied to improve the nutritional quality of plants. Broun et al. (1999) have reviewed applications on plant lipids and Grusak (2002) on phytochemicals. Several tools and useful applications already exist. Food-grade microbes have been modified to produce low-energy sugars, vitamins, prebiotics and antioxidants. Constructed strains might be used in novel fermented foods with increased nutritional value (Hugenholtz and Smid, 2002).

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Also, therapeutic use of genetically engineered lactic acid bacteria (Lactococcus lactis) against IBD in animal models has already been described. The L. lactis strain was engineered to secrete biologically active interleukin IL-10, and daily treatment with the microbe reduced significantly intestinal inflammation (Steidler et al., 2000). Van der Werf and Venema (2001) describe tools for developing improved probiotic Bifidobacterium strains or increasing their stability during processing and viability upon storage. Chronic stress initiates mucosal inflammation in rat intestine by impairing mucosal defences (SoÈderholm et al. 2002). Is it possible in future to alleviate stress disorders by special probiotics?

13.8

Sources of further information

Ageing and nutrient needs are discussed with reference to nutrition recommendations in the following articles: Russel R, Rasmussen H and Lichtenstein A (1999), `Modified food guide pyramid for people over seventy years of age', J Nutr 129: 751±753 and Lichtenstein A (1999), `Optimal nutrition for mature adult in health and disease', in Rippe J, Lifestyle Medicine, Blackwell Science, Malden MA, USA, 160±167. Nutritional effects of pre- and probiotics are reviewed in Teitelbaum J E and Walker A (2002), `Nutritional Impact of Preand Probiotics as Protective Gastrointestinal Organisms', Annu Rev Nutr 22: 107±138. Functional foods and gastrointestinal physiology and function are discussed in detail in the following article: Salminen S, Bouley M C, Boutron-Rualt M C, Cummings J, Franck A, Gibson G, Isolauri E, Moreau M-C, Roberfroid M, Rowland (1998), `Functional food science and gastrointestinal physiology and function', Br J Nutr 80 Suppl 1: 147±171. Functional foods and different aspects of physiology are extensively described in two ILSI Europe working group proceedings: British Journal of Nutrition, (1998) vol 80 (Suppl. 1) `Functional Food Science in Europe' and British Journal of Nutrition, (2002) vol 88 (Suppl. 2) `Functional Foods: Scientific and Global Perspectives'. Several new reviews discuss gut defence systems: Berkes J, Viswanathan V K, Savkovic S D, Hecht G (2003), `Intestinal epithelial responses to enteric pathogens: effects on the tight junction barrier, ion transport, and inflammation', Gut 52: 439±451; Mowat A M (2003), `Anatomical basis of tolerance and immunity to intestinal antigens', Nature Rev Immunol 3: 331±341; Fagarasan S and Honjo T (2003), `Intestinal IgA synthesis: regulation of front-line body defences', Nature Rev Immunol 3: 63±72; Baumgart D C and Dignass A U (2002), `Intestinal barrier function' Curr Opin Clin Nutr Metab Care 5: 685± 694. Effects of glutathione on gut defence has been studied in: Hoensch H,

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Morgenstern I, Petereit G, Siepmann M, Peters W H M, Roelofs H M J, Kirch W (2002) `Influence of clinical factors, diet, and drugs on the human upper gastrointestinal glutathione system', Gut 50: 235±240.

13.9

References

(2002), `Treatment of inflammatory bowel disease in a rodent model with the intestinal growth factor glucagon-like peptide2', J Pediatr Surg 35(6): 847±851. ALEXANDER J W (2002), `Nutritional pharmacology in surgical patients', Am J Surg 183: 349±352. ANDERSON M, FRITSCHE K L (2002), `(n-3) Fatty acids and infectious disease resistance', J Nutr 132: 3566±3576. ASO Y, AKAZA H. (1992), `Prophylactic effect of a Lactobacillus casei preparation on the recurrence of superficial bladder cancer', BLP Study Group. Urol Int. 49(3): 125± 129. ASO Y, AKAZA H, KOTAKE T, TSUKAMOTO T, IMAI K, NAITO S (1995), `Preventive effect of a Lactobacillus casei preparation on the recurrence of superficial bladder cancer in a double-blind trial', The BLP Study Group. Eur Urol. 27(2): 104±109. ATTELE A S, SHI Z Q, YUAN C-S (2002), `Leptin, gut, and food intake', Biochem Pharmacol 63: 1579±1583. ATUMA C, STRUGALA V, ALLEN A, HOLM L (2001), `The adherent gastrointestinal mucus gel layer: thickness and physical state in vivo', Am J Physiol Gastrointest Liver Physiol 280: G922±929. ALAVI K, SCHWARTZ M Z, PALAZZO J P, PRASAD R

AUGENLICHT L H, MARIADASON J M, WILSON A, ARANGO D, YANG WC, HEERDT B, VELCICH A

(2002), `Short chain fatty acids and colon cancer', J Nutr 132: 3804S±3808S.

BARRENETXE J, VILLARO A C, GUEMPE L, PACUAL L, MUNOZ-NAVAS M, BARBER A, LOSTAO M P

(2002), `Distribution of the long leptin reseptor isoform in brush border, basolateral membrane, and cytoplasm of enterocytes', Gut 50: 797±802. BENNO Y, MITSUOKA T (1986), `Development of intestinal microflora in humans and animals', Bifidobacteria Microflora 5: 13±25. BINGHAM S A, DAY N E, LUBEN R, FERRARI P, SLIMANI N, NORAT T, CLAVEL-CHAPELON F, KESSE E, NIETERS A, BOEING H, TJONNELAND A, OVERVAD K, MARTINEZ C, DORRONSORO M, GONZALEZ C A, KEY T J, TRICHOPOULOU A, NASKA A, VINEIS P, TUMINO R, KROGH V, BUENO-DE-MESQUITA H B, PEETERS P H, BERGLUND G, HALLMANS G, LUND E, SKEIE G,

KAAKS R, RIBOLI E (2003), `Dietary fibre in food and protection against colorectal cancer in the European Prospective Investigation into Cancer and Nutrition (EPIC): an observational study', Lancet 361(9368): 1496±1501. È RKSTEN B, SEPP E, JULGE K, VOOR T, MIKELSAAR M (2001), `Allergy development and the BJO intestinal microflora during the first year of life', J Allergy Clin Immunol 108: 516±20. È SLUND E (1999), `Glucagon-like peptide-1, satiety and appetite control BLUNDELL J E, NA (invited commentary)', Brit J Nutr 81: 259±260. BRESALIER R S (1999), `Calcium, chemoprevention, and cancer: a small step forward (a long way to go)' , Gastroenterology 116: 1261±1267. BROUN P, GETTNER S, SOMERVILLE C (1999), `Genetic engineering of plant lipids', Annu Rev Nutr 19: 197±216.

Functional foods for gut health: an overview

319

(2001), `Glucagon-like peptide 2: A nutrientresponsive gut growth factor', J Nutr 131: 709±712. BYERS T, NESTLE M, MCTIERNAN A, et al. (2002), `American Cancer Society Guidelines on nutrition and physical activity for cancer prevention: Reducing the risk of cancer with healthy food choices and physical activity', CA Cancer J Clin 52: 92±119. BURRIN D G, PETERSEN Y, STOLL B, SANGILD P

BYRNE B, MADRIGAL-ESTEBAS L, MCEVOY A, CARTON J, DOHERTY D G, WHELAN A, FEIGHERY C,

(2002), `Human duodenal epithelial cells constitutively express molecular components of antigen presentation but not costimulatory molecules', Human Immunol 63: 977±986. CALDER P C, KEW S (2002), `The immune system: a target for functional foods?', Brit J Nutr 88: S165±S176. CEBRA J J (1999), `Influences of microbiota on intestinal immune system development', Am J Clin Nutr 69 (Suppl): 1046S±1051S. CHARALAMPOPOULOS D, WANG R, PANDIELLA S S, WEBB C (2002), `Application of cereals and cereal components in functional foods: a review', Int J Food Microbiol 79: 131±141. CLINTON S K (1992), `Dietary proteins and carcinogenesis', in ed. Rowland I R, Nutrition, Toxicity and Cancer, Boca Raton, FL: CRC Press, 455±479. COTTON S C, SHARP L, LITTLE J, BROCKTON N (2000), `Glutathione S-transferase polymorphism and colorectal cancer: a HuGE review', Am J Epidemiol 151: 7±32. CUMMINS A G, THOMPSON F M (2002), `Effects of breast milk and weaning on epithelial growth of the small intestine in humans', Gut 51: 748±754. CUNNINGHAM-RUNDLES S (2003), `Is the fatty acid composition of immune cells the key to normal variations in human immune response?', Am J Clin Nutr 77: 1096±1097. DAS U N (2002), `Essential fatty acids as possible enhancers of the beneficial actions of probiotics', Nutrition 18: 786±789. DE ROOS N, KATAN M (2000), `Effects of probiotic bacteria on diarrhea, lipid metabolism, and carcinogenesis: a review of papers published between 1988 and 1998', Am J Clin Nutr 71: 405±411. DEPLANCKE B, GASKINS H R (2001), `Microbial modulation of innate defense: goblet cells and the intestinal mucus layer', Am J Clin Nutr, 73(suppl): 1131S±1141S. DONNET-HUGHES A, SCHIFFRIN E J, TURINI M E (2001), `The intestinal mucosa as a target for dietary polyunsaturated fatty acids', Lipids 36(9): 1043±1052. DONNET-HUGHES A, SCHIFFRIN E J, TURINI M E (2002), `The intestinal mucosa as a target for dietary polyunsaturated fatty acids', Lipids 36: 1043±1052. DRUCKER D J (2002), `Gut adaptation and the glucagons-like peptides', Gut 50: 428±435. DUGGAN C, CANNON J, WALKER A (2002), `Protective nutrients and functional foods for the gastrointestinal tract', Am J Clin Nutr, 74: 789±808. DUNNE C, (2001), `Adaptation of bacteria to the intestinal niche: Probiotics and gut disorder', Inflammat Bowel Dis, 7(2): 136±145. FAGARASAN S, HONJO T (2003), `Intestinal IgA synthesis: regulation of front-line body defences', Nature Rev Immunol 3: 63±71. FAVIER C, VAUGHAN E, DE VOS W, AKKERMANS A (2002), `Molecular monitoring of succession of bacterial communities in human neonates', Appl Environm Microbiol 68: 219±226. FERGUSON L R, HARRIS P J (2003), `The dietary fibre debate: more food for thought. Lancet 361(9368): 1487±1488. FONDEN R, MOGENSEN G, TANAKA R, SALMINEN S, (2000), `Culture ± containing dairy products ± effect on intestinal microflora, human nutrition and health ± current O'DONOGHUE D P, O'FARRELLY C

320

Functional foods, ageing and degenerative disease

knowledge and future perspectives', Bulletin of the International Dairy Federation 2000: 352. FULLER R (1989), `Probiotic in man and animals', Appl Bacteriol 66: 365±378. FULLER M F, REEDS P J (1998), `Nitrogen cycling in the gut', Annu Rev Nutr 18: 385±411. FURNESS J B, KUNZE W A A, CLERC N (1999), `Nutrient tasting and signaling mechanisms in the gut II. The intestine as a sensory organ: neural, endocrine, and immune responses', Am J Physiol 277 (Gastrointest Liver Physiol) G922±G928. GARSIDE P, MOWAT A M (2001), `Oral tolerance', Semin Immunol 13: 177±185. GILL H S, DARRAGH A J, CROSS M L (2001), `Optimizing immunity and gut function in the elderly', J Nutr Health Aging 5(2): 80±91. GRUSAK M A (2002), `Phytochemicals in plants: genomics-assisted plant improvement for nutritional and health benefits', Curr Opin Biotechn 13: 508±509. GUARNER F, MALAGELADA J-R (2003), `Gut flora in health and disease', Lancet 361: 512± 519. HARDMAN W E (2002), `Omega-3 Fatty Acids to Augment Cancer Therapy', J Nutr 132: 3508S±3512S. HARMSEN H J, WILDEBOER-VELOO A C, RAANGS G C, WAGENDORP A A, KLIJN N, BINDELS J G, WELLING G W (2000), `Analysis of intestinal flora development in breast-fed and formula-fed infants by using molecular identification and detection methods', J Pediatr Gastroenterol Nutr 30(1): 61±67. HASLER C M (2002), `Functional foods: benefits, concerns and challenges-a position paper from the American council on science and health', J Nutr 132(12): 3772±3781. HE F, OUWEHAND A, ISOLAURI E, HOSODA M, BENNO Y, SALMINEN S (2001), `Differences in Composition and Mucosal Adhesion of Bifidobacteria Isolated from Healthy Adults and Healthy Seniors', Curr Microbiol 43: 351±354. HEÂBUTERNE X (2003) Gut changes attributed to ageing: effects on intestinal microflora', Curr Opin Clin Nutr Metab Care 6: 49±54. HOOPER L V, WONG M H, THELIN A, HANSSON L, FALK P G, GORDON J I (2001), `Molecular analysis of commensal host-microbial relationships in the intestine', Science 291: 881±884. HOPKINS M, SHARP R, MACFARLANE G (2001), `Age and disease related changes in intestinal bacterial populations assessed by cell structure, 16S rRNA abundance, and community cellular fatty acid profiles', Gut 48: 198±205. HOULSTON R S, TOMLINSON I P M (2001), `Polymorphism and colorectal tumor risk', Gastroenterology 121: 282±301. HOWARTH G S, SHOUBRIDGE C A (2001), `Enhancement of intestinal growth and repair by growth factors', Current Opinion in Pharmacology 1: 568±574. HUGENHOLTZ J, SMID E J (2002), `Nutraceutical production with food-grade microorganisms', Curr Opin Biotechnol 13: 497±507. HUIS IN'T VELD J, HAVENAAR R (1991), `Probiotics and health in man and animal', J Chem Technol Biotechnol 51: 562±567. ISOLAURI E, KIRJAVAINEN P V, SALMINEN S (2002a), `Probiotics: a role in the treatment of intestinal infection and inflammation', Gut 50: (Suppl. III): iii54±iii59. ISOLAURI E, RAUTAVA S, KALLIOMAKI M, KIRJAVAINEN P, SALMINEN S (2002b), `Role of probiotics in food hypersensitivity', Curr Opin Allergy Clin Immunol 2: 263±71. JENKINS D J, KENDALL C W, VUKSAN V (1999), `Inulin, oligofructose and intestinal function', J Nutr 129 (7 Suppl): 1431S±1433S. KALLIOMAKI M, KIRJAVAINEN P, EEROLA E, KERO P, SALMINEN S, ISOLAURI E (2001), `Distinct patterns of neonatal gut microflora in infants in whom atopy was and was not

Functional foods for gut health: an overview

321

developing', J Allergy Clin Immunol 107: 129±134. (2001), `Modulation of human immune and inflammatory responses by dietary fatty acids', Nutrition 17: 669±673. S, BANERJEE T, MINIHANE A E, FINNEGAN Y E, WILLIAMS C M, CALDER P C (2003a), `Relation between the fatty acid composition of peripheral blood mononuclear cells and measures of immune cell function in healthy, free-living subjects aged 25±72 y', Am J Clin Nutr 77: 1278±1286.

KELLEY DS KEW

KEW S, BANERJEE T, MINIHANE A E, FINNEGAN Y E, MUGGLI R, ALBERS R, WILLIAMS C M,

(2003b), `Lack of effect of foods enriched with plant- or marinederived n-3 fatty acids on immune function', Am J Clin Nutr 77: 1287±1295. KILKKINEN A, PIETINEN P, KLAUKKA T, VIRTAMO J, KORHONEN P, ADLERCREUTZ H (2002), `Use of oral antimicrobials decreases serum enterolactone concentration', Am J Epidemiol 155: 472±477. CALDER P C

KIM H J, CAMILLERI M, MCKINZIE S, LEMPKE M B , BURTON D D, THOMFORDE G M, ZINSMEISTER

(2003), `A randomised controlled trial of a probiotic, VSL#3, on gut transit and symtoms in diarrhoea-predominant irritable bowel syndrome', Aliment Pharmacol Ther 17: 895±904. KIM Y-I (2000), `AGA Technical Review: Impact of dietary fiber on colon cancer occurrence', Gastroenterology 118: 1235±1257. KIRCHGESSNER A L (2002), `Orexins in the brain-gut axis', Endocr Rev 23(1): 1±15. KULLISAAR T, ZILMER M, MIKELSAAR M, VIHALEMM T, ANNUK H, KAIRANE C, KILK A (2002), `Two antioxidative lactobacilli strains as promising probiotics', Int J Food Microbiol 72: 215±224. È MMA È LA È J, MA È KELA È S, SALMINEN S, OUWEHAND A (2002), LAHTINEN S, SAARINEN N, A `Interactions between lignans and probiotics', Microbial ecology in Health and Disease 14: 106±109. LAIHO K, HOPPU U, OUWEHAND A C, SALMINEN S, ISOLAURI E (2002), `Probiotics: on-going research on atopic individuals', Brit J Nutr 88(Suppl. 1): S11±S18. LIDDLE R (2000), `Regulation of cholecystokinin secretion in humans', J Gastroenterol 35: 181±187. LILLY D, STILLWELL E. (1965), `Probiotics: growth promoting factors produced by microorganisms', Science 147: 747±748. LOVSHIN J, DRUCKER D J (2000), `New frontiers in the biology of GLP-2', Regul Pept 90: 27±32. MACFARLAND L (2000), `A review of the evidence of health claims for biotherapeutic agents', Micr Ecol Health Dis 12: 65±76. MACFARLANE G T, MACFARLANE S (1995), `Proteolysis and amino acid fermentation', in Gibson G R, Macfarlane G T, Human Colonic Bacteria: Role in Nutrition, Physiology, and Pathology, London: CRC Press, 75±100. MANNON P J (2002), `Peptide YY as a growth factor for intestinal epithelium', Peptides 23: 383±388. MATSUKI T, WATANABE K, TANAKA R, FUKUDA M, OYAIZU H (1999), `Distribution of Bifidobacterial species in human intestinal microflora examined with 16S rRNAgene-targeted species-specific primers', Appl Environm Microbiol 65: 4506±4512. METCHNIKOFF E (1907), Prolongation of Life, William Heinemann, London. MONTELEONE I, VAVASSORI P, BIANCONE L, MONTELEONE G, FALLONE F (2002), `Immunoregulation in the gut: succes and failures in human disease', Gut 50 (suppl III): iii60±iii64. MOORE W E, MOORE L H (1995), `Intestinal floras of populations that have a high risk of AR

322

Functional foods, ageing and degenerative disease

colon cancer', Appl Environ Microbiol 61: 3202±3207. (2003), `Anatomical basis of tolerance and immunity to intestinal antigens', Nature Rev Immunol 3: 333±341. NEGRI E, LA VECCHIA C, FRANCESHI S (2002), `Relations between vegetable, fruit and micronutrient intake. Implications for odd ratios in a case-control study', Eur J Nutr 56: 166±170. NEISH A S (2002), `The gut microflora and intestinal epithelial cells: a continuing dialogue', Microbes and infections, 4: 309±317. NEUTRA M R, MANTIS N J, KRAEHENBUHL J-P (2001), `Collaboration of epithelial cells with organized mucosal lymphoid tissues', Nature Immunol 2(11): 1004±1009. È M P M, KRAL J G (2001), `'The gut and food intake: An update for NaÈSLUND E, HELLSTRO surgeons', J Gastrointest Surg 5: 556±567. MOWAT A M

OHASHI Y, NAKAI S, TSUKAMOTO T, MASUMORI N, AKAZA H, MIYANAGA N, KITAMURA T, KAWABE K, KOTAKE T, KURODA M, NAITO S, KOGA H, SAITO Y, NOMATA K, KITAGAWA M, ASO Y (2002) `Habitual intake of lactic acid bacteria and risk reduction of bladder cancer', Urol Int. 68(4): 273±280. OTA H, KATSUYAMA T (1992), `Alternating laminated array of two types of mucin in the human gastric surface mucous layer', Histochem J 24(2): 86±92. È NLUND M-M, ISOLAURI E, SALMINEN S J (1999), OUWEHAND A C, KIRJAVAINEN K V, GRO `Adhesion of four Bifidobacterium strains to human intestinal mucus from subjects in different age groups', FEMS Microbial Lett 172: 61±64. OUWEHAND A, BIANCHI SALVADORI B, FONDEÂN R, MOGENSEN G, SALMINEN S, SELLARS R,

(2003), Health Effects of Probiotics. Bulletin of the IDF 380: 4±19. (1974), `Probiotics: the other half of the antibiotics story', Animal Nutr Health 29: 4±8.

TANAKA, R. PARKER R B

PETERS U, SINHA R, CHATTERJEE N, SUBAR A F, ZIEGLER R G, KULLDORFF M, BRESALIER R,

(2003), `Dietary fibre and colorectal adenoma in a colorectal cancer early detection programme', Lancet 361(9368): 1491±1495. PITMAN R S, BLUMBERG R S (2000), `First line of defence: the role of the intestinal epithelium as an active component of the mucosal immune system', J Gastroenterol 35: 805±807. PLAYFORD R, MACDONALD C E, JOHNSON W S (2000), `Colostrum and milk-derived peptide growth factors for the treatment of gastrointestinal disorders', Am J Clin Nutr 72: 5±14. PROSKY L (2000), `When is dietary fiber considered a functional food', Biofactors 12: 289±297. WEISSFELD J L, FLOOD A, SCHATZKIN A, HAYES B

È R, AURA A-M, OKSMAN-CALDENTEY K-M, MYLLA È RINEN P, SAARELA M, PUUPPONEN-PIMIA

(2002), `Development of functional ingredients for gut health', Trends Food Sci Technol 13: 3±11. RAY E C, AVISSAR N E, SAX H C (2002, `Growth factor regulation of enterocyte nutrient transport during intestinal adaptation', Am J Surg 183: 361±371. REEDS P J, BURRIN D G (2002), `The gut and amino acid homeostasis', Nutrition 16(7/8): 666±668. REHFELD J F (1998), 'The new biology of gastrointestinal hormones', Physiol Rev 78: 1087±1108. ROCCA A, BRUBAKER P L (1999), `Role of the vagusnerve in mediating proximal nutrient induced glucagons-like peptide-1 secretion', Endocrinology 140: 1687±1694. ROWLAND I (1996), `Endogenous production by N-nitroso compounds', Eur J Cancer MATTILA-SANDHOLM T, POUTANEN K

Functional foods for gut health: an overview

323

Prev 5 (Suppl 1): 101±102.

(1994), `Feeding of Bifidobacterium bifidum and Streptococcus thermophilus to infants in hospital for prevention of diarrhea and shedding of rotavirus', Lancet 47: 709±710.

SAAVEDRA J, BAUMAN N, OUNG I, PERMAN J, YOLKEN R

SALMINEN S, BOULEY C, BOUTRON-RUAULT M-C, CUMMINGS J H, FRANCK A, GIBSON G R, ISOLAURI E, MOREAU M-C, ROBERFROID M, ROWLAND I (1998), `Functional food science and gastro-intestinal physiology and function', Brit J Nutr 80(Suppl. I): S147±S171. SATOKARI R M, VAUGHAN E E, AKKERMANS A D, SAARELA M, DE VOS W M (2001), `Bifidobacterial diversity in human feces detected by genus-specific PCR and denaturing gradient gel electrophoresis', Appl Environ Microbiol 67(2): 504±513. SCHELL M A, KARMIRANTZOU M, SNEL B, VILANOVA D, BERGER B, PESSI G, ZWAHLEN M-C,

(2002), `The genome sequence of Bifidobacterium longum reflects its adaptation to the human gastrointestinal tract', Proc. Natl. Acad. Sci. 99: 14422±14427. SCHLEY P D, FIELD C J (2002), `The immune-enhancing effects of dietary fibres and prebiotic', Brit J Nutr 87 (Suppl 2): S221±S230. SCHNEEMAN B (2002), `Gastrointestinal physiology and functions', Brit J Nutr 88 (Suppl2): S159±S163. SHANAHAN F (2000), `Nutrient Tasting and Signaling Mechanisms in the Gut V. Mechanisms of immunologic sensation of intestinal contents', Am J Physiol Gastrointestinal Liver Physiol 278: G191±G196. SHANAHAN F (2002), `Probiotics and inflammatory bowel disease: from fads and fantasy to facts and future', Brit J Nutr 88(Suppl. 1): S5±S9. SHAO L, SERRANO D, MAYER L (2001), `The role of epithelial cells in immune regulation in the gut', Semin Immunol 13: 163±175. SHIMOTOYODOME A, MEGURO S, HASE T, TOKIMITSU I, SAKATA T (2000), `Short chain fatty acids but not lactate or succinate stimulate mucus release in the rat colon', Comparat Biochem Physiol Part A 125: 525±531. DESIERE F, BORK P, DELLEY M, PRIDMORE R D, ARIGONI F

È DERHOLM J D, YANG P C, CEPONIS P, VOHRA A, RIDDELL R, SHERMAN P M, PERDUE M H SO

(2002), `Chronic stress induces mast cell-dependent bacterial adherence and initiates mucosal inflammation in rat intestine', Gastroenterology 123(4): 1099± 1108. STEIDLER L, SCHOTTE H W, NEIRYNCK S, OBERMEIER F, FALK W, FIERS W, REMAUT E (2000), `Treatment of murine colitis by Lactococcus lactis secreting interleukin-10', Science 289: 1352±1355. STEINMAN R M, HAWIGER D, NUSSENZWEIG M C (2003), `Tolerogenic dendritic cells', Annu Rev Immunol 21: 685±711. STEVENS C V, MERIGGI A, BOOTEN K (2001), `Chemical Modification of Inulin, a Valuable Renewable Resource, and Its Industrial Applications', Biomacromolecules 2: 1±16. TAPPENDEN K A, MCBURNEY M I (1998), `Systemic short-chain fatty acids rapidly alter gastrointestinal structure, function, and expression of early response genes', Endocrinology 140: 1687±1694. TAPPENDEN K A, DROZDOWSKI L A, THOMSON A B, MCBURNEY M I (1998), `Short-chain fatty acid-supplemented total parenteral nutrition alters intestinal structure, glucose transporter 2 (GLUT2) mRNA and protein, and proglucagon mRNA abundance in normal rats', Am J Clin Nutr 68: 118±25. TEITELBAUM J E, WALKER A (2002), `Nutritional Impact of Pre- and Probiotics as Protective Gastrointestinal Organisms', Annu Rev Nutr 22: 107±138.

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THOMSEN C, RASMUSSEN O, LOUSEN T, HOLST J J, FENSELAU S, SCHREZENMEIER J, HERMANSEN K (1999), `Differential effects of saturated and monounsaturated fatty acids on postprandial lipemia and incretin responses in helathy subjects', Am J Clin Nutr 69: 1135±1143. THE DIETARY FIBER DEFINITION COMMITTEE (2001), `The definition of dietary fiber', CFW 46(3): 113±126. THULESEN J, HARTMANN B, NIELSEN C, HOLST J, POULSEN S S (1999), `Diabetic intestinal growth adaption and glucagons-like peptide 2 in the rat: effects of dietary fibre' Gut 45: 672±678. TOPPING D L, CLIFTON P M (2001), `Short-Chain Fatty Acids and Human Colonic Function: Roles of Resistant Starch and Nonstarch Polysaccharides', Physiol Rew 81(3): 1031±1064. VAN DER MEER R, BOVEE-OUDENHOVEN I (1998), `Dietary modulation of intestinal bacterial infections', Int Dairy J 8: 481±486. VAN DER WERF M J, VENEMA K (2001), `Bifidobacteria: Genetic modification and the study of their role in the colon', J Agric Food Chem 49: 378±383. VAN OMMEN B, STIERUM R (2002), `Nutrigenomics: exploiting systems biology in the nutrition and health arena', Curr Opin Biotechnol 13: 517±521. VAN POPPEL G (2002), `Nutrition and cancer ± a role for functional foods?' in Angus F and Miller C `Functional Foods', Surrey, Leatherhead Publishing, 216±225. VAUGHAN E, DE VRIES M, ZOETENDAL E, BEN-AMOR K, AKKERMANS A, DE VOS W (2002), `The intestinal LABs', Anth van Leeuwenh 82: 341±352. VERSCHUREN P M (2002), `Summary Report. Functional foods: Scientific and global perspectives', Brit J Nutr 88 (Suppl. 2): S125±S130. WESTSTRATE J A, VAN POPPEL G, VERSCHUREN P M (2002), `Functional foods, trends and future', Brit J Nutr 88 (Suppl.2): S233±S235. WILLIE J T, CHEMELLI R M, SINTON C M, YANAGISAWA M (2001), `To eat or to sleep? Orexin in the regulation of feeding and wakefulness', Annu Rev Neurosci 24: 429±458. WITTIG B M, ZEITZ M (2003), `The gut as an organ of immunology', Int J Colorectal Dis 18(3): 181±187. WOUDSTRA T, THOMSON A B R (2002), `Nutrient absorption and intestinal adaptation with ageing', Bailliere's Best Practice and Research Clinical Gastroenterology 16(1): 1±15. XIAO Q, BOUSHEY R P, DRUCKER DJ, BRUBAKER P L (1999), `Secretion of the intestinotrophic hormone glucagons-like peptide 2 is differentially regulated by nutrients in humans', Gastroenterology 117: 99±105. Â NDEZ-ESTIVARIZ C, JONES D P (2003), `Trophic and cytoZIEGLER T R, EVANS M E, FERNA protective nutrition for intestinal adaptation, mucosal repair, and barrier function', Annu Rev Nutr 23: 229±261.

14 Analysing gut microbiota M. Blaut, German Institute of Human Nutrition PotsdamRehbruecke

14.1

Introduction

The human gastro-intestinal (GI) tract is populated by a large and diverse community of up to 1014 micro-organisms which belong to an estimated 400± 500 different species (Finegold et al., 1983). Interest in gut micro-organisms has mainly been driven by the discovery that they have an impact on structure and function of the GI-tract and thereby influence host health. However, the exact role of the various bacterial population groups present in the GI-tract has been difficult to define. This requires a better knowledge of the makeup of the gut microbiota. Early investigations on the composition of the human gut microbiota missed the majority of bacteria present in the GI-tract because the techniques to grow strict anaerobes in the laboratory had not yet been fully developed. The introduction of the roll tube technique (Hungate, 1969) and of anaerobic cabinets (Aranki et al., 1969) was a major breakthrough in the cultivation of anaerobes which triggered the isolation and characterisation of previously uncultured strict anaerobes. Pioneers who laid the foundation for our present knowledge of the human gut microbiota included Moore and Holdeman (Moore and Holdeman, 1974) and Finegold and co-workers (Finegold et al., 1974). These researchers isolated a large number of bacterial species from human faeces and determined their distribution in humans. The in-depth analysis of the human gut microbiota as performed by these authors was laborious, time consuming and tedious. In particular, the identification of colonies to the species level required differentiation of large numbers of colonies on selective and differential media. This may explain why the number of studies involving the analysis of large numbers of subjects is limited. All studies indicated a high inter-individual variation in the microbiota composition, while a clear

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correlation between dietary habits and the presence of certain micro-organisms could not be found in most of these studies (Finegold, 1986). An important statement, which has been made previously by many other researchers, needs to be repeated here: The microbial composition of faeces does not necessarily reflect the microbial composition of lumenal contents from other sections of the gastro-intestinal tract. However, although quantitative differences exist there is a relatively high similarity of the bacterial composition in the colon and in the faeces (Bentley et al., 1972). Following the introduction of culture independent methods for bacterial identification it soon became clear that there is a large discrepancy between the number of micro-organisms that can be detected by virtue of their ability to form colonies on agar plates and the number of micro-organisms really present in a given habitat. Depending on the habitat, the proportion of culturable bacteria may be as low as 0.001%, as reported for seawater, and therefore the term `great plate count anomaly' was coined by Amann and co-workers (Amann et al., 1995). The application of culture independent methods to gut microbiology revealed that only 15±58% of the bacterial species resident in the human intestinal tract have been cultured (Langendijk et al., 1995; Suau et al., 1999) and that many microbiological data based on culture dependent techniques are biased because certain bacterial population groups have been underestimated while others have been overestimated. This is due to the fact that the specific growth requirements for many bacteria are not known and most media are not really selective for certain bacteria.

14.2 Molecular based methods for identifying gut micro-organisms Owing to the limited number of simple phenotypic traits, the identification of micro-organisms has always been a challenge. Moreover, most of the phenotypic characters used for bacterial identification not only lack the resolving power but also do not reflect the phylogenetic position of an organism. In recent years, ribosomal RNA (rRNA) has become the preferred marker for the identification and affiliation of new isolates because it is an excellent tool for measuring evolutionary relationships among organisms (Woese et al., 1990). Ribosomal RNAs are constituents of the ribosomes which in prokaryotes have an overall size of 70S (S=Svedberg units). They contain 2 subunits, of which the large one (50S) consists of 34 proteins, the 5S rRNA (120 bases), and the 23S rRNA (approximately 2,900 bases), while the 30S subunit contains 21 proteins and the 16S rRNA (approximately 1,500 bases). Even though all three ribosomal RNAs (5S, 16S, and 23S) have been employed in comparative sequence analysis, the 16S rRNA has become the most preferred molecule for bacterial identification (Amann et al., 1995).

Analysing gut microbiota 327 14.2.1 Generation of a database of small subunit ribosomal RNA sequences: a culture independent approach Woese was the first one to propose the use of the 16S rRNA sequence for the elucidation of bacterial phylogeny (Woese, 1987). Sequencing the 16S rRNA of a newly isolated micro-organism and subsequently comparing the retrieved sequence with the 16S rRNAs in the database is an excellent way of identifying this organism and determining its phylogenetic position. However, the 16S rRNA sequences can also be retrieved from uncultured bacteria, provided the bacterial DNA can be extracted. Owing to the fact that the 16S rRNA contains regions whose sequence is highly conserved in all bacteria and other regions whose sequence shows a high degree of variability among various organisms, it is possible to amplify the variable regions by polymerase chain reaction (PCR) by using primers that hybridise with the well-known conserved regions of the 16S rRNA gene (Olsen et al., 1986). Subsequent sequencing of the PCR product reveals the sequence of the adjacent variable region(s), the so-called signature sequence motifs, which are of diagnostic value and can be used for identification of bacteria (Amann et al., 1995). In practice, the retrieval of 16S rRNA sequences requires the isolation of nucleic acids from pure cultures of intestinal bacteria or from faecal samples. Ideally, the collection of retrieved 16S rRNA sequences should be a reflection of the microbial community composition. In other words, the frequency of a given 16S rRNA species should give an indication for the proportion of the corresponding target organism(s) in the microbial community. It is therefore important to make sure that the extraction of microbial DNA from a faecal sample is as complete as possible. Mechanic, enzymatic and/or chemical methods have been used for the disruption of the bacterial cell wall and the extraction of nucleic acids from faecal samples (Suau et al., 1999; Wilson and Blitchington, 1996). In particular humic acids, bile salts and complex polysaccharides present in faeces can inhibit PCR (Kreader, 1996). Incomplete DNA extraction or inhibition of PCR by inhibitory substances not removed during purification, may lead to a 16S rRNA gene library that is not representative of the microbial ecosystem studied. Bias in the resulting gene library may also be introduced by differences in the efficiency of the PCR amplification which may differ from template DNA to template DNA. It has been shown that the rRNA fragments of less abundant organisms tend to be underrepresented (Lee et al., 1996; Muyzer et al., 1993). Another important variable that influences the coverage of the microbial diversity is the number of amplification cycles applied in PCR: the higher the number of amplification cycles the smaller the degree of biodiversity coverage (Bonnet et al., 2002; Wilson and Blitchington, 1996). Bonnet and co-workers therefore proposed to keep the number of PCR cycles used for the amplification of ribosomal genes as small as possible (Bonnet et al., 2002). The amplified 16S rRNA genes are cloned into Escherichia coli to create a 16S rRNA gene library (Fig. 14.1). An ever-growing database of 16S rRNA sequences has substantially increased knowledge of bacterial phylogeny. The culture independent retrieval

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Fig. 14.1 Steps involved in generating a database of small subunit ribosomal RNA sequences of faecal bacteria.

of 16S rRNA sequences has been used by a number of researchers to investigate the microbial diversity in the human gut ecosystem (Suau et al., 1999; Wilson and Blitchington, 1996). Only 24% of the 16S rRNA sequences recovered in the study of Suau et al. (1999) could be assigned to described species indicating that a large proportion of the bacterial species present in the human gut microbiota have not yet been isolated and described. Analysis of the 16S rRNA-gene sequence of new faecal isolates is also the quickest and most efficient way to find out whether the isolate is a new or a previously described species. In general, bacteria exhibiting 98% sequence similarity are considered to be the same species. Based on such analyses, a number of recent isolates from human faeces have been shown to represent new diversity. These include Dorea longicatena (Taras et al., 2002), Anaerostipes caccae (Schwiertz et al., 2002a), Ruminococcus luti (Simmering et al., 2002), Roseburia intestinalis (Duncan et al., 2002) and Clostridium bolteae (Song et al., 2003). 14.2.2 Design and validation of ribosomal RNA-targeted oligonucleotide probes In recent years, the number of 16S rRNA sequences in general and of faecal micro-organisms in particular has increased considerably. A database of 16S rRNA sequences as complete as possible facilitates the design of oligonucleotide probes. They can be designed in such a way that they target bacteria at different levels of the phylogenetic hierarchy by hybridising to the target region of the

Analysing gut microbiota 329

Fig. 14.2 Alignment of the probe sequence (S-S-Acac-0194-a-A-18) and its 16S rRNA target sites in the gut anaerobe Anaerostipes caccae and closely related organisms.

RNA, where they form an RNA/DNA hybrid. While highly conserved stretches within the 16S rRNA sequence can be used to detect all bacteria, others are common to larger or smaller phylogenetic groups or even specific to a species and can therefore be used to detect the corresponding target organisms. Once it has been decided which organism or which group of organisms is to be targeted, suitable regions in the 16S rRNA sequence have to be identified that are present in the target organism but absent from the non-target organisms. A number of useful and easy-to-use computer programs for probe design have become available. These tools are often integrated into programs used for the storage, alignment and phylogenetic analysis of 16S rRNA sequences. The length of most 16S rRNA-targeted probes is in the range of 15 to 23 nucleotides. An example showing an in silico analysis for a probe targeting the gut anaerobe Anaerostipes caccae is given in Fig. 14.2. The optimal hybridisation conditions have to be determined for each probe experimentally because the dissociation temperature of an oligonucleotide probe depends on a number of parameters, including length and base composition. Whether the probe can form a hybrid with the target sequence depends primarily on the stringency of the hybridisation conditions. The stringency is determined by the hybridisation temperature and the composition of the buffer, in particular the salt concentration. Formamide is often used to increase stringency of the buffer. The specificity of a newly designed probe should be tested with rRNAs from as many faecal bacteria as possible. Optimally the organisms chosen should be representative of the ecosystem studied and include close relatives which should not be recognised by the probe. Between 45 and over 100 reference organisms have been used for testing the specificity of probes targeting human gut bacteria (Harmsen et al., 2000, 2002; Hold et al., 2003; Schwiertz et al., 2002b; Simmering et al., 1999). It is sometimes not easy to find experimental conditions under which the designed probe hybridises with the 16S rRNA of the target organism but not with the 16 rRNA of closely related bacteria with only one mismatch in their target sequence. The oligonucleotides have to be radioactively or fluorescently labelled to detect their hybridisation to the targeted 16S rRNA.

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14.2.3 Culture independent identification of gut bacteria There are several experimental approaches to identifying bacteria in a cultureindependent manner: dot blot hybridisation and whole cell in situ hybridisation. Both methods afford the determination of the relative abundance of the targeted bacterial group. Dot blot hybridisation Dot blot hybridisation and all methods derived from it do not detect bacterial cells but determine the proportion of rRNA originating from the target organism(s) relative to the amount of rRNA originating from all organisms present in the ecosystem. In practice, total RNA is isolated from an environmental sample such as human faeces and aliquots are blotted in parallel onto two nylon membranes or other suitable surfaces. One of the blots is hybridised with an oligonucleotide probe specific for the target group while the other blot is hybridised with a probe that detects all bacteria. The probes are usually radioactively labelled to quantify their binding to the blotted RNA spots. The signal intensity of a given sample on the blot hybridised with the specific probe is correlated with the signal intensity of the same sample blotted onto the other membrane and hybridised with the probe that detects all bacteria. The relative intensity of the signal may be taken as an indirect measure of the size of the targeted bacterial population group and has been referred to as RNA index (Sghir et al., 2000). Using this method, predominant bacterial groups were analysed in human faecal samples from 27 adults. The mean values determined for the following target groups were as follows: Bacteroides group, 36.8 Ô 16%; Clostridium leptum group, 16.3 Ô 7%; Clostridium coccoides group, 14.5 Ô 6%, Bifidobacterium spp., 0.7 Ô 1%; Lactobacillus spp. group, 0.6 Ô 1%; Enterobacteriaceae, 0.7 Ô 1% (Sghir et al., 2000). These signals added up to 70% of the total 16S rRNA detected by the probe targeting all bacteria. The percentages for the various groups are not comparable to the relative proportions of bacterial groups based on bacterial counts (see below). This may be explained by differences in the efficiency of the RNA recovery from the various target organisms, or by differences in the cellular RNA content. Fluorescence in situ hybridisation The most popular and widely used culture independent method is whole cell fluorescence in situ hybridisation which affords the specific detection of bacterial cells with fluorescently labelled oligonucleotide probes, often also referred to as FISH (Amann et al., 1995). Properly designed probes bind to their potential target sites in the 16S rRNA. Since one cell may contain 103 to 105 ribosomes the number of potential target sites for the labelled probe is sufficiently high to render the whole bacterial cell fluorescent so that it can be easily visualised in the epifluorescence microscope. In practice, this method requires the cells to be prepared in such a way that the target site becomes accessible to the oligonucleotide probes while preserving cell morphology. An aliquot of a homogenised faecal sample is tenfold diluted with buffer and

Analysing gut microbiota 331 thoroughly mixed. Since faecal samples contain not only bacterial cells but also particles originating from ingested food, which could interfere with the visualisation of the fluorescing bacteria at a later stage, a brief centrifugation step at reduced centrifugation force (1 min. at 300  g) is usually employed. The cells are subsequently fixed which also affords the permeabilisation of the bacterial cell envelope. Gram-negative bacteria are preferentially fixed with a freshly prepared paraformaldehyde solution whereas ethanol is used for grampositive bacteria (Amann et al., 1995). Some bacteria are more difficult to permeabilise because of their cell wall. In particular a number of grampositive bacteria additionally require lysozyme treatment to weaken the cell wall. Once the cells are fixed, they can be easily stored at ÿ20 or ÿ80ëC until analysis. For the identification and enumeration of target organisms by epifluorescence microscopy, the fixed bacterial cells have to be hybridised with the oligonucleotide probe of choice depending on which bacteria are to be targeted (see section 14.2.3). To be detectable, the oligonucleotides are labelled with a fluorochrome. Commonly used fluorochromes include tetramethylrhodamin (TMR), fluoresceinisothiocyanate (FITC), and the indocarbocyanine dyes Cy3 and Cy5. For that purpose, an aliquot of a diluted and fixed faecal sample is transferred into a well on a special type of microscopic slide (Fig. 14.3). It is important to ensure that the cells are distributed as evenly as possible to avoid a high variability between microscopic fields which are inspected for enumeration

Fig. 14.3

Enumeration of fluorescently labelled cells by epifluorescence microscopy taking advantage of custom-made slides.

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Fig. 14.4

Steps involved in culture independent detection of uncultured bacteria in faecal samples by whole cell fluorescent in situ hybridisation.

at a later stage. The cells in the well are air-dried and subsequently dehydrated by immersing the slide in 60%, 80% and 96% ethanol. After drying, the oligonucleotides are added and hybridisation is performed overnight at the optimal hybridisation temperature as determined experimentally with probe validation experiments (see section 14.2.3). After washing with buffer at 2ëC above the hybridisation temperature, mounting medium is added, which usually contains some anti-fading solution to reduce fluorescence bleaching. The cells can then be visualised by epifluorescence microscopy. It is even possible to identify several bacterial target groups simultaneously in the same sample provided each oligonucleotide employed is labelled with a different fluorochrome and the appropriate fluorescence filter is selected. A major advantage of FISH lies in the possibility of detecting bacteria that have not yet been cultured but whose 16S rRNA sequence has been retrieved in a culture independent way (14.2.1). Based on the retrieved sequence it becomes feasible to design an oligonucleotide probe which enables the detection of this particular organism by applying FISH to the original sample (Fig. 14.4). However, in this case it is not possible to test the specificity of the probe experimentally with the target organism. In particular obligate syntrophic bacteria may become detectable by this approach as shown previously for ectosymbiotic bacteria of the gastro-intestinal tract of surgeonfish (Angert et al., 1993) or of the bovine rumen (Stahl et al., 1988).

Analysing gut microbiota 333 Limitations of FISH Although FISH is a reliable technique, there are a number of limitations that have to be taken into consideration. The first limitation concerns the limit of detection. The total number of bacterial cells in 1 g human faeces (wet weight) is in the range of 1011 cells. If it is assumed that the average number of total bacterial cells per microscopic field is 1,000 it becomes clear that a species present at a concentration of 108 cells/g wet faeces will result on the average in only 1 cell per microscopic field. If a tenfold lower faecal dilution is used, the limit of detection is in the range of 107 cells/g wet faeces. If the number of cells per microscopic field is so low, a large number of microscopic fields have to be inspected to keep the statistical error small. In comparison, the detection limit for a given target organism in faeces may be considerably lower with the classical plating technique, provided less diluted samples are used and the growth of the background microbiota is suppressed by the use of selective media. Poor fluorescence may be another limitation of FISH. This may be due to an insufficient permeability of the cell envelope (Bidnenko et al., 1998) or due to a low content of ribosomes, which indicates that the cells are in a metabolically inactive state (Fegatella et al., 1998). Another explanation can be found in the fact that some probes do not bind effectively to the rRNA of the target organism although the in silico analysis indicates a perfect match with the target region. It has been demonstrated that the accessibility of the oligonucleotide probes depends on the target site within the 16S rRNA (Fuchs et al., 1998). It has been proposed that the dependence of probe binding on the target site is due to the intra- and intermolecular interactions of the ribosomal RNAs and proteins. The use of unlabelled so-called helper nucleotides, which are complementary to the neighbouring regions of the target site, may improve the accessibility of the labelled oligonucleotide probes to their target site considerably (Fuchs et al., 2000). Efforts have also been made to enhance the signal intensity in FISH. The signal-to-noise ratio not only depends on the quantum yield and molar absorbance of the fluorochrome used but also on the auto-fluorescence of the microbial cells and of the material present in the sample. Unspecific binding of the label may add to the background noise. Several alternatives for enhancing the probe signals have been tested. Indirect assays take advantage of reporter molecules such as biotin or digoxigenin bound to the oligonucleotide probe (Amann et al., 1992; Zarda et al., 1991). Following hybridisation, these reporter molecules may be detected by using avidin in the case of biotin, or specific antibodies in the case of digoxigenin. The antibodies are labelled with several molecules of a fluorescent dye or coupled to an enzyme which catalyses the formation of a product which can be visualised. A problem sometimes encountered with this labelling method stems from limitations in the accessibility of the bulky labelling complexes to their binding site in the fixed cell. It should be noted that these methods of signal enhancement have not found wide application. Even if a probe has been tested experimentally with representative faecal bacteria it is impossible to exclude the possibility that organisms other than the

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targeted one are detected. This may be the case if an unknown organism whose 16S rRNA sequence is not yet in the database happens to have the same target site and becomes fluorescently labelled although it is phylogenetically unrelated. This possibility always exists although it is probably a relatively rare event. If the targeted organisms are rod-shaped, the presence of fluorescently labelled cocci indicates that the latter are non-target organisms which have been labelled un-specifically.

14.3

Methods of characterising human gut microbiota

The human intestinal microbiota may have beneficial or adverse effects on human health. However, manipulating this complex system by dietary intervention requires better knowledge of this microbial community. To identify links between nutrition and microbiota mediated effects on health and disease, detailed microbial studies in human populations are urgently needed. On the one hand, the culture independent methods described above are ideally suited to perform such analyses, on the other hand they are time consuming as long as the enumeration is performed manually. The manual counting of bacteria is tedious and requires skilled personnel. There is therefore a need for more rapid, automated analyses of faecal samples on the basis of 16S rRNA targeted oligonucleotide probes. Two methods have been used to enumerate fluorescently labelled cells; image analysis and flow cytometry. 14.3.1 Automated microscopic detection of single cells by image analysis Image analysis aims to automate the counting of fluorescently labelled cells in faecal dilutions. In principle, the preparation of the faecal sample and the hybridisation are performed in the same way as described above. However, the fluorescent bacteria are not visualised and counted manually, but rather identified by image analysis. In principle this analysis consists of image acquisition, image storage, object recognition and object enumeration. Automated image acquisition requires a high precision motorised microscope stage whose movements in three dimensions can be programmed. Images are captured at predetermined positions using a charge coupled device (CCD) camera and stored as electronic files. The automatic image capture and the analysis routines require an optimal distribution of the bacterial cells in a faecal suspension on the slide. The slide co-ordinates for image capture may be chosen randomly or follow a programmed movement pattern within a well. In order to obtain representative cell counts, the concentration of cells on the surface should not vary excessively. One of the most difficult tasks during image capture is auto-focusing. Unfortunately, it is not sufficient to focus manually at one position of each well of a slide and to store the z-axis position, which determines the vertical position, i.e., the distance between the objective and the microscope stage, and to subsequently start the automated analysis routine. Owing to the fact that the z-axis position delivering a focused

Analysing gut microbiota 335 image may vary considerably from one x/y-axis position (movement in the horizontal plane) to another, it is necessary to recover a given z-axis position with an accuracy of only a few micrometres. Jansen and colleagues were the first ones who introduced an automated microscopy based image analysis system in gut microbiology for the analysis of intestinal bacteria in faecal samples (Jansen et al., 1999). Auto-focusing by this system involves the acquisition of a stack of images taken at various z-positions differing by 1 m. With this approach the proportion of out-of-focus images was approximately 1%. At the time when the protocol was published, the system afforded the analysis of 1,200 images within 20 h, which corresponds to 24 samples, each analysed twice, i.e., two wells per sample. In view of the improvements in computer technology since that time it is certain that the speed and accuracy of this analysis has greatly improved. Other microscopy-based operator-independent image analysis systems for the counting of multiple samples have been optimised for the analysis of marine (Pernthaler et al., 2003) or oral microbial ecosystems (Gmur et al., 2000; Singleton et al., 2001). Once the captured images have been stored in digital format, the fluorescing cells in the image have to be identified based on their fluorescence, size and shape. Commercial and non-commercial systems afford the adjustment of the recognition parameters to the type of samples analysed and to the actual oligonucleotide probe used. Potential problems may arise from the presence of weakly fluorescent non-target cells. To exclude these cells from being recognised as target cells a threshold of fluorescence intensity may be defined. The fidelity of the enumeration procedure may also be hampered by a high proportion of target cells whose fluorescence intensity is below the threshold. This problem not only impairs the automated cell recognition but also the manual identification of target cells. Poor specificity of a probe or unfavourable hybridisation conditions may account for this problem (see 14.3.3). However, poor fluorescence may also be due to reduced metabolic activity of the target cells which is associated with a lower content of ribosomal RNA. 14.3.2 Detection of single cells by flow cytometry (FCM) Flow cytometry (FCM) has found widespread application in medical diagnostics. It is used primarily for the enumeration of blood cells but has also been applied to other cells. One of the main advantages of using FCM is its unsurpassed speed of operation enabling the enumeration of up to 1,000 cells per second. However, the use of FCM in microbiology has been limited. This is primarily due to the small size of bacterial cells and the low signal intensity they produce (Steen, 1992). In spite of these disadvantages of bacterial cells in comparison to blood cells, a combination of FCM and FISH was successfully optimised for use in conjunction with rRNA targeted oligonucleotide probes (Wallner et al., 1993). While this study was done on pure cultures, FCM has also been applied to the analysis of environmental samples including faeces (Amann et al., 1990; Simon et al., 1995; Zoetendal et al., 2002).

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Fig. 14.5 Detection of fluorescently labelled bacteria with flow cytometry.

In FCM, cells in solution are passed through a small orifice so that they flow in single file through an illuminated volume where they can be registered electronically based on the light they scatter and the fluorescence they emit (Fig. 14.5). The features used for the discrimination of the cells include cell size, which is indicated by the forward angle light scatter (FSC), internal structure (granularity), which is indicated by the 90ë-angle light scatter (SSC) and intensity of fluorescence at one or several wavelengths depending on the available lasers and fluorochromes (Wallner et al., 1997). Using FISH combined with FCM (FISH-FCM), Rigottier-Gois and coworkers quantified five Bacteroides species in faecal samples from 20 healthy adults using species specific fluorescently labelled oligonucleotide probes (Rigottier-Gois et al., 2003) relative to all bacteria detected with the EUB338 probe, which essentially detects all members of the domain Bacteria. Bacteroides fragilis and Bacteroides vulgatus were detected in 90% and 85% of the individuals tested and represented on average proportions of 3.9 and 4.2% of total cells, respectively, with large variations between individuals. The other three species (B. ovatus, B. distasonis and B. putredinis) were considerably less frequent. The authors of this work used the NON338 probe, which is complementary to the EUB338 probe (Wallner et al., 1993) as a negative control because it does not bind to the 16S rRNA. Both EUB338 and NON338 were labelled with fluorescein isothiocyanate (FITC), while the oligonucleotide probes targeting the five Bacteroides species were labelled with Cy5. An argon ion laser (488 nm) was used for determining the FSC and the SSC and for exciting FITC labelled probes, while a red diode laser (635 nm) was employed to excite the Cy5 labelled probes. In addition to enumerating fluorescently labelled cells, FISH-FCM also makes it possible to determine the labelling

Analysing gut microbiota 337 efficiency for a given probe (Fuchs et al., 1998; Rigottier-Gois et al., 2003). The labelling efficiency may be deduced from the fluorescence intensity conferred by the species specific probes relative to the fluorescence intensity conferred by the EUB338 probe using the same fluorochrome for both probes. Rigottier-Gois and co-workers reported that the relative fluorescence intensity for the five Bacteroides species specific probes varied from 46 to 102% (Rigottier-Gois et al., 2003). In another study, which took advantage of FISH-FCM for investigating the faecal microbiota, an oligonucleotide probe targeting an uncultured Ruminococcus obeum-like bacterium was used to enumerate this organism in faecal samples collected from three healthy adults on three occasions during a period of four weeks (Zoetendal et al., 2002). In contrast to Rigottier-Gois et al. (2003), who determined the proportion of their target organisms relative to all bacteria as detected with the EUB338 probe, these researchers determined the cell counts. This was possible because they added fluorescent beads at known concentrations to the bacterial suspensions to be analysed. They also compared the bacterial counts obtained by FISH-FCM to the counts obtained by FISH combined with microscopic image analysis. The ratio between the two numbers varied between 0.64 and 1.67 and the mean proportions of the Clostridium coccoides-Eubacterium rectale group, which were also determined, were 16.9% as determined by microscopy and 13.5% as determined by FISH-FCM (Zoetendal et al., 2002). These results indicate that the two techniques give similar results. Taken together, FISH-FCM is a very powerful method for the enumeration of faecal bacteria, the main advantage being the speed of analysis. This feature makes FISH-FCM really suitable for high throughput analysis of faecal samples. Another advantage lies in the possibility to sort cells and thereby enrich targeted bacteria, including uncultured organisms, although this may be limited by cell aggregation. It is even possible to amplify ribosomal DNA fragments from sorted cells (Wallner et al., 1997). 14.3.3 Microbiota characterisation by DNA arrays The FISH methods described so far have the disadvantage that the number of organisms that can be targeted simultaneously is limited by the number of fluorochromes. As a consequence, the analysis of a complex microbial community like the one in the human intestinal tract is usually restricted to a panel of selected target organisms. Community analysis at a high level of resolution with FISH is very time consuming. DNA arrays or DNA chips may circumvent these limitations as they allow the parallel application of multiple probe sets. In principle, oligo- or polynucleotide probes are immobilised on a matrix such as glass, silicon or nylon membranes (Ramsay, 1998) and serve to capture labelled RNA or DNA molecules containing complementary sequences which allow the formation of DNA-DNA or DNA-RNA hybrids (Fig. 14.6).

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Fig. 14.6 Detection of 16S rRNAs with DNA arrays. Each spot on the array may target 16S rRNAs from different organisms. Labelled RNAs that hybridise with the immobilised capture probes will result in a signal.

So far, DNA chip technology has been used primarily on eukaryotic cells for monitoring the expression of target genes, large-scale gene discovery and the detection of mutations, but DNA chips have also been tested for their applicability to the analysis of microbial ecosystems (Guschin et al., 1997; Rudi et al., 2000; Small et al., 2001). However, the advantage of the array technology to detect a large number of target molecules simultaneously is seriously hampered by a number of problems that have to be solved. These include the necessity in microbial ecology to identify nucleic acids against a largely unknown genetic background (Peplies et al., 2003). Moreover, the recognition of single mismatches is crucial for the analysis of microbial ecosystems but difficult to realise as the optimal hybridisation conditions may differ between probes within a large set of probes. The adjustment of stringency for optimal discrimination as usually applied to conventional hybridisation assays is difficult to apply to DNA micro-array assays because one set of conditions cannot provide optimal target discrimination for all probes (El Fantroussi et al., 2003). Efficient labelling of the target DNA is a prerequisite for the identification of captured DNA or RNA. The use of labelled primers for the amplification by PCR of 16S rRNA genes is a possible way to label the target DNA (Peplies et al., 2003). However, it is well known that biases may be introduced by PCR (Speksnijder et al., 2001) and, therefore, the direct labelling of RNA isolated from a microbial community is preferable although the detection sensitivity may be considerably reduced. Multiple biotin labels introduced along the target RNA molecule were found to be ineffective while the use of labelled detector probes, which specifically bind to the target near the region that hybridises to immobilised capture probe was reported to be more efficient (Fig. 14.7); with

Analysing gut microbiota 339

Fig. 14.7 Detection of unlabelled 16S rRNAs by the combined use of capture probes and labelled indicator probes, which can bind to the captured target RNAs.

this strategy it was possible specifically to detect 16S rRNA from Geobacter chapellii in RNA extracts from soil (Small et al., 2001). Systematic studies have been performed to define the parameters that are crucial for specific hybridisation. The monitoring of the association/dissociation conditions would be a solution to this problem as the thermal dissociation analysis of all probe hybrids performed in parallel on a single DNA array would provide information to achieve a better discrimination between matched and mismatched duplexes (Liu et al., 2001). It was demonstrated that the analysis of the melting profile for each duplex provides information on the specificity of hybridisation, thus enabling the discrimination between target and non-target sequences (El Fantroussi et al., 2003). Applying neural network analyses, Urakawa and co-workers systematically investigated the effects of the position of a single base pair mismatch and the type of mismatch on the dissociation temperature and signal intensity of short DNA duplexes using oligonucleotide micro-arrays (Urakawa et al., 2002). The trained neural networks predicted the dissociation temperature with relatively high precision, while the prediction of the signal intensity, whose variability could partly be explained by the formamide concentration used, was poor. The analyses also indicated that the position of the mismatch at or near the 50 end is more important for determining the dissociation temperature than the type of mismatch. A systematic investigation defined a number of determinants for specific and highly discriminative DNA micro-array hybridisations and devised means for optimising the specific detection of target DNA or RNA (Peplies et al., 2003). It was shown that the standard FISH protocol can be transferred to the DNA micro-array analysis. Best hybridisation results were obtained by applying moderately stringent conditions, while discrimination of a mismatch near the terminus of a duplex was difficult to achieve. Steric hindrance was identified as an important factor that influences the binding efficiency of target molecules to

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the capture probes immobilised on a solid matrix. The size of the target DNA or RNA and the distance between the solid surface and its distance to the capture probe were demonstrated to be important determinants for the signal intensity. The application of poly(A) spacers of various lengths (6 to 24 nucleotides) and the addition of helper nucleotides that bind near the target sites of the capture probes improved the signal intensity in most cases considerably (Peplies et al., 2003). The results obtained by these researchers also suggested that fragmentation of the target DNA or RNA may lead to false positive signals. So far there is only one example for the application of the DNA array technology to the analysis of faecal microbiota (Wang et al., 2002). Three redundant 16S rRNA targeted 40-mer oligonucleotides were designed for each of 20 predominant human intestinal species and immobilised on glass slides. DNA was extracted from faecal samples of three healthy individuals and the complete 16S rRNA gene was amplified using Cy5 labelled dCTP in PCR. The labelled PCR products were applied to the oligonucleotide micro-arrays for hybridisation analysis. The results indicated some variability in the composition of the target organisms between the three individuals. In most cases, the redundant probes for a given target organism were well in agreement. Moreover, the bacteriocidal effect of an antibiotic treatment of an individual was clearly reflected in the results of the micro-array analysis performed on faecal samples from this individual. After antibiotic treatment of the targeted bacteria only Escherichia coli and Bacteroides fragilis could be detected. It may be concluded that micro-array based methods for the detection of bacteria with 16S rRNA target probe has been shown to work in principle. However, it is also clear that there are many parameters that have to be taken into consideration and optimised to avoid false positive or false negative signals. There is no doubt that an essentially error-free micro-array based detection method would greatly facilitate the microbial analysis of large numbers of samples. However, this method has to be further optimised for use on human faecal samples.

14.4 Using denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) for characterising microbiota DGGE and TGGE have become very popular tools among microbial ecologists since they allow the molecular characterisation of the diversity of microbial ecosystems and the monitoring of population dynamics (Muyzer, 1999). The technique is based on the amplification by PCR of 16S rRNA genes present in DNA extracted from environmental samples such as soil or faeces. However, in contrast to a normal PCR, one of the primers employed in the reaction differs from a normal PCR primer in that it has a long stretch of 40±50 G and C nucleotides attached to it. Following amplification, this so-called GC-clamp prevents the two DNA strands from dissociating completely, even if highly

Analysing gut microbiota 341 denaturing conditions are applied. The polyacrylamide gels employed for separating the amplification products are characterised by an increasing gradient of temperature (TGGE) or denaturing agents such as urea or formamide (DGGE). In principle, these gels afford the separation of DGGE-PCR products that differ in as little as one nucleotide (Sheffield et al., 1989). DGGE and TGGE have been employed to follow changes in microbiota composition with time and space. Each band in a D/TGGE-gel represents the population groups whose target sequence was amplified. To identify the species corresponding to a band in the gel, the DNA fragment of this band can be recovered from the gel and subjected to sequence analysis. Alternatively, bacteria isolated from the same sample are also subjected to D/TGGE-PCR and applied to the same gel. There are a number of excellent examples of the usefulness of this technique, including an investigation on the succession of microbial populations during colonisation of the intestinal tract of newborn babies (Favier et al., 2002). However, it is noteworthy that the use of a GCclamp primer may cause artefacts during annealing and that the heteroduplexes formed in PCR may lead to unspecific bands because the mismatched base pairs are less stable (Ruano and Kidd, 1992).

14.5

Future trends

14.5.1 The need for the isolation and phenotypic characterisation of new gut micro-organisms The knowledge of the identity and the numerical importance of microbial population groups in the human GI-tract are certainly important aspects for characterising this microbial ecosystem but insufficient to appreciate their possible role in host health and disease. To define the potential role of a given organism in a complex bacterial community it is necessary to culture the key organisms of this microbial community. Only then is it possible to investigate their physiology and biochemistry which are important determinants for their respective contribution to the metabolic processes catalysed by the gut microbiota. The same applies to the study of bacteria-bacteria and bacteriahost interactions. In view of the fact that the culture independent methods indicate that a large proportion of gut bacteria have eluded cultivation, it is obvious that the isolation and characterisation of new gut bacteria is still an important task. There is an urgent need for fast, reliable and easy-to-perform methods for analysing the composition of the human gut microbiota with high resolution and precision. The principal experimental approaches described above have the potential to be applied as high-throughput methods. There is no doubt that DNA arrays enable the quickest microbial analysis of faecal samples because the number of probes that can be encompassed on a microarray and hybridised with target nucleic acids is so high that a high resolution analysis is feasible. However, as mentioned above, there are still a number of serious problems to be

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solved to make this an easy-to-use method that will find widespread application in gut microbiology. It can be expected that the existing problems will be solved in the near future and that highly discriminative oligonucleotide probes targeting all relevant bacterial populations of the human gut will become available for large-scale human studies. These probes will have to be optimised for use on DNA arrays. The number of probes available for analysis of the human gut microbiota is still limited and needs to be increased. On the one hand, it will remain difficult to transform the signals of an oligonucleotide array into cell numbers of the corresponding target populations. On the other hand, the knowledge of the presence or absence of a bacterial population group is valuable information per se and, moreover, this method has the potential to be automated, which is an invaluable advantage. At present, FISH-FCM is the quickest available method for analysing human gut microbiota. The number of available and validated probes is sufficient to cover the microbial community of the gut at the group level to approximately 90%. In future, the proportion of probes targeting faecal bacteria at species level will increase and hence the resolution and the potential relevance of the results will be improved. While the FISH-FCM analysis itself is essentially automated, the sample preparation is not, but could be facilitated and accelerated by automation. It can also be expected that the possibility of sorting bacteria with FCM may lead to the isolation of previously undescribed bacteria. Future efforts will also be directed at the in situ detection of bacterial activities. The ultimate objective is to study the in vivo effects of dietary and host factors on interactions of microbial populations in the human gastrointestinal tract. Principal approaches include the in situ detection of mRNA which has been shown to work in model systems (Hodson et al., 1995; Holmstrom et al., 1999; Tolker-Nielsen et al., 1997). Since enzyme expression usually correlates with enzyme activity, the level of specific mRNAs may reflect the activity of the corresponding enzyme. However, a number of obstacles have to be overcome to make mRNA detection a widely applied method. In contrast to 16S rRNA, mRNA has a shorter lifetime and is usually present in smaller concentrations. Hence, the detection of mRNAs with fluorescently labelled oligonucleotide probes, which works well with rRNA, is difficult to achieve because the number of target mRNA molecules in a cell is too low to render the cell fluorescent (Hodson et al., 1995). The in situ amplification of mRNAs is a more promising approach, but it depends on the effective permeabilisation of the cell wall so that nucleotides, primers and enzymes can enter the cell where they catalyse the reverse transcription and subsequent linear amplification of the resulting c-DNA. To render the cells detectable, the primers or the nucleotides used in PCR have to be labelled. For example, digoxigenin (DIG)-labelled dUTP has often been used because it can be recognised by antibodies conjugated to enzymes (e.g. alkaline phosphatase) capable of converting suitable precursors into detectable molecules. Alternatively DIG may be recognised with anti-DIG antibodies labelled with a fluorescing dye such as fluorescein. The loss of label from the

Analysing gut microbiota 343

Fig. 14.8

In situ detection of metabolic activity using fluorogenic substrates.

permeabilised cells by diffusion may be a major problem of detection (Hodson, 1995). Methodological improvements in the future may help to make the in situ detection of mRNA applicable to the analysis of gene expression in gut microorganisms. The detection of enzyme activity at the cellular level may also be accomplished by the use of fluorogenic substrates whose uptake into the cell and the ensuing cleavage results in the accumulation of the cleaved-off fluorescence moiety rendering the cell detectable by FISH or FISH-FCM (Lewis et al., 1994). The formation of a fluorescent dye in the cytoplasm requires that the transport system and the enzyme catalysing the cleavage recognise the fluorogenic substrate in the same way as the natural substrate (Fig. 14.8). The loss of the fluorescent product by leakage out of the cell may be a problem which can be largely prevented by the use of mildly thiol-reactive fluorochromes which upon their cleavage from the fluorogenic substrate may react with cysteine-containing cellular proteins. Presently, the application of these functional probes is restricted to a small number of enzymes such as galactosidase, -glucosidase and -glucuronidase because the spectrum of the available fluorogenic substrates is limited. Nevertheless, some of these enzymes are of some relevance for the human gut ecosystem. The use of fluorogenic substrates has a lot of potential because their application can be combined with the 16S rRNA approach which reveals at the same time the metabolic activity and the identity of bacteria at the cellular level. It should be noted, however, that cells already fixed for the application of 16S rRNA targeted oligonucleotides, usually lose enzymatic activity. Consequently, the metabolic labelling has to be

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done with live cells first and the phylogenetic labelling has to be applied in a second step. It can be assumed that the combination of phylogenetic and metabolic labelling will find a more widespread application in the future and that the spectrum of the available fluorogenic substrates will be increased.

14.6

Sources of further information and advice

The phylogenetic analysis of ribosomal RNA sequences and the design of rRNA targeted oligonucleotide probes require the use of up-to-date sequence data sets and analysis tools. The internet sites shown below provide access to databases of 16S rRNA sequences and enable the user to perform sequence alignments, integrate new sequences into the database, obtain phylogenetic trees and design diagnostic oligonucleotide probes. These internet sites contain additional links related to phylogeny and microbial ecology. GenBank EMBL The European Ribosomal Database The Ribosomal Database Project The Arb project probeBase.net

14.7

http://www.ncbi.nlm.nih.gov http://www.ebi.ac.uk http://oberon.fvms.ugent.be:8080/ rRNA/index.html http://rdp.cme.msu.edu/html/ http://www.arb-home.de/ http://www.microbial-ecology.net/ probebase/index2.html

References

(1990) `Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations'. Appl Environ Microbiol, 56, 1919± 1925. AMANN R, ZARDA B, STAHL D A, SCHLEIFER K-H (1992) `Identification of individual prokaryotic cells with enzyme-labeled, rRNA-targeted oligonucleotide probes'. Appl Environ Microbiol, 58, 3007±3011. AMANN R I, LUDWIG W, SCHLEIFER K-H (1995) `Phylogenetic identification and in situ detection of individual microbial cells without cultivation'. Microbiol Rev, 59, 143±169. ANGERT E R, CLEMENTS K D, PACE N R (1993) `The largest bacterium'. Nature (London), 362, 239±241. ARANKI A, SYED S A, KENNEY E B, FRETER R (1969) `Isolation of anaerobic bacteria from human gingiva and mouse caecum by means of a simplified glove box procedure'. Appl Microbiol, 17, 568±576. BENTLEY D W, NICHOLS R L, CONDON R E, GORBACH S L (1972) `The microflora of the human ileum and intraabdominal colon: Results of direct needle aspiration at surgery and evaluation of the technique'. J Lab Clin Med, 79, 421±429. AMANN R I, BINDER B J, OLSON R J, CHISHOLM S W, DEVEREUX R, STAHL D A

Analysing gut microbiota 345 (1998) `Estimation of the state of the bacterial cell wall by fluorescent in situ hybridization'. Appl Environ Microbiol, 64, 3059±3062. BONNET R, SUAU A, DORE J, GIBSON G R, COLLINS M D (2002) `Differences in rDNA libraries of faecal bacteria derived from 10- and 25-cycle PCRs'. Int J Syst Evol Microbiol, 52, 757±763. DUNCAN S H, HOLD G L, BARCENILLA A, STEWART C S, FLINT H J (2002) `Roseburia intestinalis sp. nov., a novel saccharolytic, butyrate-producing bacterium from human faeces'. Int J Syst Evol Microbiol, 52, 1615±1620. BIDNENKO E, MERCIER C, TREMBLAY J, TAILLIEZ P, KULAKAUSKAS S

EL FANTROUSSI S, URAKAWA H, BERNHARD A E, KELLY J J, NOBLE P A, SMIDT H, YERSHOV G M,

(2003) `Direct profiling of environmental microbial populations by thermal dissociation analysis of native rRNAs hybridized to oligonucleotide microarrays'. Appl Environ Microbiol, 69, 2377±2382. FAVIER C F, VAUGHAN E E, DE VOS W M, AKKERMANS A D (2002) `Molecular monitoring of succession of bacterial communities in human neonates'. Appl Environ Microbiol, 68, 219±226. FEGATELLA F, LIM J, KJELLEBERG S, CAVICCHIOLI R (1998) `Implications of rRNA operon copy number and ribosome content in the marine oligotrophic ultramicrobacterium Sphingomonas sp. strain RB2256'. Appl Environ Microbiol, 64, 4433±4438. FINEGOLD S M (1986) `Normal human intestinal flora'. Ann Ist Super Sanita, 22, 731±737. FINEGOLD S M, ATTEBERY H R, SUTTER V L (1974) `Effect of diet on human fecal flora: comparison of Japanese and American diets'. Am J Clin Nutr, 27, 1456±1469. FINEGOLD S M, SUTTER V L, MATHISEN G E (1983), `Normal indigenous intestinal flora', in Hentges D J, Human intestinal microflora in health and disease, New York/ London, Academic Press, 3±31. FUCHS B M, WALLNER G, BEISKER W, SCHWIPPL I, LUDWIG W, AMANN R (1998) `Flow cytometric analysis of the in situ accessibility of Escherichia coli 16S rRNA for fluorescently labeled oligonucleotide probes'. Appl Environ Microbiol, 64, 4973± 4982. FUCHS B M, GLOCKNER F O, WULF J, AMANN R (2000) `Unlabeled helper oligonucleotides increase the in situ accessibility to 16S rRNA of fluorescently labeled oligonucleotide probes'. Appl Environ Microbiol, 66, 3603±3607. GMUR R, GUGGENHEIM B, GIERTSEN E, THURNHEER T (2000) `Automated immunofluorescence for enumeration of selected taxa in supragingival dental plaque'. Eur J Oral Sci, 108, 393±402. STAHL D A

GUSCHIN D Y, MOBARRY B K, PROUDNIKOV D, STAHL D A, RITTMANN B E, MIRZABEKOV A D

(1997) `Oligonucleotide microchips as genosensors for determinative and environmental studies in microbiology'. Appl Environ Microbiol, 63, 2397±2402. HARMSEN H J, WILDEBOER-VELOO A C, GRIJPSTRA J, KNOL J, DEGENER J E, WELLING G W (2000) `Development of 16S rRNA-based probes for the Coriobacterium group and the Atopobium cluster and their application for enumeration of Coriobacteriaceae in human feces from volunteers of different age groups'. Appl Environ Microbiol, 66, 4523±4527. HARMSEN H J, RAANGS G C, HE T, DEGENER J E, WELLING G W (2002) `Extensive set of 16S rRNA-based probes for detection of bacteria in human feces'. Appl Environ Microbiol, 68, 2982±2990. HODSON R E, DUSTMAN W A, GARG R P, MORAN M A (1995) `In situ PCR for visualization of microscale distribution of specific genes and gene products in prokaryotic communities'. Appl Environ Microbiol, 61, 4074±4082.

346

Functional foods, ageing and degenerative disease

(2003) `Oligonucleotide probes that detect quantitatively significant groups of butyrate-producing bacteria within the human fecal flora'. Applied and Environmental Microbiology, 69, 4320±4324. HOLMSTROM K, TOLKER-NIELSEN T, MOLIN S (1999) `Physiological states of individual Salmonella typhimurium cells monitored by in situ reverse transcription-PCR'. J Bacteriol, 181, 1733±1738. HUNGATE R E (1969), A roll tube method for cultivation of strict anaerobes, New York, London, Academic Press. JANSEN G J, WILDEBOER-VELOO A C, TONK R H J, FRANKS A H, WELLING G W (1999) `Development and validation of an automated, microscopy-based method of enumeration of groups of intestinal bacteria'. J Microbiol Methods, 37, 215±221. KREADER C A (1996) `Relief of amplification inhibition in PCR with bovine serum albumin or T4 gene 32 protein'. Appl Environ Microbiol, 62, 1102±1106. HOLD G L, SCHWIERTZ A, AMINOV R, BLAUT M, FLINT H J

LANGENDIJK P S, SCHUT F, JANSEN G J, RAANGS G C, KAMPHUIS G R, WILKINSON M H, WELLING

(1995) `Quantitative fluorescence in situ hybridization of Bifidobacterium spp. with genus-specific 16S rRNA-targeted probes and its application in fecal samples'. Appl Environ Microbiol, 61, 3069±3075. LEE D H, ZO Y G, KIM S J (1996) `Nonradioactive method to study genetic profiles of natural bacterial communities by PCR-single-strand-conformation polymorphism'. Appl Environ Microbiol, 62, 3112±3120. LEWIS P J, NWOGUH C E, BARER M R, HARWOOD C R, ERRINGTON J (1994) `Use of digitized video microscopy with a fluorogenic enzyme substrate to demonstrate cell- and compartment-specific gene expression in Salmonella enteritidis and Bacillus subtilis'. Mol Microbiol, 13, 655±662. LIU W T, MIRZABEKOV A D, STAHL D A (2001) `Optimization of an oligonucleotide microchip for microbial identification studies: a non-equilibrium dissociation approach'. Environ Microbiol, 3, 619±629. MOORE W E, HOLDEMAN L V (1974) `Human fecal flora: the normal flora of 20 JapaneseHawaiians'. Appl Microbiol, 27, 961±979. MUYZER G (1999) `DGGE/TGGE a method for identifying genes from natural ecosystems'. Curr Opin Microbiol, 2, 317±322. MUYZER G, DE WAAL E C, UITTERLINDEN A G (1993) `Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA'. Appl Environ Microbiol, 59, 695±700. OLSEN G J, LANE D J, GIOVANNONI S J, PACE N R, STAHL D A (1986) `Microbial ecology and evolution: a ribosomal RNA approach'. Annu Rev Microbiol, 40, 337±365. È CKNER F O, AMANN R (2003) `Optimization strategies for DNA microarrayPEPLIES J, GLO based detection of bacteria with 16S rRNA-targeting oligonucleotide probes'. Appl Environ Microbiol, 69, 1397±1407. PERNTHALER J, PERNTHALER A, AMANN R (2003) `Automated enumeration of groups of marine picoplankton after fluorescence in situ hybridization'. Appl Environ Microbiol, 69, 2631±2637. RAMSAY G (1998) `DNA chips: state-of-the art'. Nat Biotechnol, 16, 40±44. RIGOTTIER-GOIS L, ROCHET V, GARREC N, SUAU A, DORE J (2003) `Enumeration of Bacteroides species in human faeces by fluorescent in situ hybridisation combined with flow cytometry using 16S rRNA probes'. Syst Appl Microbiol, 26, 110±118. RUANO G, KIDD K K (1992) `Modeling of heteroduplex formation during PCR from mixtures of DNA templates'. PCR Methods Appl, 2, 112±116. GW

Analysing gut microbiota 347 (2000) `Application of sequencespecific labeled 16S rRNA gene oligonucleotide probes for genetic profiling of cyanobacterial abundance and diversity by array hybridization'. Appl Environ Microbiol, 66, 4004±4011.

RUDI K, SKULBERG O M, SKULBERG R, JAKOBSEN K S

SCHWIERTZ A, HOLD G L, DUNCAN A, GRUHL B, COLLINS M D, LAWSON A M, FLINT H J, BLAUT M

(2002a) `Anaerostipes caccae gen. nov., sp. nov., a new saccharolytic, acetateutilising, butyrate-producing bacterium from human faeces'. Syst Appl Microbiol, 25, 46±51. SCHWIERTZ A, LEHMANN U, JACOBASCH G, BLAUT M (2002b) `Relevance of Eubacterium species for butyrate production in the human intestine'. J Appl Microbiol, 93, 157± 162. SGHIR A, GRAMET G, SUAU A, ROCHET V, POCHART P, DOREÂ J (2000) `Quantification of bacterial groups within human fecal flora by oligonucleotide probe hybridization'. Appl Environ Microbiol, 66, 2263±2266. SHEFFIELD V C, COX D R, LERMAN L S, MYERS R M (1989) `Attachment of a 40-base-pair G + C-rich sequence (GC-clamp) to genomic DNA fragments by the polymerase chain reaction results in improved detection of single-base changes'. Proc Natl Acad Sci U S A, 86, 232±236. SIMMERING R, KLEESSEN B, BLAUT. M (1999) `Quantification of the flavonoid-degrading bacterium Eubacterium ramulus in human fecal samples with a species-specific oligonucleotide hybridization probe'. Appl Environ Microbiol, 65, 3705±3709. SIMMERING R, TARAS D, SCHWIERTZ A, LE BLAY G, GRUHL B, LAWSON P A, COLLINS M D, BLAUT

(2002) `Ruminococcus luti sp. nov, isolated from a human faecal sample'. Syst Appl Microbiol, 25, 189±193. SIMON N, LEBOT N, MARIE D, PARTENSKY F, VAULOT D (1995) `Fluorescent in situ hybridization with rRNA-targeted oligonucleotide probes to identify small phytoplankton by flow cytometry'. Appl Environ Microbiol, 61, 2506±2513. M

SINGLETON S, CAHILL J G, WATSON G K, ALLISON C, CUMMINS D, THURNHEER T, GUGGENHEIM B, GMUR R (2001) `A fully automated microscope bacterial enumeration system for studies of oral microbial ecology'. J Immunoassay Immunochem, 22, 253±274. SMALL J, CALL D R, BROCKMAN F J, STRAUB T M, CHANDLER D P (2001) `Direct detection of 16S rRNA in soil extracts by using oligonucleotide microarrays'. Appl Environ Microbiol, 67, 4708±4716. SONG Y, LIU C, MOLITORIS D R, TOMZYNSKI T J, LAWSON P A, COLLINS M D, FINEGOLD S M

(2003) `Clostridium bolteae sp. nov., isolated from human sources'. Syst Appl Microbiol, 26, 84±89. SPEKSNIJDER A G, KOWALCHUK G A, DE JONG S, KLINE E, STEPHEN J R, LAANBROEK H J (2001) `Microvariation artifacts introduced by PCR and cloning of closely related 16S rRNA gene sequences'. Appl Environ Microbiol, 67, 469±472. STAHL D A, FLESHER B, MANSFIELD H R, MONTGOMERY L (1988) `Use of phylogenetically based hybridization probes for studies of ruminal microbial ecology'. Appl Environ Microbiol, 54, 1079±1084. STEEN H B (1992) `Noise, sensitivity, and resolution of flow cytometers'. Cytometry, 13, 822±830. SUAU A, BONNET R, SUTREN M, GODON J J, GIBSON G R, COLLINS M D, DORe J (1999) `Direct analysis of genes encoding 16S rRNA from complex communities reveals many novel molecular species within the human gut'. Appl Environ Microbiol, 65, 4799± 4807. TARAS D, SIMMERING R, COLLINS M D, LAWSON P A, BLAUT M (2002) `Reclassification of

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Eubacterium formicigenerans Holdeman and Moore 1974 as Dorea formicigenerans gen. nov., comb. nov., and description of Dorea longicatena sp. nov., isolated from human faeces'. Int J Syst Evol Microbiol, 52, 423±428. TOLKER-NIELSEN T, HOLMSTROM K, MOLIN S (1997) `Visualization of specific gene expression in individual Salmonella typhimurium cells by in situ PCR'. Appl Environ Microbiol, 63, 4196±4203. URAKAWA H, NOBLE P A, EL FANTROUSSI S, KELLY J J, STAHL D A (2002) `Single-base-pair discrimination of terminal mismatches by using oligonucleotide microarrays and neural network analyses'. Appl Environ Microbiol, 68, 235±244. WALLNER G, AMANN R, BEISKER W (1993) `Optimizing fluorescent in situ hybridization with rRNA-targeted oligonucleotide probes for flow cytometric identification of microorganisms'. Cytometry, 14, 136±143. WALLNER G, FUCHS B, SPRING S, BEISKER W, AMANN R (1997) `Flow sorting of microorganisms for molecular analysis'. Appl Environ Microbiol, 63, 4223±4231. WANG R F, BEGGS M L, ROBERTSON L H, CERNIGLIA C E (2002) `Design and evaluation of oligonucleotide-microarray method for the detection of human intestinal bacteria in fecal samples'. FEMS Microbiol Lett, 213, 175±182. WILSON K H, BLITCHINGTON R B (1996) `Human colonic biota studied by ribosomal DNA sequence analysis'. Appl Environ Microbiol, 62, 2273±2278. WOESE C R (1987) `Bacterial evolution'. Microbiol Rev, 51, 221±271. WOESE C R, KANDLER O, WHEELIS M L (1990) `Towards a natural system of organisms: proposal for the domains Archaea, Bacteria and Eucarya'. Proc Natl Acad Sci USA, 87, 4576±4579. ZARDA B, AMANN R, WALLNER G, SCHLEIFER K-H (1991) `Identification of single bacterial cells using digoxigenin-labelled, rRNA-targeted oligonucleotides'. J General Microbiol, 137, 2823±2830. ZOETENDAL E G, BEN-AMOR K, HARMSEN H J, SCHUT F, AKKERMANS A D, DE VOS W M (2002) `Quantification of uncultured Ruminococcus obeum-like bacteria in human fecal samples by fluorescent in situ hybridization and flow cytometry using 16S rRNAtargeted probes'. Appl Environ Microbiol, 68, 4225±4232.

15 Dietary lipids and immune function P. C. Calder, University of Southampton, UK

15.1 Introduction: the immune system in health, disease and ageing 15.1.1 The immune system in health The immune system acts to protect the host from infectious agents that exist in the environment (bacteria, viruses, fungi, parasites). It serves to distinguish `non-self ' from `self '. In addition the immune system plays an important role in the identification and elimination of tumour cells. Thus, an effective and efficient immune system is central to host defence against infectious diseases and cancer. A detailed description of the components of the immune system, how they work and how they interact is beyond the scope of this chapter. However, details may be found in standard immunology textbooks (e.g. Abbas et al., 1994). Suffice it to say that the immune system responds to challenge (e.g. a bacterial infection) with an increase in the activity of certain components that act to eliminate the source of the challenge. The human immune system has the capacity to respond to millions of antigens (these are the precise peptide components that are recognised as `non-self ' by the immune system and which trigger an immune response). This response will include both non-specific and specific actions that will involve a variety of cell types, mediators and chemical agents. The exact nature of the response will depend upon the origin and nature of the antigen (and so the origin and nature of the challenge). The immune response is, by definition, highly destructive and therefore it must be regulated in order that it does not proceed for a longer duration than is required and that it does not cause excessive damage to host tissues. Clearly, because of the many differing components involved in the immune response its regulation will be highly complex. However, one paradigm that has developed

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Fig. 15.1 The Th1, Th2 paradigm. Solid arrows indicate producers; broken arrows indicate regulators. IFN, interferon; IL, interleukin; Th, T helper cell.

to explain the promotion and regulation of an appropriate response is the socalled Th1, Th2 paradigm (Mossman and Sad, 1996). This describes the differentiation of the helper T lymphocyte (Th cell; defined by the appearance of the protein CD4 on its surface) along one of two phenotypic pathways (Fig. 15.1). The pathway followed depends upon the nature of the antigen being presented to the undifferentiated T helper cell and the cytokines present; cytokines are peptide mediators that have a hormone-like regulatory role within the immune response. An intracellular antigen, which will result from the phagocytic uptake of a bacterium, and the presence of interleukin (IL)-12, produced by the antigen-presenting cell, will promote differentiation along the Th1 pathway (Fig. 15.1). The Th1 cells thus produced will generate IL-2 and interferon (IFN)- . IL-2 will promote the proliferation of antigen-specific T lymphocytes while IFN- will activate the cells involved in elimination of bacteria, viruses, fungi and tumour cells (e.g. monocytes, macrophages, cytotoxic T lymphocytes, natural killer cells) and will promote the generation of antigen-specific immunoglobulin (Ig) G by B lymphocytes. Such antibodies can coat pathogens to facilitate their recognition and uptake by phagocytes. Extracellular antigens, for example due to infection with helminthic worms, and the presence of IL-4 will promote differentiation along the Th2 pathway (Fig. 15.1). The Th2 cells thus produced will generate IL-4, IL-5 and IL-13 (amongst other cytokines). IL-4 will promote the generation of antigen-specific IgE by B lymphocytes. The IgE will bind to and cross-link IgE receptors on the

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surface of mast cells and induce the extracellular release of the contents of mast cell granules. IL-5 promotes the activation of eosinophils. Mast cells and eosinophils are the mediators of host defence against extracellular parasites. Thus, Th1 and Th2 cells are involved in promoting an appropriate immune response to different types of infectious organisms. The final element of the Th1, Th2 paradigm is cross-regulation; IFN- inhibits the differentiation and activity of Th2 cells while IL-4 inhibits the differentiation and activity of Th2 cells (Fig. 15.1). Clearly this serves to cause a transient `skewing' of the immune response while an infectious agent is present. However, once the source of the infection is eliminated the system will return to the basal state in preparation for any subsequent infections. 15.1.2 The immune system in disease Whilst the capacity to mount vigorous Th1 or Th2 type responses is central to effective host defence against the range of possible infectious organisms, a number of human diseases are associated with inappropriate activation or activity of the immune response. These diseases appear to be associated with an inherent, inappropriate `skewing' towards either a Th1 or Th2 type response. There is a genetic predisposition towards such skewing, with susceptibility genes frequently being associated with antigen presentation (Howell et al., 2002). It is likely that the combination of genetic predisposition and certain environmental and/or lifestyle factors allows the disease to manifest itself. In some individuals the immune system responds to presentation of a host antigen (or a normally benign foreign one) rather than a foreign antigen; this triggers a Th1±type response with the destructive actions of the immune system targeted at those body sites where the antigen (or similar ones) is expressed. An example of such a disease is rheumatoid arthritis. In rheumatoid arthritis susceptibility genes map to antigen presentation proteins and this somehow results in host antigen being presented to T cells, which differentiate along the Th1 path. The Th1-type cytokines promote the activity of macrophages and the generation of auto-antibodies by B lymphocytes. The Th1type cytokines also suppress the Th2-type response and so skew the system towards the Th1 phenotype (Panayi, 1999). The host's synovial joints become infiltrated by activated T cells, macrophages and B cells. The macrophages produce a range of cytokines including tumour necrosis factor (TNF)-, IL-1 and IL-6 (Feldmann and Maini, 1999). Some effects of these so-called inflammatory cytokines are listed in Table 15.1. Clearly they have a role in innate immunity, in activating cellular immune responses and in co-ordinating the whole body response to infection and injury. However, in excess, they are involved in causing a range of local and systemic responses that are detrimental to the host. Macrophages will also produce the matrix proteases that degrade the cartilage and bone of the host. Other diseases in which an inappropriate Th1-type response appears to be involved include Crohn's disease, multiple sclerosis and atherosclerosis. Together these diseases are termed chronic inflammatory diseases.

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Table 15.1 List of some effects of the classic pro-inflammatory cytokines TNF, IL-1 and IL-6. Note that not all cytokines affect all responses listed equipotently Site of action

Biological effect

Hyothalamus Liver Vascular endothelium

Induction of fever Synthesis of acute phase proteins Activation ÿ! upregulation of adhesion molecules, chemokines, inducible nitric oxide synthase Activation ÿ! bone resorption Activation ÿ! matrix metalloproteinase synthesis ÿ! extracellular matrix degradation Activation of proteolysis ÿ! muscle wasting Activation of lipolysis; induction of insulin resistance Synthesis of TNF, IL-1, IL-6 and IL-8

Osteoclasts Fibroblasts Skeletal muscle Adipose tissue Monocytes and macrophages T cells B cells Bone marrow Monocytes, macrophages Neutrophils Kidney mesangial cells

Activation ÿ! proliferation and synthesis of IFN- Activation ÿ! antibody production Induction of haematopoiesis Induction of phospholipase A2 and COX-2 ÿ! synthesis of prostaglandins Chemotaxis Activation ÿ! degranulation, respiratory burst Chemotaxis Reactive oxygen species production

An inappropriate predisposition to skewing towards a Th2 response also occurs. Here the antigens involved are frequently normally benign foreign antigens, such as those present in cow's milk, egg, house dust mite, tree or grass pollen and are referred to as allergens since they can trigger allergy in sensitised individuals (Romagnani, 2000). The Th2 response results in production of allergen-specific IgE, which, when and where allergen is present, will induce mast cell degranulation, and in activation of eosinophils. This process can be termed allergic inflammation. 15.1.3 Ageing and the immune system Changes in the immune system appear to be associated with ageing. A full description of these is beyond the scope of this chapter and may be found elsewhere (Lesourd and Mazari, 1999; Lesourd et al., 2002). It appears that these age-related changes are not a general decline but rather a dysregulation. There is a decrease in the number of T cells, Th cells and cytotoxic T cells in the bloodstream with increasing age, and there appears to be a decrease in some T cell functions such as proliferation and production of IL-2. Whether IFN- production declines with age is not clear at present, although there are some reports that it does. Primary antibody responses to vaccination decline with age and the antibodies produced have lower antigen affinity. These changes are in part due to changes in B cell phenotypes; ageing is associated with a decline in

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the B cells that make antibodies against foreign antigens and with an increase in those that make auto-antibodies. There are a number of reports of increased production of Th2-type cytokines with age. Together these observations suggest that ageing is characterised by a progressive switch towards Th2-type responses. This may mean that elderly subjects are more susceptible to bacterial, viral and fungal infections, since they may not be able to mount a sufficiently strong Th1type response, and that they become progressively more responsive to allergens. Despite the apparent switch towards a Th2-type phenotype with ageing, monocyte/macrophage responses, particularly production of inflammatory cytokines, appear enhanced with ageing. For example, TNF- concentrations were higher in the plasma of 81-year-old subjects than in plasma from 18 to 30 year olds (Bruunsgaard et al., 2000). Likewise, there was a significant positive linear correlation between age and plasma TNF- concentration among subjects aged 20 to 100 years (Paolisso et al., 1998). Endotoxin-stimulated blood monocytes from older subjects produce greater amounts of IL-1 and IL-6 than those from young subjects (Meydani et al., 1991). Thus, ageing appears to be associated with an increased tendency towards inflammatory cytokine production. Given the effects of these cytokines (Table 15.1), this proinflammatory tendency might contribute to age-related chronic diseases and might also promote adverse responses to injury and surgery. 15.1.4 Adhesion molecules in health, disease and ageing Adhesion molecules are cell surface proteins involved in the cell-to-cell communication which contributes to the movement of leukocytes between body compartments. Such interactions involve adhesion molecules on the vascular endothelial cell surface and their ligand adhesion molecules on the leukocyte surface. These molecules serve to bind circulating leukocytes and to promote their movement into the sub-endothelial space. While this movement of leukocytes is important in mounting appropriate inflammatory and immune responses and in the homing of leukocytes to lymphoid organs, it also appears to play an important role in the development of atherosclerosis and chronic inflammatory diseases (Munro, 1993; Faull, 1995). Among the key adhesion molecules are vascular cell adhesion molecule 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1) and E-selectin. The expression of these molecules is up-regulated by inflammatory stimuli such as TNF- and reactive oxygen species. Soluble forms of VCAM-1, ICAM-1 and E-selectin (termed sVCAM-1, sICAM-1 and sE-selectin) are found in plasma, most likely as the result of shedding from the surface of activated endothelial cells. Thus, soluble adhesion molecule concentrations are believed to reflect endothelial inflammation. Indeed, the plasma concentrations of sVCAM-1, sICAM-1 and sE-selectin are elevated in inflammatory conditions (e.g. rheumatoid arthritis) in which there is increased expression of the cellular forms on endothelial and other cells (Gearing and Newman, 1993). Plasma concentrations of sICAM-1, sVCAM-1

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and sE-selectin are higher in individuals with cardiovascular disease than in controls and there is a positive correlation between the extent of atherosclerosis and the plasma concentrations of sICAM-1 (Rohde et al., 1998) and sVCAM-1 (Peter et al., 1997; de Caterina et al., 1997; Rohde et al., 1998). It was reported that sICAM-1 concentration predicts future myocardial infarction (Ridker et al., 1998) and that sICAM-1 concentration is a molecular marker of atherosclerosis and of other forms of coronary heart disease that is independent of other risk factors (Hwang et al., 1997). Ageing appears to be associated with increased endothelial inflammation as indicated by circulating soluble adhesion molecule concentrations. Positive correlations between age and the concentrations of sVCAM-1, sICAM-1 and sEselectin have been reported in Japanese (Morisaki et al., 1997), American (Hwang et al., 1997; Rohde et al., 1999) and British (Miles et al., 2001) subjects. For example, plasma concentrations of sICAM-1, sVCAM-1 and sEselectin were on average 60%, 38% and 14% higher in the blood of apparently healthy British males aged 55 to 75 years than in the blood of those aged 18 to 40 years (Miles et al., 2001).

15.2

Dietary fatty acids: nomenclature, sources and intakes

In Western countries an adult eats on average 75 to 150 g of fat each day and fat contributes 30 to 45% of dietary energy. By far the most important component of dietary fat in quantitative terms is triacylglycerol, which in most diets constitutes > 95% of dietary fat. Each triacylglycerol molecule is composed of three fatty acids esterified to a glycerol backbone. Other components of dietary fat, such as phospholipids, also contain fatty acids in their structure. Thus, fatty acids are major constituents of dietary fat. Because of the wide range of foods consumed, the human diet contains a great variety of fatty acids. The most abundant fatty acids have straight chains of an even number of carbon atoms. The chain lengths vary from 4 (e.g. in milk) to 30 (e.g. in some fish oils) and may contain double bonds (unsaturated fatty acids; Fig. 15.2). It is the nature of the constituent fatty acids (their chain length and degree of unsaturation) that gives a fat its physical properties. Fatty acids are often referred to by their common names, but are more correctly identified by a systematic nomenclature (Table 15.2). This nomenclature indicates the number of carbon atoms and the number and position of double (unsaturated) bonds in the chain (see Fig. 15.2). It is the position of the first double bond in the hydrocarbon chain that is indicated by the n-7, n-9, n-6 or n-3 part of the shorthand notation for a fatty acid. Note that n-6 and n-3 are sometimes referred to as omega-6 and omega-3. Mammalian cells are able to synthesise (from non-fat precursors) saturated fatty acids and unsaturated fatty acids of the n-9 and n-7 series but lack the delta12 and delta-15 desaturase enzymes (found in most plants) for insertion of a double bond at the n-6 or n-3 position (Figs 15.2 and 15.3). Thus, mammalian

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Fig. 15.2

355

The structure of some fatty acids.

cells cannot synthesise n-6 or n-3 polyunsaturated fatty acids (PUFAs) de novo. The n-6 and n-3 fatty acids are essential substrates for many of the major regulatory lipids in the body and as they cannot be synthesised in the body, they must be obtained from the diet. The commonly consumed PUFAs are linoleic acid (18:2n-6) and -linolenic acid (18:3n-3; ALA). Plant tissues and plant oils tend to be rich sources of linoleic and -linolenic acids. For example, linoleic acid contributes over 50% and often up to 80% of the fatty acids fond in corn, sunflower, safflower and soybean oils. Rapeseed and soybean oils are also good sources of ALA since this fatty acid contributes between 5 and 15% of the fatty acids present. However, the richest source of ALA is linseed oil (also known as flaxseed oil) in which ALA can contribute as much as 60% of the fatty acids present. Once consumed in the diet, linoleic acid and ALA can be converted to the longer chain, more unsaturated derivatives (Fig. 15.3). This process occurs mainly in the liver. Thus linoleic acid is converted via -linolenic (GLA; 18:3n-6) and dihomo- -linolenic (DGLA; 20:3n-6) acids to arachidonic acid (20:4n-6) (Fig. 15.2). Likewise, ALA is converted to eicosapentaenoic acid (EPA; 20:5n-3) (Fig. 15.2). There is some controversy about the extent to which docosahexaenoic acid (DHA; 22:6n-3) can be synthesised from EPA in humans. According to the United Kingdom Adult Survey conducted in 1986, the daily diet of the average adult male in the United Kingdom contains 42 g saturated fatty acids, 31 g monounsaturated fatty acids (mainly oleic acid) and 15.8 g

Table 15.2 Fatty acid nomenclature and sources Systematic name

Trivial name

Shorthand notation

Sources

Decanoic Dodecanoic Tetradecanoic Hexadecanoic

Capric Lauric Myrsitic Palmitic

10:0 12:0 14:0 16:0

Octadecanoic 9-Hexadecenoic 9-Octadecenoic

Stearic Palmitoleic Oleic

18:0 16:1n-7 18:1n-9

9,12-Octadecadienoic

Linoleic

18:2n-6

9,12,15-Octadecatrienoic

-Linolenic

18:3n-3

6,9,12-Octadecatrienoic 11,14,17-Eicosatrienoic

-Linolenic Mead

18:3n-6 20:3n-9

8,11,14-Eicosatrienoic 5,8,11,14-Eicosatetraenoic

Dihomo- -linolenic Arachidonic

20:3n-6 20:4n-6

5,8,11,14,17-Eicosapentaenoic 7,10,13,16,19-Docosapentaenoic 4,7,10,13,16,19-Docosahexaenoic

Eicosapentaenoic Docosapentaenoic Docosahexaenoic

20:5n-3 22:5n-3 22:6n-3

De novo synthesis; coconut oil De novo synthesis; coconut oil De novo synthesis; milk De novo synthesis; milk; eggs; Animal fats; meat; cocoa butter; Palm oil (other vegetable oils contain lesser amounts); fish oils De novo synthesis; milk; eggs; animal fats; meat; cocoa butter Desaturation of palmitic acid; fatty fish; fish oils Desaturation of stearic acid; milk; eggs; animal fats; meat; cocoa butter; Most vegetable oils especially olive oil Cannot be synthesised in mammals; some milks; eggs; animal fats; meat; most vegetable oils especially corn, sunflower, safflower and soybean oils; green leaves Cannot be synthesised in mammals; green leaves; some vegetable oils especially rapeseed, soybean and linseed oils Synthesised from linoleic acid; borage and evening primrose oils Synthesised from oleic acid; indicator of essential fatty acid deficiency Synthesised from -linolenic acid Synthesised from linoleic acid via -linolenic and dihomo- -linolenic acids; meat Synthesised from -linolenic acid; fatty fish; fish oils Synthesised from -linolenic acid via eicosapentaenoic acid Synthesised from -linolenic acid via eicosapentaenoic acid; fatty fish; fish oils

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Fig. 15.3

357

The biosynthesis of unsaturated fatty acids; CLA, conjugated linoleic acid.

PUFAs (Department of Health, 1994). Table 15.3 shows intakes of various PUFAs in adult men and women in the United Kingdom. The main PUFA in the diet is linoleic acid followed by ALA. Longer chain PUFAs are consumed in lower amounts than linoleic and -linolenic acids (Table 15.3; British Nutrition Foundation, 1999). Estimates of the intake of arachidonic acid intakes in Western populations vary between 50 and 300 mg/day for adults (Table 15.3). Table 15.3 Habitual intakes of PUFAs in adults in the United Kingdom (g/day). Data are from Kew et al. (2003a) Men (n = 88) Median Linoleic acid Arachidonic acid Total n-6 PUFAs ALA EPA Docosapentaenoic acid DHA Total n-3 PUFAs

Women (n = 62)

10th 90th percentile percentile

Median

10th 90th percentile percentile

13.2 0.21 13.7 1.4 0.15 0.09

7.2 0.10 7.5 0.1 0.04 0.04

24.1 0.35 24.7 2.5 0.42 0.17

11.1 0.17 11.5 1.3 0.11 0.08

6.8 0.05 7.1 0.1 0.06 0.02

18.1 0.31 18.5 1.9 0.39 0.15

0.23 2.0

0.07 1.2

0.45 3.5

0.17 1.7

0.09 1.1

0.42 2.9

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EPA and DHA are found in high quantities in `fatty' fish (e.g. herring, mackerel, fresh tuna, sardines, salmon) and in the commercially available products known as fish oils. The latter have two origins: the body oils of fatty fish or the oils extracted from the livers of fish which live in warmer waters (e.g. cod). EPA and DHA comprise 20% to 30% of the fatty acids in a typical preparation of fish oil, which means that a one gram fish oil capsule can provide 200 to 300 mg of EPA plus DHA. In the absence of fatty fish or fish oil consumption, ALA is the main dietary n-3 PUFA (Table 15.3). Average intake of the long-chain n-3 PUFAs in the United Kingdom is estimated at < 250 mg per day (British Nutrition Foundation, 1999). Conjugated linoleic acid (CLA) is a term used to describe the mixture of isomers of linoleic acid with conjugated double bonds (i.e. the two double bonds are separated by only one single bond; see Fig. 15.2). The double bonds can be in either the cis or trans configuration (all double bonds in the fatty acids described above and in Table 15.1 are in the cis configuration) and can be in any position in the carbon chain. Thus, there are a large number of isomers of CLA. The most frequently encountered isomers have the double bonds in positions 8 and 10, 9 and 11, 10 and 12 or 11 and 13. CLA is formed as a result of the metabolism of linoleic acid and ALA in the rumen, and so CLA is found in the milk and meat of ruminants. The predominant (> 90%) CLA in cow's milk is the cis-9, trans-11 isomer. Estimates of intakes of CLA from the Western diet range from 15 to several hundred mg/day. However, average intakes of CLA appear to be 100 to 200 mg/day, with milk and dairy products contributing > 60% of this (Ens et al., 2001; Ritzenthaler et al., 2001; Voorrips et al., 2002). 15.2.1 The fatty acid composition of cells of the immune system can be modulated by dietary fatty acids The principal role of fatty acids in cells of the immune system is as components of cell membrane phospholipids, and human inflammatory and immune cells tend to be rather rich in arachidonic acid. However, there is some variation in the fatty acid composition of human immune cells, which may reflect, in part, variation in the dietary intake of various fatty acids. Table 15.4 presents the fatty acid composition of blood mononuclear cells (an 85:15 mixture of lymphocytes and monocytes) taken from 67 healthy males aged 20 to 40 years living in the southern United Kingdom. As expected, arachidonic acid comprises about 20% of total fatty acids but varies from 12.6 to 27.6%. In contrast to the high content of arachidonic acid, EPA is a fairly minor constituent of human mononuclear cells comprising an average of < 1% of total fatty acids. The data in Table 15.4 are consistent with those recently reported for 150 men and women living in the southern United Kingdom (Kew et al., 2003a). If some of the variation in the fatty acid composition of human immune cells is related to dietary variation, then the fatty acid composition of human immune cells should be amenable to dietary manipulation, as seen with experimental and farm animal studies. A number of studies have investigated this possibility.

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Table 15.4 Fatty acid composition of human peripheral blood mononuclear cells. Data are for 67 males aged 20 to 40 years and are previously unpublished g/100 g total fatty acids Fatty acid

Mean

Standard deviation

10th percentile

90th percentile

Myristic acid Palmitic acid Stearic acid Oleic acid Linoleic acid GLA ALA DGLA Arachidonic acid EPA Doco sapentaenoic acid DHA

0.55 19.9 21.3 17.2 8.6 0.08 0.17 2.7 19.5 0.8 2.9 2.3

0.33 2.5 3.1 3.1 0.9 0.22 0.38 1.1 3.0 0.9 2.1 1.1

0.18 16.5 18.9 14.1 7.3 0 0 1.7 15.4 0.1 1.2 1.2

1.02 22.4 23.8 20.7 9.9 0.35 0.66 4.6 23.7 1.5 4.7 3.9

A dietary intervention in which healthy human volunteers consumed a diet providing extra oleic acid at the expense of saturated fatty acids for eight weeks resulted in a significant increase in the content of oleic acid in peripheral blood mononuclear cells: 20.3 Ô 1.0% of total fatty acids vs. 24.5 Ô 0.8% of fatty acids (Yaqoob et al., 1998). However, in another study, increased intake of oleic acid (6.5 g/day for 12 weeks) from olive oil capsules did not significantly alter the oleic acid content of human blood mononuclear cells (Yaqoob et al., 2000). The different findings of these two studies may relate to the amount of oleic acid provided. In the dietary intervention study oleic acid intake was increased from 11 to 18% of total energy intake, representing an increased intake of 15 to 20 g/day. An increased intake of linoleic acid (6.5 g/day for 12 weeks) from sunflower oil capsules did not significantly alter the fatty acid composition of human blood mononuclear cells (Yaqoob et al., 2000). Increased intake of GLA (1 g/day for 12 weeks) from evening primrose oil capsules increased the DGLA content of human blood mononuclear cells by about 50% but this was not statistically significant (Yaqoob et al., 2000). Arachidonic acid content did not increase. A more modest intake of GLA (0.7 g/day for 12 weeks) by elderly humans increased the DGLA content of mononuclear cells by about 50%, again without an increase in arachidonic acid content (Thies et al., 2001a). DGLA increased in human neutrophils following consumption of 3 or 6, but not 1.5, g GLA/day for three weeks (Johnson et al., 1997). Increased GLA consumption and appearance of DGLA was not associated with a change in the arachidonic acid content of human neutrophils (Johnson et al., 1997). Increased intake of arachidonic acid (0.7 g/day for 12 weeks) by elderly humans increased the arachidonic acid content of mononuclear cells from about 20% to about 23% of fatty acids (Thies et al., 2001a).

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Kelley et al. (2001) reported that intake of 3.9 g CLA/day in the form of mixed isomers in capsules resulted in significant incorporation of CLA isomers into blood mononuclear cells (total CLA content increased from about 0.12 to about 1% of total fatty acids) over nine weeks. In comparison, Albers et al. (2003) reported CLA contents of 0.9% of total fatty acids and about 1.3% of fatty acids in mononuclear cells before and after 85 days of supplementation of the diet with 1.6 g CLA/day. Supplementing the diet of human volunteers with fish oil in capsules results in incorporation of both EPA and DHA into blood neutrophils (Lee et al., 1985; Sperling et al., 1993; Gibney and Hunter, 1993; Luostarinen and Saldeen, 1996; Healy et al., 2000), monocytes (Lee et al., 1985; Fisher et al., 1990; Gibney and Hunter, 1993), T lymphocytes (Gibney and Hunter, 1993), B lymphocytes (Gibney and Hunter, 1993) and mononuclear cells (Endres et al., 1989; Molvig et al., 1991; Caughey et al., 1996; Yaqoob et al., 2000; Thies et al., 2001a). This incorporation is again largely at the expense of arachidonic acid. Both EPA and DHA are readily taken up, with near-maximal incorporation occurring within four weeks (Yaqoob et al., 2000; Thies et al., 2001a). Incorporation of both fatty acids occurs in a dose response manner (Healy et al., 2000). Once dietary supplementation ceases, EPA is lost from the cells within eight weeks, but DHA is better retained (Yaqoob et al., 2000). An ALA intake of about 18 g/day for eight weeks resulted in an increased content of ALA in human mononuclear cells (from 0.2 to 0.6% of fatty acids) as well as a small increase in the content of EPA and docosapentaenoic acid (Kelley et al., 1993). There was no change in content of arachidonic acid or DHA. In another study, an ALA intake of about 14 g/day for four weeks resulted in an increased content of ALA in human mononuclear cells (from 0.1 to 0.3% of fatty acids; Caughey et al., 1996) and neutrophils (from 0.4 to 0.6% of fatty acids; Mantzioris et al., 1994). This was associated with a small decrease in the content of arachidonic acid in mononuclear cells, but not in neutrophils, and with an increase in the content of EPA but not DHA (Mantzioris et al., 1994; Caughey et al., 1996). In contrast to these studies using large amounts of ALA, increased intake of smaller amounts of ALA from capsules (2 g/day for 12 weeks) by elderly human volunteers did not alter the fatty acid composition of blood mononuclear cells (Thies et al., 2001a). Likewise, an increase in ALA intake from capsules of 4 g/day for 12 weeks had little impact on the fatty acid composition of human neutrophils (Healy et al., 2000). A recent study showed that consumption of a spread providing a daily ALA intake of 4.5 g for six months did not influence the fatty acid consumption of blood mononuclear cells (Kew et al., 2003b). However, consuming a spread providing a daily intake of 9.5 g ALA/day significantly increased the EPA, but not the DHA, content of mononuclear cells by 30% (Kew et al., 2003b). Taken together these studies suggest that quite significant changes in fatty acid consumption might be required to alter the fatty acid composition of cells of the human immune system. Consistent patterns that have emerged from human studies are:

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· the oleic and linoleic acid contents of human immune cells are difficult to manipulate · increased intake of GLA increases DGLA content · markedly increased intake of arachidonic acid increases arachidonic acid content · markedly increased intake of CLA increases CLA content · fish oil intake increases EPA and DHA contents (and decreases arachidonic acid content) · an increase in intake of ALA of at least 4 g/day is necessary to alter fatty acid composition · a substantial increase in ALA intake increases EPA, but not DHA, content.

15.3 Fatty acid composition of immune cells and immune function: eicosanoids 15.3.1 Arachidonic acid as an eicosanoid precursor The principal functional role for arachidonic acid in inflammatory and immune cells is as a substrate for synthesis of the eicosanoid family of bioactive mediators. These include the prostaglandins (PG), thromboxanes, leukotrienes (LT), hydroxy-eicosatetraenoic acids and lipoxins. Arachidonic acid in cell membranes can be mobilised by various phospholipase enzymes, especially phospholipase A2, and the free arachidonic acid can subsequently act as a substrate for the enzymes which synthesise eicosanoids (Fig. 15.4). Metabolism by cyclooxygenase enzymes (COX) gives rise to the 2-series prostaglandins and thromboxanes (Fig. 15.4). There are two isoforms of COX: COX-1 is a constitutive enzyme and COX-2 is induced in inflammatory cells as a result of stimulation and is responsible for the markedly elevated production of prostaglandins that occurs upon cellular activation. Prostaglandins are formed in a cell-specific manner. For example, upon activation monocytes and macrophages produce large amounts of PGE2 and PGF2, neutrophils produce moderate amounts of PGE2, and mast cells produce PGD2. Lymphocytes are a poor source of prostaglandins, but may release arachidonic acid extracellularly for use by other cell types (Goldyne and Stobo, 1982). Metabolism of arachidonic acid by the 5-lipoxygenase (5-LOX) pathway gives rise to hydroxy and hydroperoxy derivatives, and the 4-series leukotrienes, LTA4, B4, C4, D4 and E4 (Fig. 15.4). 5±LOX is found in mast cells, monocytes, macrophages and granulocytes. Arachidonic acid-derived eicosanoids are involved in modulating the intensity and duration of inflammatory responses and in regulating immune responses (see Lewis et al., 1990; Tilley et al., 2001 for reviews). The effects of PGE2 and LTB4 are summarised in Table 15.5. LTB4 has a range of proinflammatory effects. PGE2 also has a number of pro-inflammatory effects. Note however, that PGE2 also acts to down-regulate the production of the classic inflammatory cytokines (TNF, IL-1 and IL-6) and inhibits the production of

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Fig. 15.4 Metabolism of 20 carbon polyunsaturated fatty acids to yield eicosanoids. Cox, cyclooxygenase; LOX, lipoxygenase; LT, leukotrienes; PG, prostaglandins.

4-series leukotrienes. PGE2 also induces the production of lipoxins (Levy et al., 2001), which are now recognised to be inflammation `stop' signals (Gewirtz et al., 2002; Vachier et al., 2002). Thus, PGE2 is both a mediator and a regulator of inflammation, and exerts both pro- and anti-inflammatory actions. PGE2 also promotes IgE production by B lymphocytes and so in this respect PGE2 is proallergic. Finally, PGE2 inhibits T cell proliferation and the production of the Th1-type cytokine IFN- . In these resects PGE2 is immunosuppressive. Table 15.5 Effects of PGE2 and LTB4 on inflammation and immunity PGE2

LTB4

Pro-inflammation Induces fever Increases vascular permeability Increases vasodilatation Causes pain Enhances pain caused by other agents

Pro-inflammation Increases vascular permeability Enhances local blood flow Chemotactic agent for leukocytes Induces release of lysosomal enzymes Induces release of reactive oxygen species by granulocytes Increases production of TNF, IL-1 and IL-6

Anti-inflammation Inhibits production of TNF, IL-1 and IL-6 Inhibits 5-LOX Induces lipoxin production Immunosuppression Inhibits production of IL-2 and IFN- Inhibits lymphocyte proliferation Pro-allergy Promotes IgE production

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The capacity to generate eicosanoids from arachidonic acid appears to be influenced by age. Production of PGE2 and TXA2 was higher by macrophages, splenocytes and lung tissue from 24-month old compared with three-month old mice (Meydani et al., 1988, 1992; Hayek et al., 1994; Wu et al., 1998). Furthermore, PGE2 production by blood mononuclear cells from older women was greater than by cells from younger women (Meydani et al., 1991). The agerelated increase in production of arachidonic acid-derived eicosanoids is related to increased COX activity (Hayek et al., 1997; Wu et al., 1998), which in turn is due to increased expression of the COX-2 gene (Hayek et al., 1997). The latter study showed that peak COX-2 mRNA levels in endotoxin-stimulated macrophages from old mice were twice those seen in young mice. This correlated with twice the peak COX-2 activity and twice the peak PGE2 production in these mice (Hayek et al., 1997; Wu et al., 1998). This age-related increase in the production of PGE2 and related eicosanoids may play a role in the immune and inflammatory changes seen in the elderly. 15.3.2 Di-homo- -linolenic acid and eicosanoid production DGLA is a substrate for COX-2 and for 5-LOX giving rise to derivatives which have a different structure from those produced from arachidonic acid (i.e. 1series PG and 3-series LT) (Fig. 15.4). PGE1 has a number of anti-inflammatory effects including inhibition of superoxide, elastase and myeloperoxidase production by neutrophils, and inhibition of TNF, IL-1 and IL-6 production by monocytes and macrophages. DGLA is also a substrate for 15-LOX giving rise to 15-hydroxy-DGLA, which is a 5-LOX inhibitor. DGLA levels in inflammatory cells are increased by GLA supplementation of the diet (see section 15.2) and GLA supplementation has been shown to result in increased production of PGE1 and decreased production of PGE2, LTB4, and LTC4 (Johnson et al., 1997; Wu et al., 1999). 15.3.3 Long-chain n-3 PUFAs and eicosanoid production Since significantly increased consumption of long-chain n-3 PUFAs results in a decrease in the amount of arachidonic acid in the membranes of inflammatory cells, there will be less substrate available for synthesis of eicosanoids from arachidonic acid. In accordance with this, fish oil feeding results in a decreased capacity of inflammatory cells to synthesise COX- and 5-LOX-derived eicosanoids from arachidonic acid (e.g. Lee et al., 1985; Endres et al., 1989; Meydani et al., 1991; Sperling et al., 1993; Caughey et al., 1996). However, the effects of n-3 PUFAs on eicosanoid production extend beyond simply decreasing the amount of substrate available. For example, EPA competitively inhibits the oxygenation of arachidonic acid by COX (Obata et al., 1999). Recent cell culture studies have demonstrated that n-3 PUFAs suppress cytokine-induction of COX-2 and 5-LOX gene expression (Curtis et al., 2000, 2002). It is the net result of these various actions, that accounts for the decreased

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generation of arahidonic-acid derived eicosanoids by n-3 PUFAs. The reduction in generation of arachidonic acid-derived mediators that accompanies fish oil consumption has led to the idea that fish oil is anti-inflammatory. In addition to inhibiting metabolism of arachidonic acid, EPA is able to act as a substrate for both COX and 5-LOX (Fig. 15.4), giving rise to derivatives which have a different structure from those produced from arachidonic acid (i.e. 3series PG and TX and 5-series LT). Thus, the EPA-induced suppression in the production of arachidonic acid-derived eicosanoids may be accompanied by an elevation in the production of EPA-derived eicosanoids. This is most evident for the 5-LOX products of EPA metabolism (Lee et al., 1985; Sperling et al., 1993). The eicosanoids produced from EPA are considered to be less biologically potent than the analogues synthesised from arachidonic acid, although the full range of biological activities of these compounds has not been investigated. Therefore, it is possible that EPA gives rise to eicosanoids that are less inflammatory, less pro-allergic and less immunosuppressive than those produced from arachidonic acid (see Miles et al., 2002, 2003 for further discussion). Interestingly recent studies have revealed that n-3 PUFAs give rise to novel anti-inflammatory eicosanoids generated via COX-2 (Levy et al., 2001).

15.4 Dietary fatty acids and immune function: mechanisms of action There is a large literature based upon cell culture and animal feeding studies investigating the effects of various fatty acids on inflammation and immune function. These studies have established a basis for effects that might be observed following manipulation of the fatty acid composition of the human diet and have been very useful in identifying mechanisms of action of different fatty acids. A description of these studies is beyond the scope of this chapter, which will focus upon results from human studies. However, a number of review articles serve to summarise, evaluate and discuss these studies (Calder 1996, 1997, 1998a,b, 2001b, 2002; Calder et al., 2002; Yaqoob, 1998, 2003; Kelley and Erickson, 2003; Harbige, 2003). 15.4.1 Oleic acid A dietary intervention study in which healthy middle-aged men consumed diets providing 11.3 (control) or 18.4% energy from oleic acid for eight weeks was performed by Yaqoob et al. (1998). There was no significant effect on the proportion of T cells, B cells, T helper cells, cytotoxic T cells, monocytes or natural killer cells in the circulation or on natural killer cell activity or mitogenstimulated proliferation of lymphocytes. There was however a significant reduction in the proportion of mononuclear cells (most likely monocytes) expressing ICAM-1 (Yaqoob et al., 1998). In another study providing 9 g encapsulated olive oil per day for 12 weeks did not affect the proportion of T

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cells, B cells, T helper cells, cytotoxic T cells, monocytes, or natural killer cells in the circulation; on natural killer cell activity, mitogen-stimulated proliferation of lymphocytes, or stimulated production of a range of cytokines by lymphocytes and monocytes (Yaqoob et al., 2000). 15.4.2 N-6 PUFAs Surprisingly few human studies have been conducted investigating the immunological effects of linoleic acid. The most detailed of those reported were those of Kelley et al. (1989, 1992) that involved providing volunteers with low fat diets (25% energy as fat) that were rich (12.9% of energy) or poor (3.5% of energy) in linoleic acid. No differences were observed in the response of lymphocytes to various T cell mitogens, in circulating concentrations of IgM, IgG, IgE or IgA, or in delayed-type hypersensitivity, an in vivo measure of cellmediated immunity. Yaqoob et al. (2000) included a group consuming 9 g encapsulated sunflower oil/day for 12 weeks in their study. This had no effect on T lymphocyte proliferation, natural killer cell activity, or production of TNF-, IL-1, IL-1 , IL-6, IL-2 and IFN- by mononuclear cells. These studies suggest a limited effect of linoleic acid (at a level  3.5% of dietary energy) on human immune function. In another study, 11.2 g linoleic acid/day from safflower oil for 12 weeks had no effect on the serum concentrations of IL-6, C-reactive protein or amyloid A (Rallidis et al., 2003). The habitual intake of linoleic acid in the subjects studied was an average of 11 g/day; thus the linoleic acid intake was substantially increased (by an average of about 100%) in these subjects, without any adverse effects on inflammatory markers. Supplementation studies using GLA-rich oils such as borage oil and providing volunteers with > 2.4 g GLA/day report decreased T lymphocyte proliferation (Rossetti et al., 1997), decreased production of TNF-, IL-1 and IL-6 by monocytes (de Luca et al., 1999), and decreased production of platelet-activating factor by neutrophils (Johnson et al., 1997). Recent studies using 1 or 0.8 g GLA/ day for 12 weeks report no effect on circulating immune cell numbers and types, T lymphocyte proliferation, natural killer cell activity, neutrophil and monocyte phagocytosis, neutrophil and monocyte respiratory burst, or production of a range of cytokines by lymphocytes and monocytes (Yaqoob et al., 2000; Thies et al., 2001a,b,c). Furthermore there was no effect of 0.8 g GLA/day on plasma soluble adhesion molecule concentrations (Thies et al., 2001c). These studies suggest that the lowest amount of GLA required to influence inflammation and immune function is somewhere between 1 and 2.4 g per day. Two studies of the influence of dietary arachidonic acid on human immune function have been performed. In the first of these 1.5 g arachidonic acid/day was included as part of a low-fat diet (27% energy from fat) consumed for eight weeks by healthy males aged 20 to 38 years (Kelley et al., 1997, 1998a). This level of arachidonic acid did not alter the proliferation of T cells in response to mitogens, natural killer cell activity, the production of TNF-, IL-1 , IL-6 or IL-2 by mononuclear cells, the delayed-type hypersensitivity response, or

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antibody responses to immunisation with vaccines against three strains of influenza virus (Kelley et al., 1997, 1998a). However, there was an increase in the production of PGE2 and LTB4 by endotoxin-stimulated mononuclear cells (Kelley et al., 1998a). The second study involved supplementing the diet of healthy elderly (aged 55 to 75 years) subjects with 0.7 g arachidonic acid/day for 12 weeks (Thies et al., 2001a,b,c). There was no significant effect on circulating immune cell numbers or on any of the immune parameters measured. The latter included T lymphocyte proliferation, natural killer cell activity, neutrophil and monocyte phagocytosis, neutrophil and monocyte respiratory burst, and the production of TNF-, IL-1 , IL-6, IL-2 and IFN- (Thies et al., 2001a,b,c). There was also no effect on plasma sICAM-1, sVCAM-1 or sE-electin concentrations (Thies et al., 2001c). These studies suggest that increasing arachidonic acid intake in healthy humans may not have adverse immunological effects. However, these studies had durations of 8 and 12 weeks, respectively, and the longer-term effects of arachidonic acid on inflammation and immune function in humans are not known. 15.4.3 Conjugated linoleic acid Two studies of CLA and human immune function have been reported to date (Kelley et al., 2000, 2001; Albers et al., 2003). Kelley et al. (2000, 2001) provided an encapsulated mix of CLA isomers (total CLA intake 3.9 g/day) to young women consuming a 30% energy from fat diet for nine weeks. The mix contained a number of isomers with no single isomer contributing more than 23.6% of total CLA; cis-9, trans-11 CLA and trans-10, cis-12 CLA contributed 17.6% (690 mg per day) and 22.6% (880 mg per day), of CLA, respectively. CLA did not affect the numbers of T lymphocytes, T helper cells, cytotoxic T cells, B cells, monocytes or natural killer cells in the circulation (Kelley et al., 2000). There was also no effect of CLA on T or B lymphocyte proliferation, on the production of TNF-, IL-1 , IL-2 and IFN- , on the serum antibody response to immunisation with vaccines against three strains of influenza virus, or on the delayed-type hypersensitivity response (Kelley et al., 2000, 2001). Finally there was no effect of CLA on production of PGE2 or LTB4 by endotoxin-stimulated mononuclear cells (Kelley et al., 2001). Although this study could be taken to indicate that CLA (at an intake of 3.9 g/day) does not affect human immune function, it is important to note that the study used a rather crude mixture of CLA isomers. Thus, the amount of a CLA isomer that does affect immune function may have been insufficient. Furthermore, different isomers may have opposing actions so that overall a mixture of isomers is without effect. Albers et al. (2003) provided about 1.6 g CLA/day as an 80:20 or a 50:50 mix of the cis-9, trans-11 and trans-10, cis-12 isomers to healthy males aged 31 to 69 years for 12 weeks; sunflower oil was used as the control. There was no effect of either mix on T lymphocyte or monocyte proliferation, natural killer cell activity, production of IL-2, IL-4 and IFN- by lymphocytes, and production of TNF-, IL-1 , IL-6 and PGE2 by monocytes. Furthermore the delayed-type

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hypersensitivity response was not affected. Subjects were immunised with a vaccine for hepatitis B. Although the mean concentration of anti-hepatitis B vaccine antibodies was not different between the groups, more subjects mounted a response to the vaccine in the 50:50 group. The lack of effect in the 80:20 group suggests that the trans-10, cis-12 CLA isomer is responsible for the improved response in the 50:50 group. Clearly this requires further investigation with studies using pure CLA isomers. Thus, at this stage the precise effects of different CLA isomers on human immune function remain unknown. 15.4.4 N-3 PUFAs Fish oil, EPA and DHA Studies investigating the effect of fish oil on human inflammatory and immune cell functions and responses date back to the early 1980s and there is a great deal of literature on this area. Effects on a number of cellular functions and on a range of mediators have been investigated. Although most studies have used fish oil which contains both EPA and DHA, some studies have tried to distinguish between the effects of these two long-chain n-3 PUFAs. The literature on n-3 PUFAs and inflammation and immune function in humans has been collated and reviewed a number of times and the reader is referred to such reviews for a full description and discussion of these studies (e.g. Calder 2001a,b). Chemotaxis, respiratory burst, phagocytosis and adhesion Fish oil, providing between 2.3 and 14.5 g EPA plus DHA/day has been reported to decrease neutrophil chemotaxis (Lee et al., 1985; Schmidt et al., 1989, 1992; Luostarinen et al., 1992; Sperling et al., 1993), neutrophil respiratory burst (Thompson et al., 1991; Varming et al., 1995; Luostarinen and Saldeen, 1996) and neutrophil binding to endothelial cells (Lee et al., 1985). Fish oil, providing 4.5 to 5.3 g EPA plus DHA/day, decreased monocyte chemotaxis (Schmidt et al., 1989, 1992; Endres et al., 1989) and respiratory burst (Fisher et al., 1990). However, lower doses of long-chain n-3 PUFAs (0.55 to 2.3 g/day) did not affect neutrophil or monocyte phagocytosis or respiratory burst (Schmidt et al., 1996; Healy et al., 2000; Thies et al., 2001c; Kew et al., 2003b) or neutrophil chemotaxis (Healy et al., 2000). Although Hughes et al. (1996) reported that 1.6 g EPA plus DHA/day for 3 weeks decreased ICAM-1 on the surface of blood monocytes, this effect was not confirmed in a recent study using 0.77 and 1.7 g EPA plus DHA/day for six months (Kew et al., 2003b). Natural killer cell activity There are relatively few studies of long-chain n-3 PUFAs and human natural killer cell activity. Fish oil providing 1.1 g EPA plus DHA/day significantly decreased natural killer cell activity in subjects aged 55 to 75 years (Thies et al., 2001b). In contrast, 3.2 g EPA plus DHA/day had no effect on natural killer cell activity (Yaqoob et al., 2000). It is possible that the age of the subjects studied may be a factor accounting for these differences.

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Monocyte-derived cytokines Fish oil providing > 2.4 g EPA plus DHA/day decreased the production of TNF (Endres et al., 1989; Meydani et al., 1991; Gallai et al., 1993; Caughey et al., 1996), IL-1 (Endres et al., 1989; Meydani et al., 1991; Gallai et al., 1993; Caughey et al., 1996) and IL-6 (Meydani et al., 1991) by mononuclear cells. Another study, providing subjects consuming a low-fat diet that included fatty fish (daily intake of 1.2 g EPA plus DHA), reported decreased production of TNF, IL-1 and IL-6 (Meydani et al., 1993). Intake of 4.55 g EPA plus DHA as ethyl esters/day for four weeks significantly decreased plate-derived growth factor (PDGF)-A and -B and monocyte chemotactic peptide-1 mRNA in monocytes (Baumann et al., 1999). This is consistent with the significant decrease in plasma PDGF concentration observed in women consuming 4 g fish oil/day (1.2 g EPA plus DHA/day) for four weeks (Wallace et al., 1995). In contrast to the above studies are a number reporting no effect of 0.55 to 3.4 g EPA plus DHA/day on production of TNF (Molvig et al., 1991; Cooper et al., 1993; Schmidt et al., 1996; Blok et al., 1997; Yaqoob et al., 2000; Thies et al., 2001c; Kew et al., 2003b; Wallace et al., 2003), IL-1 (Molvig et al., 1991; Cooper et al., 1993; Cannon et al., 1995; Schmidt et al., 1996; Blok et al., 1997; Yaqoob et al., 2000; Thies et al., 2001c; Kew et al., 2003b; Wallace et al., 2003) and IL-6 (Cooper et al., 1993; Schmidt et al., 1996; Thies et al. 2001c; Kew et al., 2003b). Possible reasons for the discrepancies in the literature are discussed in detail elsewhere (Calder, 2001a), but may relate to the dose of n-3 PUFAs used, the duration of the study, the age of the subjects studied, sample size, and differences in background diet. Duration appears not to be a factor because some relatively short studies report effects (e.g. Caughey et al., 1996), whereas other short (Cooper et al., 1993) and longer term (Blok et al., 1997; Kew et al., 2003b) studies report no effect. However, the dose of long-chain n-3 PUFAs provided is likely to be important; a recent study reported for the first time a dose-response relationship between EPA plus DHA and IL-6 production (Wallace et al., 2003). This study reported that the threshold for an effect of long-chain n-3 PUFAs on IL-6 production is between 0.44 and 0.94 g/day. However, dose cannot be the sole explanation for differences in the literature, because some studies providing as much as 3.2 g EPA plus DHA per day report no effect on cytokine production (e.g. Yaqoob et al., 2000). Another recent study has highlighted a further possible explanation. This study reports that polymorphisms in the promoter regions of the TNF- and TNF- genes play a role in conferring sensitivity of TNF- production to fish oil intervention (Grimble et al., 2002). Antigen presentation Hughes et al. (1996) reported that 1.6 g EPA plus DHA/day for three weeks decreased the level of expression of MHC-II (HLA-DP, -DQ and -DR) on the surface of blood monocytes. Lymphocyte proliferation and production of lymphocyte derived cytokines Supplementation of the diet with fish oil providing 2.4 g EPA plus DHA/day decreased the proliferation of lymphocytes from older (aged 51 to 68 years) but

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not young (aged 21 to 33 years) women (Meydani et al., 1991). IL-2 production was also decreased in the older, but not younger, group (Meydani et al., 1991). Molvig et al. (1991) reported decreased lymphocyte proliferation after providing 1.7 or 3.4 g EPA plus DHA/day to men, while Gallai et al. (1993) reported that 5.2 g EPA plus DHA/day decreased IL-2 and IFN- production. Inclusion of fatty fish providing 1.2 g EPA plus DHA/day to elderly subjects consuming a low fat diet decreased lymphocyte proliferation and IL-2 production (Meydani et al., 1993). Fish oil providing 1.1 g EPA plus DHA/day significantly decreased proliferation of T lymphocytes from subjects aged 55 to 75 years, although there was no effect on IL-2 or IFN- production (Thies et al., 2001a). In contrast to these observations there are several reports of no effect of 0.77 to 3.2 g EPA plus DHA/day on T lymphocyte proliferation or production of various T cell derived cytokines including IL-2 and IFN- (Yaqoob et al., 2000; Wallace et al., 2003; Kew et al., 2003b). These latter studies investigated mainly younger subjects. Putting these studies together suggests that it is difficult to influence production of cytokines by T lymphocytes except by using very high doses of long-chain n3 PUFAs (e.g. Gallai et al., 1993). Furthermore, proliferation of lymphocytes from older subjects appears to be more sensitive to increased availability of long-chain n-3 PUFAs than that of those from younger subjects. Delayed-type hypersensitivity The delayed-type hypersensitivity response to seven recall antigens was decreased by inclusion of fatty fish (1.2 g EPA plus DHA/day) in a low fat diet (Meydani et al., 1993). In another study providing 0.77 or 1.7 g EPA plus DHA/day for six months did not affect this response (Kew et al., 2003b). Soluble adhesion molecules The concentrations of sICAM-1, sVCAM-1 and sE-selectin were significantly negatively correlated with the concentration of non-esterfied EPA in the bloodstream of elderly males at high risk of coronary heart disease (Yli-Jama et al., 2002). Furthermore the concentrations of sICAM-1 and sVCAM-1 were significantly negatively correlated with the concentration of non-esterfied DHA in the bloodstream (Yli-Jama et al., 2002). These observations suggest that an increase in long-chain n-3 PUFAs may decrease endothelial inflammation, as indicated by soluble adhesion molecule concentrations. In accordance with this, consumption of 1.1 g EPA plus DHA/day for 12 weeks by subjects aged 55 to 75 years significantly decreased sVCAM-1 concentration, with non-significant decreases in sICAM-1 and sE-selectin concentrations (Thies et al., 2001c). Average decreases were 26% (sVCAM-1), 14% (sICAM-1) and 23% (sEselectin). A reduction in soluble adhesion molecule concentrations was not observed in young male subjects consuming 1.2 g EPA plus DHA/day for 12 weeks (Miles et al., 2001), suggesting that older subjects may be more sensitive to the effects of long-chain n-3 PUFAs. In contrast to the observation of Thies et al. (2001c), three studies report increases in these soluble adhesion molecules following fish oil administration (Seljeflot et al., 1998; Abe et al., 1998;

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Johansen et al., 1999). However, these studies used high doses of fish oil providing 3.3 to 5.1 g EPA plus DHA/day, studied subjects with much higher cardiovascular risk and used different study designs from that used by Thies et al. (2001c) (see Miles et al., 2001 for a discussion). C-reactive protein C-reactive protein (CRP) is an acute phase protein synthesised by the liver in response to infections and to certain inflammatory stimuli, most notably IL-6. Thus, plasma CRP concentrations are elevated in chronic inflammatory conditions and in individuals who are obese or with advanced cardiovascular disease. Since n-3 fatty acids affect inflammatory processes they might be expected to affect CRP concentrations. Indeed, the concentration of CRP was significantly negatively correlated with the proportion of DHA in the membranes of granulocytes of patients undergoing coronary angiography (Madsen et al., 2001). Fish oil (3.6 g EPA plus DHA/day for 12 weeks) significantly lowered (by 20%) plasma CRP concentrations in patients with rheumatoid arthritis, who have elevated concentrations (Nielsen et al., 1992). However, 4.4 g EPA plus DHA/day for six weeks did not lower CRP concentrations in obese or non-obese subjects (Chan et al., 2002). Furthermore, a recent study reported no effect of either 2 or 6.6 g EPA plus DHA/day for 12 weeks on serum CRP in healthy subjects (Madsen et al., 2003). EPA vs. DHA and Fish oil vs. DHA Recent studies have compared effects of EPA and DHA or of fish oil and DHA and have attempted to identify whether the effects of fish oil are due to EPA or to DHA. There was no effect of 3.8 g of either EPA or DHA/day for seven weeks on phagocytosis of E. coli by human monocytes (Halvorsen et al., 1997). Kelley et al. (1998b, 1999) reported the effects in men aged 20 to 40 years of including 6 g DHA/day in a 30% energy from fat diet for 90 days. There was no effect of DHA on lymphocyte proliferation, serum immunoglobulin G concentrations, the delayed-type hypersensitivity response or the serum antibody response to immunisation against three strains of influenza virus (Kelley et al., 1998b, 1999). Natural killer cell was unaffected at day 55 but was significantly decreased at day 80 (Kelley et al., 1999). Similarly, the production of TNF- and IL-1 tended to decrease at day 55 but was significantly decreased at day 80 (Kelley et al., 1999). More recently, 0.75 g DHA/day for 12 weeks did not affect phagocytosis or respiratory burst by neutrophils and monocytes, natural killer cell activity, lymphocyte proliferation, the production of cytokines by lymphocytes and monocytes, or the concentrations of circulating adhesion molecules (Thies et al., 2001a,b,c). Thus a low dose of DHA ( 0.75 g/day) does not affect inflammation or immune function even in elderly subjects. However a very high dose of DHA (6 g/day) exerts some anti-inflammatory and immunosuppressive effects. The limited nature of these effects at such a high dose is suggestive that DHA does not mediate the effects of fish oil upon inflammatory and immune processes.

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-Linolenic acid A high dose of ALA (13.7 g/day for four weeks) decreased the production of TNF and IL-1 by human monocytes by an average of 27 and 30%, respectively (Caughey et al., 1996). The effect of this dose of ALA compares with reductions of 70 and 78%, respectively, after four weeks of 2.7 g EPA plus DHA/day (Caughey et al., 1996). Adding linseed oil providing about 15 g ALA/day as part of a low-fat diet (29% of energy from fat) resulted in a decrease in T lymphocyte proliferation in response to some, but not all, mitogens and a decrease in the delayed-type hypersensitivity response (Kelley et al., 1991). Circulating antibody concentrations were not affected (Kelley et al., 1991). Supplementing the diet of healthy young males with encapsulated linseed oil providing 3.5 g ALA/day for 12 weeks did not alter superoxide production by neutrophils or neutrophil chemotaxis (Healy et al., 2000). Furthermore, the number of T lymphocytes, helper T lymphocytes, cytotoxic T lymphocytes, B lymphocytes and natural killer cells in the bloodstream was not affected (Wallace et al., 2003). Likewise there was no effect on the proliferation of T lymphocytes in response to a mitogen, the production of IL-2, IL-4, IFN- and IL-10 by lymphocytes, or the production of TNF-, IL-1 and IL-6 by monocytes (Wallace et al., 2003). A similar lack of effect of ALA (2 g/day for 12 weeks) on circulating immune cell numbers, T cell proliferation, production of cytokines by lymphocytes and monocytes and natural killer cell activity was reported in elderly subjects (Thies et al., 2001a,b,c). There was however, a significant decrease in sVCAM-1 and sE-selectin, but not sICAM-1, concentrations in these subjects (Thies et al., 2001c). The average decreases were 15% and 27%, respectively. The decrease in sE-selectin concentration was similar to that seen in subjects consuming 1.1 g EPA plus DHA/day (23%; Thies et al., 2001c), but the decrease in sVCAM-1 concentration was less than that seen in the subjects consuming long-chain n-3 PUFAs (26%; Thies et al., 2001c). A recent intervention study used margarines to provide dietary intakes of 4.5 or 9.5 g ALA/day for six months, largely at the expense of linoleic acid (Kew et al., 2003b). ALA did not affect the proportions of T lymphocytes, helper T lymphocytes, cytotoxic T lymphocytes, B lymphocytes or monocytes in the circulation. There was no effect of ALA on the phagocytic activity of neutrophils and monocytes, on the ability of neutrophils and monocytes to undergo respiratory burst, on T lymphocyte proliferation, or on the production of cytokines (IL-2, IL-4, IFN- , TNF-, IL-1 , IL-6) by mononuclear cells (Kew et al., 2003b). Finally, ALA did not alter the delayed-type hypersensitivity response (Kew et al., 2003b). In another dietary intervention study using margarine and other strategies to provide an average of 6.3 g ALA/day for one year, plasma fibrinogen concentration was lower (by approximately 5%) than in the control group (Bemelmans et al., 2002). In another study, 8.1 g ALA/day from linseed oil for 12 weeks significantly decreased the serum concentrations of IL-6 (average decrease 23%), C-reactive protein (25%) and amyloid A (26%) (Rallidis et al., 2003). The habitual intake of ALA in the subjects studied was an average of 1 g/day. Thus, a substantial

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increase in ALA intake in these subjects appears to have induced a marked antiinflammatory effect.

15.5 Other mechanisms of action of dietary fatty acids not involving eicosanoids Altered synthesis of eicosanoids is a biologically plausible and readily accepted mechanism by which to explain the actions of certain fatty acids, especially n-3 PUFAs upon inflammation and immunity. However, many of the effects reported are different from those that would be predicted if EPA was acting as a PGE2 antagonist. For example, n-3 PUFAs decrease production of TNF- by monocytes and macrophages, but so does PGE2. One explanation for this is that focusing on PGE2 is too simplistic. It may be that it is the overall impact of n-3 PUFAs on the entire range of arachidonic acid-derived mediators coupled with the increased production of EPA-derived mediators (whose effects are not known and some of which are not yet even discovered) that accounts for the observed effects. Thus, the effects of n-3 PUFAs may still be related to eicosanoids. Another explanation is that n-3 PUFAs, and other active fatty acids, work through eicosanoid-independent mechanisms. There is some evidence for this from cell culture studies (Santoli and Zurier, 1989; Calder et al., 1992; Soyland et al., 1993). These other mechanisms also rely upon an altered fatty acid composition of membrane phospholipids (Fig. 15.5) and include effects on: · the physical nature of the membrane (often referred to as fluidity) · the ability of the membrane to undergo structural and functional changes in response to a cellular stimulus · the ability to generate intracellular signalling molecules. As will be seen below, these mechanisms are closely related to one another, since cell membrane composition, fluidity and function and the generation of signalling molecules following a cellular stimulus are interlinked. The fluidity of the plasma membrane, or of regions of the plasma membrane, is important in the functioning of cells (Stubbs and Smith, 1984). The fluidity of a membrane is determined by its lipid components and by their fatty acid composition (Stubbs and Smith, 1984). Membrane fluidity is an important regulator of phagocytosis (Calder et al., 1990). Cell culture experiments have demonstrated that changes in fatty acid composition of immune cells alter membrane fluidity (e.g. Calder et al., 1994), and that this is related to altered cell function (Calder et al., 1990, 1994). However, an alteration in membrane fluidity of cells of the immune system has been less easy to demonstrate after dietary manipulations (e.g. Yaqoob et al., 1995; Tappia et al., 1997). This may be because the fatty acid composition changes induced by dietary changes are less extreme than those seen in cell culture. In addition, in the intact animal, mechanisms to counter the fluidising effect of increasing the PUFA content of membranes (e.g. insertion of cholesterol) can be achieved more readily than in

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Fig. 15.5 Potential mechanisms by which fatty acids can affect inflammation and immune function.

culture. Therefore, alterations in the fluidity of the immune cell membranes may not be a mechanism by which fatty acids affect immune function. However, techniques used to determine membrane fluidity tend to measure `average' fluidity over a large surface and it may be that, while this is unaffected, fluidity of smaller regions of the membrane does change but is undetected. Lipid rafts are microenvironments in the outer leaflet of the phospholipid bilayer of plasma membranes. Lipid rafts are rich in unsaturated fatty acids, and they are considered to be more fluid than other regions of the membrane. Many proteins involved in cell signalling are located in lipid rafts (Brown and London, 1998; Simons and Toomre, 2000), and they appear to be particularly important in the signalling processes within immune cells (Katagiri et al., 2001). For example, the T cell receptor clusters within lipid rafts upon contact with an antigen presenting cell forming a contact zone within which intracellular signalling is initiated. Several signalling proteins including members of the src family of protein kinases such as lyk and fyn are concentrated on the cytoplasmic side of lipid rafts and become activated in response to signalling through the T cell receptor. Cell culture studies have demonstrated that provision of EPA to T cells results in marked enrichment of EPA in lipid rafts and in the displacement of certain proteins from those rafts (Stulnig et al., 2001). These displaced proteins included lck and the protein known as linker of activated T cells (LAT). LAT is involved in signalling subsequent to src kinases and among its substrates in T cells is phospholipase C 1. A recent study showed that EPA decreased the phosphorylation of LAT and of phospholipase C 1 in

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cultured T cells and the authors suggested that this was caused by selective displacement of LAT from lipid rafts (Zeyda et al., 2002). Interestingly, feeding fish oil to rats resulted in decreased PLC 1 phosphorylation in T lymphocytes subsequently stimulated in culture (Sanderson and Calder, 1998). Taken together these findings suggest that dietary n-3 PUFAs might modify the composition of lipid rafts in T cells and that this alters the subsequent cellular response to a stimulus. Many of the established cell signalling molecules are generated directly from membrane phospholipids. Examples of these signalling molecules include inositol-1,4,5±trisphosphate, diacylglycerol, phosphatidic acid, choline, and ceramide. These have important roles in regulating the activity of proteins involved in immune cell responses. The concentration and/or composition of lipid-derived signalling molecules have been shown to be sensitive to n-3 PUFA availability either in cell culture or through the diet (Miles and Calder, 1998 for references). This may be due to either altered activity of the enzymes that generate the signals or to altered composition of the substrate molecules. There is evidence to support each of these possibilities (Miles and Calder, 1998; Yaqoob, 2003). For example, lymphocyte phospholipase C 1 activity is reduced after feeding a diet rich in fish oil, which might account for the decreased generation of signalling molecules observed (Sanderson and Calder, 1998). A change in the generation of intracellular signalling molecules may have rapid effects on cellular responses. However, there may also be longer term effects such as alterations in the pattern of gene expression. The effects of fatty acids, especially PUFAs, on expression of genes coding for key regulatory proteins in various metabolic pathways has been most clearly described in hepatocytes and adipocytes (Jump et al., 1994). These effects of fatty acids are mediated by both indirect mechanisms (e.g. by eicosanoids, hormones) and direct effects on gene expression. There is now emerging evidence that PUFAs regulate the expression of genes involved in inflammation and immunity (Calder, 2002). Among the genes that are down-regulated by n-3 PUFAs are those encoding TNF-, IL-1, COX-2, 5-LOX, 5-LOX activating protein, certain matrix metalloproteinases, and VCAM-1. Since the expression of many of these genes is regulated by the transcription factor nuclear factor kappa B (NFB), these observations suggest that n-3 PUFAs might somehow affect the activity of this transcription factor. This might be through effects on cell signalling leading to NFB activation. There is recent evidence that dietary fish oil affects NFB activity (Lo et al., 1999; Xi et al., 2001), in a manner that is consistent with its ability to down-regulate the production of inflammatory mediators. A second group of transcription factors currently undergoing scrutiny for their role in inflammation are the peroxisome proliferator activated receptors (PPARs). The main members of this family are PPAR and PPAR . Although PPAR and play important roles in liver and adipose tissue, respectively (Schoonjans et al., 1996), they are also found in inflammatory cells (Chinetti et al., 1998; Ricote et al., 1998). PPARs can bind, and appear to be regulated by, PUFAs and eicosanoids (Kliewer et al., 1995; Devchand et al., 1996). PPAR

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deficient mice have a prolonged response to inflammatory stimuli (Devchand et al., 1996), suggesting that PPAR activation might be anti-inflammatory. More recently activators of both PPAR and have been shown to inhibit the activation of a number of inflammatory genes, including TNF-, IL-1 , IL-6, IL-8, COX-2 and VCAM-1 (see Calder, 2002 for references). Two mechanisms for the anti-inflammatory actions of PPARs have been proposed (Chinetti et al., 2000; Delerive et al., 2001). The first is that PPARs might stimulate the breakdown of inflammatory eicosanoids through induction of peroxisomal oxidation. The second is that PPARs might interfere with/antagonise the activation of other transcription factors, including NFB. Although the effect of fish oil on PPAR expression in inflammatory cells has not been reported, studies in other tissues (e.g. Berthou et al., 1995) suggest that n-3 PUFAs might act by increasing the level of these anti-inflammatory transcription factors in such cells.

15.6

Dietary fatty acids and inflammatory diseases

As indicated in section 15.1, inappropriate immune activity or immune dysregulation is a feature of a range of degenerative human diseases. Furthermore, ageing can be associated with diminished acquired immune function, with altered balances within the T cell phenotypes, and with increased inflammation. These changes might predispose the elderly to infections and/or to specific diseases including rheumatoid arthritis, allergic diseases, and inflammatory bowel disease. It is now recognised that atherosclerosis is an inflammatory disease (Ross et al., 1999). Furthermore, acute cardiovascular events are driven by inflammatory activities within the vessel wall (Plutzky, 1999). Given the effects of different fatty acids described in section 15.4, it is possible that the status of certain fatty acids may play a role in influencing the risk of inappropriate immune activity, immune dysregulation, immune decline and inflammation. It follows from this that decreased consumption of those fatty acids that increase risk and increased consumption of those that decrease risk should be associated with improved health. As far as diseases with an immunologic basis are concerned, there is evidence that oleic acid, GLA and long-chain n-3 PUFAs consumption is associated with improvements in outcome. An extrapolation from this is that they may lower the risk of developing the disease in the first place 15.6.1 Oleic acid Evidence for a protective effect of oleic acid comes from epidemiological and clinical studies involving rheumatoid arthritis. Linos et al. (1991, 1999) compared the relative risk of development of rheumatoid arthritis to lifelong consumption of olive oil in a Greek population. They found that individuals in the lowest category of consumption had 2.5±times higher risk of having arthritis

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than those in highest category of consumption. This study has several limitations including its retrospective nature and the difficulty of assessing lifelong consumption accurately. Nevertheless, Mediterranean populations have a lower prevalence of rheumatoid arthritis than those in Northern Europe (Cimmino et al., 1998) and where it occurs it appears to be less severe (Drosos et al., 1992). In a clinical trial investigating the potential benefit of fish oil in patients with rheumatoid arthritis, Kremer et al. (1990) used olive oil (9 g/day) as the placebo. They identified that olive oil improved five out of the 45 clinical measures made, and suggested that olive oil itself may be of some benefit in these patients. 15.6.2 GLA GLA has been used in a variety of diseases involving immune dysregulation or inflammation. It has been found to be efficacious in some of these. Indeed, in the United Kingdom certain preparations of GLA are licensed as medicines for use in atopic dermatitis, a disease in which it is particularly effective (see Burton, 1990 for a review). GLA also appears to be effective in rheumatoid arthritis. At least five double-blind, placebo-controlled studies of GLA in rheumatoid arthritis have been reported. These are reviewed elsewhere (Zurier, 1998; Belch and Muir, 2000) and so will only be summarised here. These studies provided between 0.36 and 2.8 g GLA/day for between 12 and 52 weeks. Each study reported some form of clinical improvement with GLA including a reduction in duration of morning stiffness and in the number and painfulness of tender and swollen joints. Three of the studies reported decreased use of non-steroidal antiinflammatory drugs (NSAIDs). 15.6.3 Long-chain n-3 PUFAs Aspirin and NSAIDs are widely used for symptom relief in inflammatory disease. These drugs act as COX inhibitors specifically targeting 2-series prostaglandins and thromboxanes. The discovery that long-chain n-3 PUFAs also act to reduce formation of these mediators promoted studies in animal models and clinical trials in a range of human diseases. Dietary fish oil has been shown to increase survival and decrease proteinuria and anti-DNA antibody formation in mice with autoimmune glomerulonephritis (a model of lupus), to decrease joint inflammation in rodent models of arthritis, and to decrease inflammation in rat models of colitis and of type-1 diabetes (see Calder 1997, 2001b for references). The efficacy of fish oil has been studied in several inflammatory diseases including rheumatoid arthritis, Crohn's disease, ulcerative colitis, psoriasis, lupus, multiple sclerosis, cystic fibrosis and asthma. Although there are clinical benefits reported from trials in each of these diseases (e.g. see Belluzzi and Miglio, 1998; Rodgers, 1998; Ziboh, 1998; Beckles Willson et al., 2003), the only one for which there is really strong evidence of benefit is rheumatoid arthritis. This may be a reflection of the large number of well designed and well

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conducted studies in arthritis; there have been at least 14 randomised, placebocontrolled, double-blind studies of fish oil in rheumatoid arthritis. These trials have been reviewed in some detail elsewhere (James and Cleland, 1997; Geusens, 1998; Calder, 2001c; Calder and Zurier, 2001), and so will be discussed only briefly here. They used between 1 and 7.1 g EPA plus DHA per day (average dose was 3.3 g/day) with a duration of 12 to 52 weeks. A variety of improvements in clinical outcome were reported. These include reduced duration of morning stiffness, reduced number of tender or swollen joints, reduced joint pain, reduced time to fatigue, increased grip strength and decreased use of NSAIDs. It has been concluded that the evidence for benefit from long-chain n-3 PUFAs in rheumatoid arthritis is robust (Cleland and James, 2000). There is currently considerable interest in the relative effects of n-3 and n-6 PUFAs in asthma (and other atopic diseases) (Hodge et al., 1994; Black and Sharp, 1997; Calder and Miles, 2000). The discussion centres on the roles of various eicosanoids produced from arachidonic acid in mediating allergic inflammation and in programming T lymphocytes to a phenotype that predisposes to such inflammation. Arachidonic acid-derived eicosanoids such as PGD2, LTC4, D4 and E4 are produced by the cells that mediate pulmonary inflammation in asthma (e.g. mast cells) and are believed to be the major mediators of asthmatic bronchoconstriction. Thus, it is considered that provision of n-3 PUFAs to asthmatics might be beneficial because of the resulting decrease in production of 4-series leukotrienes and other arachidonic acidderived mediators. However, the situation is complicated by the fact that different eicosanoids have different effects, some antagonising others. For example, the observations that PGE2 inhibits 5-LOX and promotes generation of lipoxins that act as inflammation `stop signals', indicate that PGE2 could, in fact, be protective in active asthma. Thus, interventions that aim to suppress PGE2 production could be counterproductive, at least in some asthmatics. Nevertheless, a number of trials of fish oil in asthma and related atopic diseases have been performed (Calder and Miles, 2000). Most of these studies reveal limited clinical impact, despite significant biochemical changes, although some have shown clinical improvements at least in some patient groups (Hodge et al., 1996; Broughton et al., 1997; Nagakura et al., 2000). A recent meta-analysis of fish oil in asthma concluded that there was no evidence of benefit (Woods et al., 2003). However, trying to intervene in the disease once it has developed may be the wrong approach, and n-3 PUFAs may still have a role in prevention. Since, PGE2 regulates T lymphocyte differentiation promoting the development of the Th2-type phenotype that underlies sensitisation to environmental allergens, it is possible that early exposure to long-chain n-3 PUFAs may be protective towards allergy, asthma and related diseases. There is some epidemiological evidence in support of this (Calder, 2003). However, despite a biologically plausible mechanism and supportive biochemical and epidemiological data, the key to demonstrating a protective effect of increased long-chain n-3 PUFA consumption towards allergic-type

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diseases must come from well-designed placebo-controlled intervention studies. It is now recognised that sensitisation to allergens occurs early in life, and so the characteristics of the maternal diet may be very important in determining predisposition to these diseases. Therefore, studies addressing this question need to be performed in pregnant women. Several such studies are currently under way and their findings are eagerly anticipated. Consumption of long-chain n-3 PUFAs lowers the risk of mortality from cardiovascular disease (see Calder and Yaqoob, 2003 for references). Since inflammation within the vessel wall contributes to the development of atherosclerosis (Ross, 1999), anti-inflammatory effects might play a role in the observed protective effect of n-3 PUFAs. N-3 PUFAs lower risk of myocardial infarction (Calder and Yaqoob, 2003) which is induced by inflammatory activity within the vessel wall (Plutzky, 1999). A recent study has provided evidence that long-chain-3 PUFAs from fish oil act to decrease inflammation within advanced atherosclerotic plaques (Thies et al., 2003). Therefore, the anti-inflammatory effects of n-3 PUFAs may make an important contribution to their cardioprotective effects. The improved appetite, increased dietary intake and weight gain in advanced pancreatic patients supplementing their diet with 2 g EPA/day (Barber et al., 1999) appears to relate to decreased production of mediators that give rise to cachexia such as IL-6 (Barber et al., 1999, 2000, 2001). Thus, the applications of the anti-inflammatory effects of n-3 PUFAs may extend beyond those disorders tradionally considered as `inflammatory'.

15.7 Targeting the immune function and inflammation: fatty acid-enriched functional foods Most experimental studies investigating dietary fatty acids, inflammation and immunity have used encapsulated oils, although a small number have used dietary change and/or specially modified foodstuffs. One of the great challenges in using functional foods to modulate the immune system will be to deliver sufficient amounts of the active fatty acids to have the desired effects. From the foregoing discussion it is clear that oleic acid, GLA, CLA and n-3 PUFAs are each of interest and it may be that different approaches will need to be used for each of these. 15.7.1 Oleic acid Western populations consume a significant amount of oleic acid (see section 15.1) with meat, cereals, milk and milk products, spreads and vegetables each supplying significant amounts. Olive oil is especially rich in oleic acid and other oleic acid rich oils such as high-oleic sunflower oil have been developed. Spreads that include a substantial proportion of oleic acid have become available in some countries. The use of oleic acid-rich oils and spreads in cooking and

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baking can lead to a substantial increase in oleic acid intake at the expense of either saturated or n-6 polyunsaturated fatty acids, or both, depending upon the exact nature of the replacement. However, the increase in oleic acid intake that appears to be required in order to influence immune function and inflammatory processes is quite large (Yaqoob et al., 1998), although this approach may have a significant impact on cardiovascular risk factors (Williams et al., 1999). 15.7.2 GLA GLA is found in oils that are commonly sold as dietary supplements, such as evening primrose oil and borage (also called starflower) oil. GLA is also found in blackcurrant seed oil. The dose of GLA required to elicit changes in cell fatty acid composition appears to be of the order of 1 to 1.5 g/day, and this may not be associated with changes in cell function. The incorporation of GLA into foods to deliver a biologically effective dose may be difficult. 15.7.3 CLA Preparations of CLA that are available in capsules are almost exclusively mixtures of several isomers, some of which will be biologically inactive and some of which may even be detrimental to human health (see Gaullier et al., 2002). The cis-9, trans-11 isomer of CLA is naturally found in ruminant milks, and is by far the most commonly consumed of the CLA isomers. Current intake of CLA is < 250 mg with most of this coming from milk and dairy products and much of the rest from meat. The cis-9, trans-11 CLA content of cows' milk and of beef can be increased by altering dairy feeding strategies (Lawson et al., 2001). Therefore, CLA-rich milk, dairy products and meat can be produced. Through consumption of a range of such modified products it will be possible for intake of cis-9, trans-11 CLA to be significantly increased. However, it is not yet clear whether this isomer exerts immunologic effects (see section 15.4.3). There are suggestions that it is the trans-10, cis-12, rather than the cis-9, trans11, isomer that is immunologically active. Currently it is not possible to produce milk containing trans-10, cis-12 CLA (Lawson et al., 2001), so that dairy products enriched with this isomer do not seem to be a probability. However, it is possible to produce trans-10, cis-12 CLA in pure form chemically, so this may be a viable alternative for use in foodstuffs, such as spreads. However, the whole range of health effects of this isomer need be examined in humans before it can be considered for widespread use. To date there are no studies of CLA-enriched foods and immune function in humans. 15.7.4 N-3 PUFAs From the foregoing discussion it is evident that long-chain n-3 PUFAs are more biologically potent than the precursor ALA. The only food sources that are naturally rich in long-chain n-3 PUFAs are the meat and blubber of marine

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mammals such as whale and seal; the flesh of fatty fish such as herring, mackerel, tuna, salmon, trout and sardines; and the DHA-rich brains and neural tissue of mammals. Of these sources, only fatty fish present a suitable option for most consumers. An intake of long-chain n-3 PUFAs of at least 1 g/day, and probably more (perhaps as much as 2 g/day), is required to exert immunologic effects. Fatty fish provide 1.5 to 3.5 g long-chain n-3 PUFAs per portion (British Nutrition Foundation, 1999). Thus, regular consumption of fatty fish would deliver amounts of these fatty acids to exert immunologic effects. Whitefish and shellfish are also sources of long-chain n-3 PUFAs, but one portion of these delivers between 0.1 and 0.5 g long-chain n-3 PUFAs (British Nutrition Foundation, 1999). Some consumers are unable to access or prepare fatty fish or dislike it. Therefore, although fatty fish is an excellent mode of delivery of long-chain n-3 PUFAs, it is not suitable for all consumers. Therefore, there is a need for alternative strategies for delivery of these fatty acids. One strategy is to enrich foods that do not normally contain long-chain n-3 PUFAs. There are two approaches to this. The first is to enrich existing foods with fish oil through food-processing technologies. One difficulty with this is that fish oil has a characteristic smell and taste that may not be appealing to consumers. More importantly, long-chain n-3 PUFAs oxidise very readily on exposure to air and so the enriched foods may be unstable and, at best, they will have a fairly short shelf life. One way to circumvent these problems is to use `microencapsulated' fish oil, in which the n-3 PUFAs are protected from air. There is still a limitation to how much n-3 PUFA can be incorporated through this route. However, this approach was recently used to prepare a spread that provided 0.8 g EPA plus DHA/day when consumed as part of the habitual diet (Kew et al., 2003b). The spread contained 0.56 g EPA plus DHA per 25 g. The increased intake of longchain n-3 PUFAs did not affect either immune cell fatty acid composition or function (Kew et al., 2003b). This is most likely because the dose of n-3 PUFAs provided was insufficient. The second approach to providing long-chain n-3 PUFAs is to enrich traditional foods with n-3 PUFAs through farming practices. For example, feeding pigs on a diet containing fish oil results in a marked increase in the amounts of EPA, DPA and DHA in the meat and fat (Leskanich et al., 1997; Irie and Sakimoto, 1992). Furthermore, feeding pigs on linseed, a source of ALA, resulted in increased amounts of EPA and DPA, but not DHA, in muscle, liver and kidney (Matthews et al., 2000). However, again one problem that arises when the level of enrichment of these tissues with n-3 PUFAs gets too high is lipid peroxidation, spoiling, and poor consumer acceptance. The immunologic effect of meats enriched in long-chain n-3 PUFAs, in the absence of other fatty acid interventions, has not been examined. Since markedly increasing the long-chain n-3 PUFA content of single foods presents a technical difficulty, an alternative approach may be to enrich many foods but to a lesser extent, such that consumption of the combination of foods will increase long-chain n-3 PUFA intake. Candidate foods would be those

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mentioned above (spreads, animal meats), eggs, bread, yoghurt, and milk-based drinks. An alternative to this could be to increase ALA consumption, since ALA can be converted to the longer chain fatty acids (Fig. 15.3). The study of Kelley et al. (1991, 1993) described earlier provided 18 g ALA/d from linseed oil mixed into salads, yoghurts, spreads and vegetables. Mantzioris et al. (1994) and Caughey et al. (1996) provided 14 g ALA/day through spreads and cooking oil. Both of these studies reported increases in the EPA content of immune cells and alterations in immune cell function (see sections 15.2.4 and 15.4.6). Therefore, foods enabling high ALA consumption may be useful to modifying immune function in humans. Kew et al. (2003b) used spreads to increase ALA intake by human volunteers to 4.5 or 9 g/day for a six-month period. The lower level of intake did not alter mononuclear cell fatty acid composition. However, the higher level of intake increased the EPA content of mononuclear cells by 30% (Kew et al., 2003b). This did not induce any alteration in any of the wide range of immune parameters studied. This observation, coupled with similar negative findings from studies with encapsulated linseed oil (Healy et al., 2000; Thies et al., 2001a,b,c; Wallace et al., 2003) suggests that quite marked increases in ALA intake are required to affect immune function, and that the only way to achieve these will be to produce a range of foods with increased ALA content. One factor restricting the effectiveness of ALA may be its limited conversion to longer chain-3 PUFAs (Burdge et al., 2002; Burdge and Wootton, 2002). This is believed to be due to the low activity of the -6 destaurase enzyme (Fig. 15.3). If this really is a limitation then a novel approach to increasing EPA status of human immune cells may to consume an increased amount of the product of 6 destaurase, stearidonic acid (Fig. 15.3). Very recently, James et al. (2003) reported that consumption of 0.75 or 1.5 g stearidonic acid/day (from capsules) increased the EPA and DPA content of red blood cells and plasma phospholipids, whereas consumption of the same amounts of ALA did not. They also stated that stearidonic acid increased the amount of EPA and DPA in mononuclear cells, although that data was not presented. However, stearidonic acid did not influence the production of TNF-, IL-1 or PGE2 by LPS-stimulated whole blood (James et al., 2003). This study highlights a novel approach to enriching human immune cells with long-chain n3 PUFAs, but it reinforces the conclusion that increases in intake of fatty acids that will induce functional changes are difficult to achieve. One final approach to increasing intake of n-3 PUFAs has been the combined use of an ALA-rich spread, salad dressing and mayonnaise made with linseed oil, linseed oil for cooking, canned fatty and fresh lean fish, and sausages and French onion dip containing microencapsulated fish oil (Mantzioris et al., 2000). This combination was used to increase the average intakes of ALA, EPA and DHA to 9.2, 0.8 and 1 g/day, respectively. The average proportion of ALA in blood mononuclear cells increased from 0.02 to 0.1% of fatty acids after two weeks, while the average proportion of EPA increased from 0.4 to 1.1% of fatty acids after four weeks. The proportions of DPA and DHA also increased

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significantly (Mantzioris et al., 2000). The production of PGE2, TXB2, TNF- and IL-1 by LPS-stimulated whole blood decreased by 26%, 34%, 40% and 20%, respectively. Thus, this study indicates that food vehicles can be used to deliver n-3 PUFAs to human immune cells in sufficient quantities to induce functional changes. 15.7.5 Combinations of nutrients A recent study used a novel semi-skimmed milk formulated to provide 5.1 g oleic acid, 0.13 g EPA, 0.2 g DHA, as well as vitamin E, vitamin B12 and folic acid, in a daily portion of 500 ml (Baro et al., 2003). The milk contained more linoleic acid than normal milk and contained only 30% of the normal amount of saturated fatty acids. Young adult volunteers consumed the milk (500 ml/day) for eight weeks. After this time the plasma concentrations of sICAM-1 and sVCAM-1 were significantly decreased by 10% and 15%, respectively, perhaps an indication of decreased inflammation.

15.8

Conclusions

Cells of the human immune system are amenable to altered fatty acid composition via the diet. This can lead to altered function. The effects of the long-chain n-3 PUFAs are the most studied and the best described. These fatty acids have been demonstrated to be efficacious in the treatment of some diseases related to immune dysfunction and to induce effects that might promote healthier ageing. Other fatty acids of interest in this context are oleic acid, GLA, CLA and n-3 PUFAs that are precursors to EPA (e.g. ALA, stearidonic acid). The levels of dietary intake at which these fatty acids modify immune cell function are quite high and this presents a challenge for the development of functional foods.

15.9

References

ABBAS A K, LICHTMAN A H, POBER J S

Philadelphia, WB Saunders.

(1994), Cellular and Molecular Immunology,

ABE Y, EL-MASRI B, KIMBALL K T, POWNALL H, REILLY C F, OSMUNDSEN K, SMITH C W,

(1998), Soluble adhesion molecules in hypertriglyceridaemia and potential significance on monocyte adhesion, Arterioscler Thromb Vasc Biol, 18, 723±731.

BALLANTYNE C M

ALBERS R, VAN DER WIELAN R P J, BRINK E J, HENDRIKS H F J, DOROVSKA-TARAN V N, MOHEDE I

(2003), Effects of cis-9, trans-11 and trans-10, cis-12 conjugated linoleic acid (CLA) isomers on immune function in healthy men, Eur J Clin Nutr, 57, 595±603. BARBER M D, ROSS J A, PRESTON T, SHENKIN A, FEARON K C H (1999), Fish oil-einriched nutritional supplement attenuates progression of the acute-phase response in weight-losing patients with advanced pancreatic cancer, J Nutr, 129, 1120±1125. CM

Dietary lipids and immune function

383

(2000), Metabolic response to feeding in weight-losing pancreatic cancer patients and its modulation by a fish oil-enriched nutritional supplement, Clin Sci, 98, 389±399. BARBER M D, FEARON K C H, TISDALE M J, MCMILLAN D C, ROSS J A (2001), Effect of a fish oilenriched nutritional supplement on metabolic mediators in patients with pancreatic cancer cachexia, Nutr Canc, 40, 118±124. BARBER M D, MCMILLAN D C, PRESTON T, ROSS J A, FEARON K C H

BARO L, FONOLLA J, PENA J L, MARYINEZ-FEREZ A, LUCENA A, JIMENEZ J, BOZA J J, LOPEZ-

HUERTAS E (2003), n-3 Fatty acids plus oleic acid and vitamin supplemented milk consumption reduces total and LDL cholesterol, homocysteine and levels of endothelial adhesion molecules in healthy humans, Clin Nutr, 22, 175±182. BAUMANN K H, HESSEL F, LARASS I, MULLER T, ANGERER P, KIEFL R, VON SCHACKY C (1999), Dietary !-3, !-6, and !-9 unsaturated fatty acids and growth factor and cytokine gene expression in unstimulated and stimulated monocytes, Arterioscler Thromb Vasc Biol, 19, 59±66. BECKLES WILLSON N, ELLIOTT T M, EVERARD M L (2003) Omega-3 fatty acids (from fish oils) for cystic fibrosis (Cochrane Review), in The Cochrane Library, Issue 2, Oxford, Update Software. BELCH J J F, MUIR A (2000), Evening primrose oil and borage oil in rheumatologic conditions, Am J Clin Nutr, 71, 352S±356S, BELLUZI A, MIGLIO F (1998) n-3 Fatty acids in the treatment of Crohn's disease, in Kremer J M, Medicinal Fatty Acids in Inflammation, Basel, Birkhauser, 91±101. BEMELMANS W J, BROER J, FESKENS E J, SMIT A J, MUSKIET F A, LEFRANDT J D, BOM V J, MAY J F,

(2002), Effect of increased intake of alpha-linolenic acid and group nutritional education on cardiovascular risk factors: the Mediterranean Alpha-linolenic Enriched Groningen Dietary Intervention (MARGARIN), Am J Clin Nutr, 75, 221±227.

MEYBOOM-DE JONG B

BERTHOU L, SALADIN R, YAQOOB P, BRANELLEC D, CALDER P, FRUCHART J-C, DENEFLE D,

AUWERX J, STAELS B (1995), Regulation of rat liver apolipoprotein A-I, apolipoprotein A-II and acyl CoA oxidase gene expression by fibrates and dietary fatty acids, Eur J Biochem, 232, 179±187. BLACK P N, SHARP S (1997), Dietary fat and asthma: is there a connection?, Eur Resp J, 10, 6±12. BLOK W L, DESLYPERE J-P, DEMACKER P N M, VAN DER VEN-JONGEKRIJG J, HECTORS M P C, VAN

(1997), Pro- and anti-inflammatory cytokines in healthy volunteers fed various doses of fish oil for 1 year, Eur J Clin Invest, 27, 1003±1008. BRITISH NUTRITION FOUNDATION (1999), Briefing Paper: n-3 Fatty Acids and Health, London, British Nutrition Foundation. BROUGHTON K S, JOHNSON C S, PACE B K, LIEBMAN M, KLEPPINGER K M (1997), Reduced asthma symptoms with n-3 fatty acid ingestion are related to 5-series leukotriene production, Am J Clin Nutr, 65, 1011±1017. BROWN D A, LONDON E (1998), Function of lipid rafts in biological membranes, Ann Rev Cell Dev Cell Biol, 14, 111±136. BRUUNSGAARD H, PEDERSEN A N, SCHROLL M, SKINHOJ P, PEDERSEN B K (2000), TNF-, leptin, and lymphocyte function in human ageing, Life Sci, 67, 2721±2731. BURDGE G C, WOOTTON S A (2002), Conversion of -linolenic acid to eicosapentaenoic, docosapentaenoic and docosahexaenoic acids in young women, Brit J Nutr, 88, 411±420. BURDGE G C, JONES A E, WOOTTON S A (2002), Eicosapentaenoic and docosapentaenoic DER MEER J M W, KATAN M B

384

Functional foods, ageing and degenerative disease

acids are the principal products of -linolenic acid metabolism in young men, Brit J Nutr, 88, 335±363. BURTON J L (1990) Essential fatty acids in atopic eczema: clinical studies, in Horrobin D F, Omega-6 Essential Fatty Acids: Pathophysiology and Roles in Cinical Medicine, New York, Alan R Liss, 67±73. CALDER P C (1996), Sir David Cuthbertson Medal Lecture: Immunomodulatory and antiinflammatory effects of omega-3 polyunsaturated fatty acids, Proc Nutr Soc, 55, 737±774. CALDER P C (1997), N-3 polyunsaturated fatty acids and cytokine production in health and disease, Ann Nutr Metab, 41, 203±234. CALDER P C (1998a), N-3 fatty acids and mononuclear phagocyte function, in Kremer J M, Medicinal Fatty Acids in Inflammation, Basel, Birkhauser, 1±27. CALDER P C (1998b), Dietary fatty acids and lymphocyte functions, Proc Nutr Soc, 57, 487±502. CALDER P C (2001a), N-3 polyunsaturated fatty acids, inflammation and immunity: pouring oil on troubled waters or another fishy tale? Nutr Res, 21, 309±341. CALDER P C (2001b), Polyunsaturated fatty acids, inflammation and immunity, Lipids, 36, 1007±1024. CALDER, P C (2001c), N-3 fatty acids and rheumatoid arthritis, in Ransley J K, Donnelly J K and Read N W, Food and Nutritional Supplements in Health and Disease, London, Springer Verlag, 175±197. CALDER P C (2002), Dietary modification of inflammation with lipids, Proc Nutr Soc, 61, 345±358. CALDER P C (2003), Polyunsaturated fatty acids and cytokine profiles: a clue to the changing prevalence of atopy?, Clin Exp Allergy, 33, 412±415. CALDER P C, MILES E A (2000), Fatty acids and atopic disease, Pediat All Immunol, 11 (Suppl.), 29±36. CALDER P C, YAQOOB P (2003), Cardiovascular benefit of long chain n-3 fatty acids ± the evidence gets stronger, Curr Top Nutraceut Res, 1, 105±112. CALDER P C, ZURIER R B (2001), Polyunsaturated fatty acids and rheumatoid arthritis, Curr Opin Clin Nutr Metab Care, 4, 115±121. CALDER P C, BOND J A, HARVEY D J, GORDON S, NEWSHOLME E A (1990), Uptake of saturated and unsaturated fatty acids into macrophage lipids and their effect upon macrophage adhesion and phagocytosis, Biochem J, 269, 807±814. CALDER P C, BEVAN S J, NEWSHOLME E A (1992), The inhibition of T-lymphocyte proliferation by fatty acids is via an eicosanoid-independent mechanism, Immunology, 75, 108±115. CALDER P C, YAQOOB P, HARVEY D J, WATTS A, NEWSHOLME E A (1994), The incorporation of fatty acids by lymphocytes and the effect on fatty acid composition and membrane fluidity, Biochem J, 300, 509±518. CALDER P C, YAQOOB P, THIES F, WALLACE F A, MILES E A (2002), Fatty acids and lymphocyte functions, Brit J Nutr, 87, S31±S48. CANNON J G, FIATARONE M A, MEYDANI M, GONG J, SCOTT L, BLUMBERG J B, EVANS W J (1995), Ageing and dietary modulation of elastase and interleukin-1 beta secretion, Am J Physiol, 268, R208±R213. CAUGHEY G E, MANTZIORIS E, GIBSON R A, CLELAND L G, JAMES M J (1996), The effect on human tumor necrosis factor a and interleukin 1b production of diets enriched in n3 fatty acids from vegetable oil or fish oil, Am J Clin Nutr, 63, 116±122. CHAN D C, WATTS G F, BARRETT P H R, BEILIN L J, MORI T A (2002) Effect of atorvastatin and

Dietary lipids and immune function

385

fish oil on plasma high-sensitivity C-reactive protein concentrations in individuals with visceral obesity, Clin Chem, 48, 877±883. CHINETTI G, GRIGLIO S, ANTONUCCI M, TORRA I P, DELERIVE P, MAJD Z, FRUCHART J C,

(1998), Activation of peroxisome-activated receptors alpha and gamma induces apoptosis of human monocyte-derived macrophages, J Biol Chem, 273, 25573±25580. CHINETTI G, FRUCHART J C, STAELS B (2000), Peroxisome proliferator-activated receptors (PPARs): nuclear receptors at the crossroads between lipid metabolism and inflammation, Inflamm Res, 49, 497±505. CIMMINO M A, PARISI M, MOGGIANA G, MELA G S, ACCARDO S (1998), Prevalence of rheumatoid arthritis in Italy: the Chivari study, Ann Rheum Dis, 57, 315±318. CLELAND L G, JAMES M J (2000), Fish oil and rheumatoid arthritis: antiinflammatory and collateral health benefits, J Rheumatol, 27, 2305±2307. COOPER A L, GIBBONS L, HORAN M A, LITTLE R A, ROTHWELL N J (1993), Effect of dietary fish oil supplementation on fever and cytokine production in human volunteers, Clin Nutr, 12, 321±328. CURTIS C L, HUGHES C E, FLANNERY C R, LITTLE C B, HARWOOD J L, CATERSON B (2000), N-3 fatty acids specifically modulate catabolic factors involved in articular cartilage degradation, J Biol Chem, 275, 721±724. CHAPMAN J, NAJIB J, STAELS B

CURTIS C L, REES S G, LITTLE C B, FLANNERY C R, HUGHES C E, WILSON C, DENT C M, OTTERNESS

(2002), Pathologic indicators of degradation and inflammation in human osteoarthritic cartilage are abrogated by exposure to n-3 fatty acids, Arth Rheum, 46, 1544±1553.

I G, HARWOOD J L, CATERSON B

DE CATERINA R, BASTA G, LAZZERINI G, DELL'OMO G, PETRUCCI R, MORALE M, CARMASSI F,

(1997), Soluble vascular cell adhesion molecule-1 as a bihumoral correlate of atherosclerosis, Arteriscler Thromb Vasc Biol, 17, 2646±2654. DELERIVE P, FRUCHART J C, STAELS B (2001), Peroxisome proliferator-activated receptors in inflammation control, J Endocrinol, 169, 453±459. DE LUCA P, ROSSETTI R G, ALAVIAN C, KARIM P, ZURIER R B (1999), Effects of gammalinolenic acid on interleukin-1 beta and tumor necrosis factor-alpha secretion by stimulated human peripheral blood monocytes: studies in vitro and in vivo, J Invest Med, 47, 246±250. DEPARTMENT OF HEALTH (1994), Nutritional Aspects of Cardiovascular Disease, London, HMSO. DEVCHAND P R, KELLER H, PETERS J M, VAZQUEZ M, GONZALEZ F J, WAHLI W (1996), The PPAR-leukotriene B4 pathway to inflammation control, Nature, 384, 39±43. DROSOS A A, LANCHBURY J S, PANAYI G S, MOUTSOPOULOS H M (1992) Rheumatoid arthritis in Greek and British patients, Arth Rheum, 35, 745±748. PEDRINELLI R

ENDRES S, GHORBANI R, KELLEY V E, GEORGILIS K, LONNEMANN G, VAN DER MEER J M W, CANNON J G, ROGERS T S, KLEMPNER M S, WEBER P C, SCHAEFFER E J, WOLFF S M,

(1989), The effect of dietary supplementation with n-3 polyunsaturated fatty acids on the synthesis of interleukin-1 and tumor necrosis factor by mononuclear cells, N Eng J Med, 320, 265±271. ENS J G, MA D W L, COLE K S, FIELD C J, CLANDININ M T (2001), An assessment of c9,t11 linoleic acid intake in a small group of young Canadians, Nutr Res, 21, 955±960. FAULL R J (1995), Adhesion molecules in health and disease, Aust NZ J Med, 25, 720±730. FELDMAN M, MAINI R N (1999), The role of cytokines in the progression of rheumatoid arthritis, Rheumatology, 38 (Suppl 2), 3±7. FISHER M, LEVINE P H, WEINER B H, JOHNSON M H, DOYLE E M, ELLIS P A, HOOGASIAN J J (1990), DINARELLO C A

386

Functional foods, ageing and degenerative disease Dietary n-3 fatty acid supplementation reduces superoxide production and chemiluminescence in monocyte enriched preparation of leukocytes, Am J Clin Nutr, 51, 804±808.

GALLAI V, SARCHIELLI P, TREQUATTRINI A, FRANCESCHINI M, FLORIDI A, FIRENZE C, ALBERTI A, DI BENEDETTO D, STRAGLIOTTO E (1993), Cytokine secretion and eicosanoid production in the peripheral blood mononuclear cells of MS patients undergoing dietary supplementation with n-3 polyunsaturated fatty acids, J Neuroimmunol, 56, 143±153. GAULLIER J-M, BERVEN G, BLANKSON H, GUDMUNDSEN O (2002), Clinical trial results support a preference for using CLA preparations enriched with two isomers rather than four isomers in human studies, Lipids, 37, 1019±1025. GEARING A J H, NEWMAN W (1993), Circulating adhesion molecules, Immunol Today, 14, 506±512. GEUSENS P P (1998), N-3 fatty acids in the treatment of rheumatoid arthritis, in Kremer J M, Medicinal Fatty Acids in Inflammation, Basel, Birkhauser, 111±123. GEWIRTZ A T, COLLIER-HYAMS L S, YOUNG A N, KUCHARZIK T, GUILFORD W J, PARKINSON J F,

(2002), Lipoxin A4 analogs attenuate induction of intestinal epithelial proinflammatory gene expression and reduce the severity of dextran sodium sulfate-induced colitis, J Immunol, 168, 5260±5267. GIBNEY M J, HUNTER B (1993), The effects of short- and long-term supplementation with fish oil on the incorporation of n-3 polyunsaturated fatty acids into cells of the immune system in healthy volunteers, Eur J Clin Nutr, 47, 255±259. GOLDYNE M E, STOBO J D (1982), Human monocytes synthesize eicosanoids from T lymphocyte-derived arachidonic acid, Prostaglandins, 24, 623±630. WILLIAMS I R, NEISH A S, MADARA J L

GRIMBLE R F, HOWELL W M, O'REILLY G, TURNER S J, MARKOVIC O, HIRRELL S, EAST J M,

CALDER P C (2002), The ability of fish oil to suppress tumor necrosis factor-a production by peripheral blood mononuclear cells in healthy men is associated with polymorphisms in genes that influence tumor necrosis factor a production, Am J Clin Nutr, 76, 454±459. HALVORSEN D A, HANSEN J-B, GRIMSGAARD S, BONNA K H, KIERULF P, NORDOY A (1997), The effect of highly purified eicosapentaenoic and docosahexaenoic acids on monocyte phagocytosis in man. Lipids, 32, 935±942. HARBIGE L S (2003), Fatty acids, the immune response, and autoimmunity: a question of n6 essentiality and the balance between n-6 and n-3, Lipids, 38, 323±341. HAYEK M G, MEYDANI S N, MEYDANI M, BLUMBERG J B (1994), Age differences in eicosanoid production of mouse splenocytes ± effects on mitogen-induced T-cell proliferation, J Gerontol, 49, B197±B207. HAYEK M G, MURA C, WU D Y, BEHARKA A A, HAN S N, PAULSEN K E, HWANG D, MEYDANI S N

(1997), Enhanced expression of indicible cyclooxygenase with age in murine macrophages, J Immunol, 159, 2445±2451. HEALY D A, WALLACE F A, MILES E A, CALDER P C, NEWSHOLME P (2000), Effect of low to moderate amounts of dietary fish oil on neutrophil lipid composition and function, Lipids, 35, 763±768. HODGE L, PEAT J K, SALOME C (1994), Increased consumption of polyunsaturated oils may be a cause of increased prevalence of childhood asthma, Aust NZ J Med, 24, 727. HODGE L, SALOME C M, PEAT J K, HABY M M, XUAN W, WOOLCOCK A J (1996) Consumption of oily fish and childhood asthma risk, Med J Aust, 164, 137±140. HOWELL W M., CALDER P C, GRIMBLE R F (2002), Gene polymorphisms, inflammatory diseases and cancer, Proc Nutr Soc, 61, 447±456.

Dietary lipids and immune function

387

(1996), Fish oil supplementation inhibits the expression of major histocompatibility complex class II molecules and adhesion molecules on human monocytes, Am J Clin Nutr, 63, 267±272.

HUGHES D A, PINDER A C, PIPER Z, JOHNSON I T, LUND E K

HWANG S-J, BALLANTYNE C M, SHARRETT A R, SMITH L C, DAVIS C E, GOTTO A M, BOERWINKLE

(1997), Circulating adhesion molecules VCAM-1, ICAM-1 and E-selectin in carotid atherosclerosis and incident coronary heart disease cases, Circulation, 96, 4219±4225. IRIE M, SAKAMOTO M (1992), Fat characteristics of pigs fed fish oil containing eicosapentaenoic acid and docosahexaenoic acid, J An Sci, 70, 470±477. JAMES M L, CLELAND L G (1997) Dietary n-3 fatty acids and therapy for rheumatoid arthritis, Seminars in Arthritis and Rheumatism, 2, 85±97. JAMES M J, URSIN V M, CLELAND L G (2003) Metabolism of stearidonic acid in human subjects: comparison with the metabolism of other n-3 fatty acids, Am J Clin Nutr, 77, 1140±1145. JOHANSEN O, SELJEFLOT I, HOSTMARK A T, ARNESEN H (1999), The effect of supplementation with omega-3 fatty acids on soluble markers of endothelial function in patients with coronary heart disease, Arterioscler Thromb Vasc Biol, 19, 1681±1686. E

JOHNSON M M, SWAN D D, SURETTE M E, STEGNER J, CHILTON T, FONTEH A N, CHILTON F H

(1997), Dietary supplementation with gamma-linolenic acid alters fatty acid content and eicosanoid production in healthy humans, J Nutr, 127, 1435±1444. JUMP D B, CLARKE S D, THELAN A, LIIMATTA M, REN B, BADIN M (1996), Dietary polyunsaturated fatty acid regulation of gene transcription. Prog Lipid Res, 35, 227±241. KATAGIRI Y U, KIYOKAWA N, FUJIMOTO J (2001), A role for lipid rafts in immune cell signalling, Microbiol Immunol, 45, 1±8. KELLEY D S, ERICKSON K L (2003), Modulation of body composition and immune cell functions by conjugated linoleic acid in humans and animal models: benefits vs. risks, Lipids, 38, 377±386. KELLEY D S, BRANCH L B, IACONO J M (1989), Nutritional modulation of human immune status, Nutr Res, 9, 965±975. KELLEY D S, BRANCH L B, LOVE J E, TAYLOR P C, RIVERA Y M, IACONO J M (1991), Dietary alpha-linolenic acid and immunocompetence in humans, Am J Clin Nutr, 53, 40± 46. KELLEY D S, DOUGHERTY R M, BRANCH L B, TAYLOR P C, IACONO J M (1992), Concentration of dietary n-6 polyunsaturated fatty acids and the human immune status, Clin Immunol Immunopathol, 62, 240±244. KELLEY D S, NELSON G J, LOVE J E, BRANCH L B, TAYLOR P C, SCHMIDT P C, MACKEY B E, IACONO J M (1993), Dietary -linolenic acid alters tissue fatty acid composition, but not blood lipids, lipoproteins or coagulation status in humans, Lipids, 28, 533±537. KELLEY D S, TAYLOR P C, NELSON G J, SCHMIDT P C, MACKEY B E, KYLE D (1997), Effects of dietary arachidonic acid on human immune response, Lipids, 32, 449±456. KELLEY D S, TAYLOR P C, NELSON G J, MACKEY B E (1998a), Arachidonic acid supplementation enhanses synthesis of eicosanoids without suppressing immune functions in young healthy men, Lipids, 33, 125±130. KELLEY D S, TAYLOR P C, NELSON G J, MACKEY B E (1998b), Dietary docosahexaenoic acid and immunocompetence in young healthy men, Lipids, 33, 559±566. KELLEY D S, TAYLOR P C, NELSON G J, SCHMIDT P C, FERRETTI A, ERICKSON K L, YU R, CHANDRA R K (1999), Docosahexaenoic acid ingestion inhibits natural killer cell activity and production of inflammatory mediators in young healthy men, Lipids, 34, 317±324.

388

Functional foods, ageing and degenerative disease

KELLEY D S, TAYLOR P C, RUDOLPH I L, BENITO P, NELSON G J, MACKEY B E, ERICKSON K L

(2000), Dietary conjugated linoleic acid did not alter immune status in young healthy women, Lipids, 35, 1065±1071. KELLEY D S, SIMON V A, TAYLOR P C, RUDOLPH I L, BENITO P, NELSON G J, MACKEY B E,

KEW

ERICKSON K L (2001), Dietary supplementation with conjugated linoleic acid increased its concentration in human peripheral blood mononuclear cells, but did not alter their function, Lipids, 36, 669±674. S, BANERJEE T, MINIHANE A M, FINNEGAN Y E, WILLIAMS C M, CALDER P C (2003a), Relation between the fatty acid composition of peripheral blood mononuclear cells and measures of immune cell function in healthy, free-living subjects aged 25±72 y, Am J Clin Nutr, 77, 1278±1286.

KEW S, BANERJEE T, MINIHANE A M, FINNEGAN Y E, MUGGLI R, ALBERS R, WILLIAMS C M,

(2003b), Lack of effect of foods enriched with plant- or marine-derived n-3 fatty acids on human immune function, Am J Clin Nutr, 77, 287±1295. KLEIWER S A, LENHARD J M, WILLSON T M, PATEL I, MORRIS D C, LEHMAN J M (1995), A prostaglandin J2 metabolite binds peroxisome proliferator-activated receptor and promotes adipocyte differentiation, Cell, 83, 813±819. CALDER P C

KREMER J M, LAWRENCE D A, JUBIZ W, DIGIACOMO R, RYNES R, BARTHOLOMEW L E, SHERMAN M (1990), Dietary fish oil and olive oil supplementation in patients with rheumatoid arthritis, Arth Rheum, 33, 810±820. LAWSON R E, MOSS A R, GIVENS D I (2001), The role of dairy products in supplying conjugated linoleic acid to man's diet: a review, Nutr Res Rev, 14, 153±172. LEE T H, HOOVER R L, WILLIAMS J D, SPERLING R I, RAVALESE J, SPUR B W, ROBINSON D R,

(1985), Effects of dietary enrichment with eicosapentaenoic acid and docosahexaenoic acid on in vitro neutrophil and monocyte leukotriene generation and neutrophil function, N Eng J Med, 312, 1217±1224. LESKANICH C O, MATTHEWS K R, WARKUP C C, NOBLE R C, HAZZLEDINE M (1997), The effect of dietary oil containing (n-3) fatty acids on the fatty acid, physicochemical, and organoleptic characteristics of pig meat and fat, J An Sci, 75, 673±683. LESOURD B, MAZARI L (1999), Nutrition and immunity in the elderly, Proc Nutr Soc, 58, 685±695. LESOURD B, RAYNAUD-SIMON A, MAZARI L (2002), Nutrition and ageing of the immune system, in Calder P C, Field C J, Gill H S. Nutrition and the Immune System, Wallingford, CABI, 357±374. LEVY B D, CLISH C B, SCHMIDT B, GRONERT K, SERHAN C N (2001), Lipid mediator class switching during acute inflammation: signals in resolution, Nature Immunol, 2, 612±619. LEWIS R A, AUSTEN K F, SOBERMAN R J (1990), Leukotrienes and other products of the 5lipoxygenase pathway: biochemistry and relation to pathobiology in human diseases, N Eng J Med, 323, 645±655. COREY E J, LEWIS R A, AUSTEN K F

LINOS A, KAKLAMANI E, KONTOMERKOS A, KOUMANTAKI Y, GAZI S, VAIOPOULOS G, TSOKOS G

C, KAKLAMANIS P H (1991), The effect of olive oil and fish consumption on rheumatoid arthritis ± a case control study, Scand J Rheumatol, 20, 419±426.

LINOS A, KAKLAMANI V G, KAKLAMANI E, KOUMANTAKI Y, GIZIAKI E, PAPAZOGLOU S,

(1999), Dietary factors in relation to rheumatoid arthritis: a role for olive oil and cooked vegetables?, Am J Clin Nutr, 70, 1077±1082. LO C J, CHIU K C, FU M J, CHU A, HELTON S (2000), Fish oil modulates macrophage P44/42 mitogen-activated protein kinase activity induced by lipopolysaccharide, J Parent MANTZOROS C S

Dietary lipids and immune function

389

Ent Nutr, 24, 159±163. (1996), Dietary fish oil decreases superoxide generation by human neutrophils: relation to cyclooxygenase pathway and lysosomal enzyme release, Prost Leuk Essent Fatty Acids, 55, 167±172. LUOSTARINEN R, SIEGBAHN A, SALDEEN T (1992), Effect of dietary fish oil supplemented with different doses of vitamin E on neutrophil chemotaxis in healthy volunteers, Nutr Res, 12, 1419±1430. MADSEN T, SKOU HA, HANSEN V E, FOG L, CHRISTENSEN J H., TOFT E, SCHMIDT E B (2001), Creactive protein, dietary n-3 fatty acids, and the extent of coronary artery disease, Am J Cardiol, 88, 1139±1142. MADSEN T, CHRISTENSEN J H, BLOM M, SCHMIDT E B (2003), The effect of dietary n-3 fatty acids on serum concentrations of C-reactive protein: a dose-response study, Brit J Nutr, 89, 517±522. MANTZIORIS E, JAMES M J, GIBSON R A, CLELAND L G (1994) Dietary substitution with an linolenic acid-rich vegetable oil increases eicosapentaenoic acid concentrations in tissues, Am J Clin Nutr, 59, 1304±1309. MANTZIORIS E, CLELAND L G, GIBSON R A, NEUMANN M A, DEMASI M, JAMES M J (2000) Biochemical effects of a diet containing foods enriched with n-3 fatty acids, Am J Clin Nutr, 72, 42±48. MATTHEWS K R, HOMER D B, THIES F, CALDER P C (2000), Effect of whole linseed (Linum usitatissimum) in the diet of finishing pigs on growth and performance and on the quality and fatty acid composition of various tissues, Brit J Nutr, 83, 637±643. MEYDANI S N, YOGEESWARAN G, LIU S, BASKAR S, MEYDANI M (1988), Fish oil and tocopherol-induced changes in natural killer cell-mediated cytotoxicity and PGE2 synthesis in young and old mice, J Nutr, 118, 1245±1252. LUOSTARINEN R, SALDEEN T

MEYDANI S N, ENDRES S, WOODS M M, GOLDIN B R, SOO C, MORRILL-LABRODE A, DINARELLO C, GORBACH S L (1991), Oral (n-3) fatty acid supplementation suppresses cytokine production and lymphocyte proliferation: comparison between young and older women, J Nutr, 121, 547±555. MEYDANI S N, SHAPIRO A C, MEYDANI M, BLUMBERG J B (1992), Lung eicosanoid synthesis is affected by age, dietary fat and vitamin E, J Nutr, 122, 1627±1633. MEYDANI S N, LICHTENSTEIN A H, CORNWALL S, MEYDANI M, GOLDIN B R, RASMUSSEN H,

(1993), Immunologic effects of National Cholesterol Education Panel Step-2 Diets with and without fish-derived n-3 fatty acid enrichement, J Clin Invest, 92, 105±113. MILES E A, CALDER P C (1998), Modulation of immune function by dietary fatty acids, Proc Nutr Soc, 57, 277±292. MILES E A, THIES F, WALLACE F A, POWELL J R, HIRST T L, NEWSHOLME E A, CALDER P C (2001), Influence of age and dietary fish oil on plasma soluble adhesion molecule concentrations, Clin Sci, 100, 91±100. MILES E A., ALLEN E, CALDER P C (2002), In vitro effects of eicosanoids derived from different 20±carbon fatty acids on production of monocyte-derived cytokines in human whole blood cultures, Cytokine, 20, 215±223. MILES E A, ASTON L, CALDER P C (2003), In vitro effects of eicosanoids derived from different 20±carbon fatty acids on T helper type 1 and T helper type 2 cytokine production in human whole-blood cultures, Clin Exp Allergy, 33, 624±632. DINARELLO C A, SCHAEFER E J

MOLVIG J, POCIOT F, WORSAAE H, WOGENSEN L D, BAEK L, CHRISTENSEN P, MANDRUPPOULSEN T, ANDERSEN K, MADSEN P, DYERBERG J, NERUP J (1991), Dietary supplementation with omega 3 polyunsaturated fatty acids decreases mononuclear cell proliferation

390

Functional foods, ageing and degenerative disease and interleukin 1 beta content but not monokine secretion in healthy and insulin dependent diabetic individuals, Scand J Immunol, 34, 399±410.

MORISAKI N, SAITO I, TAMURA K, TASHIRO J, MASUDA M, KANZAKI T, WATANABE S, MASUDA Y, SAITO Y (1997), New indices of ischemic heart disease and ageing: studies on the serum levels of intercellular adhesion molecule-1 (ICAM-1) and soluble vascular cell adhesion molecule-1 (VCAM-1) in patients with hypercholesterolemia and ischemic heart disease, Atherosclerosis, 131, 43±48. MOSSMAN T R, SAD S (1996), The expanding universe of T-cell subsets: Th1, Th2 and more, Immunol Today, 17, 138±146. MUNRO J M (1993), Endothelial-leukocyte adhesive interactions in inflammatory diseases, Eur Heart J, 14 (Suppl K), 72±77. NAGAKURA T, MATSUDA S, SHICHIJYO K, SUGIMOTO H, HATA K (2000), Dietary supplementation with fish oil rich in omega-3 polyunsaturated fatty acids in children with bronchial asthma, Eur Resp J, 16, 861±865. NIELSEN G L, FAARVANG K L, THOMSEN B S, TEGLBJAERG K L, JENSEN L T, HANSEN T M,

LERVANG H H, SCHMIDT E B, DYERBERG J, ERNST E (1992), The effects of dietary supplementation with n-3 polyunsaturated fatty acids in patients with rheumatoid arthritis: a randomized, double blind trial, Eur J Clin Invest, 22, 687±691. OBATA T, NAGAKURA T, MASAKI T, MAEKAWA K, YAMASHITA K (1999), Eicosapentaenoic acid inhibits prostaglandin D2 generation by inhibiting cyclo-oxygenase-2 in cultured human mast cells, Clin Exp Allergy, 29, 1129±1135. PANAYI G S (1999), Targeting of cells involved in the pathogenesis of rheumatoid arthritis, Rheumatology, 38 (Suppl. 2), 8±10. PAOLISSO G, RIZZO M R, MAZZIOTTI G, TAGLIAMONTE M R, GAMBARDELLA A, ROTONDI M,

(1998), Advancing age and insulin resistance: role of plasma tumor necrosis factor-alpha, Am J Physiol, 275, E294±E299.

CARELLA C, GIUGLIANO D, VARRICCHIO M, DONOFRIO F

PETER K, NAWROTH P, CONRADT C, NORDT T, WEISS T, BOEHME M, ALLENBERG J, KUBLER W,

(1997), Circulating vascular adhesion molecule-1 correlates with the extent of human atherosclerosis in contrast to circulating intercellular adhesion molecule1, E-selectin, P-selectin, and thrombomodulin, Arterioscler Thromb Vasc Biol, 17, 505±512. PLUTZKY J (1999), Athreosclerotic plaque rupture: emerging insights and opportunities, Am J Cardiol, 84, 15J±20J. BODE C

RALLIDIS L S, PASCHOS G, LIAKOS G K, VELISSARIDOU A H, ANASTASIADIS G, ZAMPELAS A

(2003), Dietary -linolenic acid decreases C-reactive protein, serum amyloid A and interkeukin-6 in dyslipidaemic patients, Atherosclerosis, 167, 237±242. RICOTE M, LI A C, WILLSON T M, KELLY C J, GLASS C K (1998), The peroxisome proliferatoractivated receptor-gamma is a negative regulator of macrophage activation, Nature, 391, 79±82. RIDKER P M, HENNEKENS C H, ROITMAN-JOHNSON B, STAMPFER M J, ALLEN J (1998), Plasma concentration of soluble adhesion molecule 1 and risks of future myocardial infarction in apparently healthy men, Lancet, 351, 88±92. RITZENTHALER K L, MCGUIRE M K, FALEN R, SHULTZ T D, DASGUPTA N, MCGUIRE M A (2001), Estimation of conjugated linoleic acid intake by written dietary assessment methodologies underestimates actual intake evaluated by food duplicate methodology, J Nutr, 131, 1548±1554. RODGERS J B (1998), N-3 fatty acids in the treatment of ulcerative colitis, in Kremer J M, Medicinal Fatty Acids in Inflammation, Basel, Birkhauser, 103±109.

Dietary lipids and immune function

391

ROHDE L E, LEE R T, RIVERO J, JAMACOCHIAN M, ARROYO L H., BRIGGS W, RIFAI N, LIBBY P,

(1998), Circulating cell adhesion molecules are correlated with ultrasound-based assesment of carotid atherosclerosis, Arterioscler Thromb Vasc Biol, 18, 1765±1770. ROHDE L E P, HENNEKENS C H, RIDKER P M (1999), Cross-sectional study of soluble intercellular adhesion molecule-1 and cardiovascular risk factors in apparently healthy men, Arterioscler Thromb Vasc Biol, 19, 1595±1599. ROMAGNANI S (2000), The role of lymphocytes in allergic disease, J Allergy Clin Immunol, 105, 399±408. ROSS R (1999), Atherosclerosis ± An inflammatory disease, N Eng J Med, 340, 115±126. ROSSETTI R G, SEILER C M, DE LUCA P, LAPOSATA M, ZURIER R B (1997), Oral administration of unsaturated fatty acids: effects on human peripheral blood T lymphocyte proliferation, J Leuk Biol, 62, 438±443. SANDERSON P, CALDER P C (1998), Dietary fish oil appears to prevent the activation of phospholipase C-c in lymphocytes, Biochim Biophys Acta, 1392, 300±308. SANTOLI D, ZURIER R B (1989), Prostaglandin E precursor fatty acids inhibit human IL-2 production by a prostaglandin-E-independent mechanism, J Immunol, 143, 1303± 1309. SCHMIDT E B, PEDERSEN J O, EKELUND S, GRUNNET N, JERSILD C, DYERBERG J (1989), Cod liver oil inhibits neutrophil and monocyte chemotaxis in healthy males, Atheroslerosis, 77, 53±57. CREAGER M A, RIDKER P M

SCHMIDT E B, VARMING K, PEDERSEN J O, LERVANG H H, GRUNNET N, JERSILD C, DYERBERG J

(1992), Long term supplementation with n-3 fatty acids. ii. effect on neutrophil and monocyte chemotaxis, Scand J Clin Lab Invest, 52, 229±236. SCHMIDT E B, VARMING K, MOLLER J M, BULOW PEDERSON I, MADSEN P, DYERBERG J (1996), No effect of a very low dose of n-3 fatty acids on monocyte function in healthy humans, Scand J Clin Invest, 56, 87±92. SCHOONJANS K, STAELS B, AUWERX J (1996), The peroxisome proliferator activated receptors (PPARs) and their effects on lipid metabolism and adipocyte differentiation, Biochim Biophys Acta, 1302, 93±109. SELJEFLOT I, ARNESEN H, BRUDE I R, NENSETER M S, DREVON C A, HJERMANN I (1998), Effects of omega-3 fatty acids and/or antioxidants on endothelial cell markers, Eur J Clin Invest, 28, 629±635. SIMONS K, TOOMRE D (2000), Lipid rafts and signal transduction, Nature Rev, 1, 31±39. SOYLAND E, NENSETER M S, BRAATHEN L, DREVON C A (1993), Very long-chain n-3 and n-6 polyunsaturated fatty acids inhibit proliferation of human T lymphocytes in vitro, Eur J Clin Invest, 23, 112±121. SPERLING R I, BENINCASO A I, KNOELL C T, LARKIN J K, AUSTEN K F, ROBINSON D R (1993), Dietary !-3 polyunsaturated fatty acids inhibit phosphoinositide formation and chemotaxis in neutrophils, J Clin Invest, 91, 651±660. STUBBS C D, SMITH A D (1984), The modification of mammalian membrane polyunsaturated fatty acid composition to membrane fluidity and function, Biochim Biophys Acta, 779, 89±137. STULNIG T M, HUBER J, LEITINGER N, IMRE E M, ANGELISOVA P, NOWOTNY P, WALDHAUS W

(2001), Polyunsaturated eicosapentaenoic acid displaces proteins from membrane rafts by altering raft lipid composition, J Biol Chem, 276, 37335±37340. TAPPIA P S, LADHA S, CLARK D C, GRIMBLE R F (1997), The influence of membrane fluidity, TNF receptor binding, cAMP production and GTPase activity on macrophage cytokine production in rats fed a variety of fat diets, Mol Cell Biochem, 166, 135±143.

392

Functional foods, ageing and degenerative disease

(2001a), Dietary supplementation with gamma linolenic acid or fish oil decreases T lymphocyte proliferation in healthy older humans, J Nutr, 131, 1918±1927. THIES F, NEBE-VON-CARON G, POWELL J R, YAQOOB P, NEWSHOLME E A, CALDER P C (2001b), Dietary supplementation with eicosapentaenoic acid, but not with other long chain n-3 or n-6 polyunsaturated fatty acids, decreases natural killer cell activity in healthy subjects aged > 55 years, Am J Clin Nutr, 73, 539±548. THIES F, NEBE-VON-CARON G, POWELL J R, YAQOOB P, NEWSHOLME E A, CALDER P C

THIES F, MILES E A, NEBE-VON-CARON G, POWELL J R, HURST T L, NEWSHOLME E A, CALDER P C

(2001c), Influence of dietary supplementation with long-chain n-3 or n-6 polyunsaturated fatty acids on blood inflammatory cell populations and functions and on plasma soluble adhesion molecules in healthy humans, Lipids, 36, 1183± 1193. THIES F, GARRY J M C, YAQOOB P, RERKASEM K, WILLIAMS J, SHEARMAN C P, GALLAGHER P J,

(2003), Association of n-3 polyunsaturated fatty acids with stability of atherosclerotic plaques: a randomised controlled trial, Lancet, 361, 477±485. THOMPSON P J, MISSO N L A, PASSARELLI M, PHILLIPS M J (1991), The effect of eicosapentaenoic acid consumption on human neutrophil chemiluminescence, Lipids, 26, 1223±1226. TILLEY S L, COFFMAN T M, KOLLER B H (2001), Mixed messages: modulation of inflammation and immune responses by prostaglandins and thromboxanes, J Clin Invest, 108, 15±23. VACHIER I, CHANEZ P, BONNANS C, GODARD P, BOUSQUET J, CHAVIS C (2002), Endogenous anti-inflammatory mediators from arachidonate in human neutrophils, Biochem Biophys Res Commun, 290, 219±224. CALDER P C, GRIMBLE R F

VARMING K, SCHMIDT E B, SVANEBORG N, MOLLER J M, LERVANG H H, GRUNNET N, JERSILD C,

(1995), The effect of n 3 fatty acids on neutrophil chemiluminescence. Scand J Clin Lab Invest, 55, 47±52.

DYERBERG J

VOORRIPS L E, BRANTS H A M, KARDINAAL A F M, HIDDINK G J, VAN DEN BRANDT P A, GOLDBOHM R A (2002), Intake of conjugated linoleic acid, fat, and other fatty acids in relation to postmenopausal breast cancer: the Netherlands Cohort Study on Diet and Cancer, Am J Clin Nutr, 76, 873±882. WALLACE F A, MILES E A, CALDER P C (2003) Comparison of the effects of linseed oil and different doses of fish oil on mononuclear cell function in healthy subjects, Brit J Nutr, 89, 679±689. WALLACE J M W, TURLEY E, GILMORE W S, STRAIN J J (1995), Dietary fish oil supplementation alters leukocyte function and cytokine production in healthy women, Arterioscler Thromb Vasc Biol, 15, 185±189.

WILLIAMS C M, FRANCIS-KNAPPER J A, WEBB D, BROOKES C A, ZAMPELAS A, TREDGER J A, WRIGHT J, MEIJER G, CALDER P C, YAQOOB P, ROCHE H, GIBNEY M J (1999), Cholesterol reduction using manufactured foods high in monounsaturated fatty acids: a randomized crossover study, Brit J Nutr, 81, 439±446. WOODS R K, THIEN F C K, ABRAMSON M J (2003) Dietary marine fatty acids (fish oil) for asthma in adults and children, in The Cochrane Library, Issue 2, Oxford, Update Software. WU D, MURA C, BEHARKA A A, HAN S N, PAULSEN K E, HWANG D, MEYDANI S N (1998), Ageassociated increase in PGE(2) synthesis and COX activity in murine macrophages is reversed by vitamin E, Am J Physiol, 275, C661±C668. WU D, MEYDANI M, LEKA L S, NIGHTINGALE Z, HANDELMAN G J, BLUMBERG J B, MEYDANI S N

Dietary lipids and immune function

393

(1999), Effect of dietary supplementation with blackcurrant seed oil on the immune response of healthy elderly subjects, Am J Clin Nutr, 70, 536±543. XI S, COHEN D, BARVE S, CHEN L H (2001), Fish oil suppressed cytokines and nuclear factor kappaB induced by murine AIDS virus infection, Nutr Res, 21, 865±878. YAQOOB P (1998), Monounsaturated fats and immune function, Proc Nutr Soc, 57, 511± 520. YAQOOB P (2003), Lipids and the immune response: from molecular mechanisms to clinical applications, Curr Opin Clin Nutr Metab Care, 6, 133±150. YAQOOB P, NEWSHOLME E A, CALDER P C (1995), Influence of cell culture conditions on diet-induced changes in lymphocyte fatty acid composition, Biochim Biophys Acta, 1255, 333±340. YAQOOB P, KNAPPER J, WEBB D, WILLIAMS C, NEWSHOLME E A, CALDER P C (1998), Effect of olive oil on immune function in middle-aged men, Am J Clin Nutr, 67, 129±135. YAQOOB P, PALA H S, CORTINA-BORJA M, NEWSHOLME E A, CALDER P C (2000), Encapsulated fish oil enriched in -tocopherol alters plasma phospholipid and mononuclear cell fatty acid compositions but not mononuclear cell functions, Eur J Clin Invest, 30, 260±274. YLI-JAMA P, SELJEFLOT I, MEYER H E, HJERKINN E M, ARNESEN H, PEDERSEN J I (2002) Serum non-esterified very long-chain PUFA are associated with markers of endothelial dysfunction, Atherosclerosis, 164, 275±281. ZEYDA M, STAFFLER G, HOREJSI V, WALDHAUSL W, STULNIG T (2002) LAT displacement from lipid rafts as a molecular mechanism for the inhibition of t cell signaling by polyunsaturated fatty acids, J Biol Chem, 277, 28418±28423. ZIBOH V A (1998), The role of n-3 fatty acids in psoriasis, in Kremer J M, Medicinal Fatty Acids in Inflammation, Basel, Birkhauser, 45±53. ZURIER R B (1998), Gammalinolenic acid treatment of rheumatoid arthritis, in Kremer J M, Medicinal Fatty Acids in Inflammation, Basel, Birkhauser, 29±43.

16 Improving gut health in the elderly K. M. Tuohy, E. Likotrafiti, K. Manderson, G. R. Gibson and R. A. Rastall, The University of Reading, UK

16.1

Introduction

World wide, the population is ageing, with declining birth rates (especially in developed countries) and increasing life expectancy, contributing to the growing of an aged population. The impact of this ageing population is likely to be felt most keenly in Europe, where the proportion of older people will increase from 20% in 1998 to 35% in 2050 where one in every three people will be over 60 years (Ho, 1996). According to the World Health Organisation (WHO) there are currently 580 million people in the world aged 60 or over and this figure is expected to rise to 1 billion in the next 20 years (Kalache, 1999). Up to 85% of the elderly population are likely to undergo medical intervention for single or multiple diseases at any given time (Garibaldi and Nurse, 1986). Thus, an ageing population is accompanied by a significant rise in health costs and the need for socially acceptable care facilities is likely to impact greatly on socioeconomic parameters within Westernised countries. With ageing comes a reduction in overall health and an increase in morbidity and mortality due to infectious disease, many associated with the gastrointestinal tract. It is estimated that mortality due to gastrointestinal infections is up to 400 times higher in the elderly compared to younger adults (HeÂbuterne, 2003). The scope of this chapter is to describe the changes within the ageing gastrointestinal microflora, which may account, in part, for the increase in severity of gastrointestinal infections with age, and discuss how dietary interventions with functional foods may fortify gastrointestinal health in the elderly. More chronic disease states associated with old age (e.g. colon cancer) and their interaction with the gastrointestinal microflora will also be discussed.

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Successional development of gastrointestinal microflora

Normally, the gastrointestinal tract is sterile at birth and we derive our gut microflora during conventional delivery from the faecal and vaginal microflora of our mother and from the post-natal environment. Early colonisers, taking advantage of readily available nutrients within the first few days of life, (e.g. facultative streptococci/enterococci, lactobacilli and the Enterobacteriaceae) lower lumenal redox potential, allowing subsequent colonisation by more strictly anaerobic species of bacteria such as Bacteroides spp., Clostridium spp. and the bifidobacteria (Finegold et al., 1983). Upon breast feeding, a microflora dominated by bifidobacteria establishes. Indeed, human breast milk contains an array of bifidogenic oligosaccharides (e.g. N-acetylated aminosugars), and peptides (e.g. glycomacropeptide) which have been shown to selectively encourage the growth of bifidobacteria within the gastrointestinal tract. Breast milk also contains a significant immunological component, which also impacts on the composition and activity of the gut microflora (Mountzouris et al., 2002). A much more diverse collection of bacterial species has been shown to colonise the gastrointestinal tract of formula-fed infants, with the dominance of bifidobacteria often supplanted by Bacteroides spp. and the clostridia (Mackie et al., 1999). Fortification of infant milk formula feeds with bifidogenic agents such as prebiotics (see below) is an active area of research and development within many formula feed and health care companies. The gut microflora becomes more diverse during weaning onto solid foods, and it has been proposed that a microflora resembling that of the adult becomes established by the age of two, although it is likely that successional development continues until much later into childhood. Indeed, a lower microbial diversity in children aged 16 months to seven years compared to adults was observed using proportionality of 16S rRNA species compared to total faecal 16S rRNA using dot-blot oligonucleotide probing (Hopkins et al., 2001, 2002). It is likely that as bacterial numbers and competition for nutrients increase, ecological niches become occupied by more specialised groups of bacteria, eventually resulting in climax microbial populations where potentially all ecological niches are occupied (Falk et al., 1998). Such species and ecological diversity affords the adult gut microflora a high degree of homeostasis and greatly limits the ability of invading bacteria to colonise the gut. In health, the adult gut microflora has been shown to be remarkably stable in species composition within individuals over time (Zoetendal et al., 1998). It has been estimated that this microflora is made up of some 500 different species of bacteria with maybe 50 different species representing the dominant populations within the gut microflora (Moore and Holdeman, 1974). Other bacterial species occupy specific and often unique ecological niches within this complex microbial ecosystem, often cross-feeding on the by-products of more dominant members of the microflora.

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16.2.1 The microflora of the gastrointestinal tract and interactions with host physiology As well as changing temporally from birth to adulthood, the gut microflora changes spatially within the gastrointestinal tract. The human stomach is relatively sparsely colonised by bacteria with numbers of facultative anaerobes such as lactobacilli, streptococci and yeast occurring in numbers as low as 102 colony-forming units (CFU) per ml contents. Gastric acid is the chief factor limiting microbial colonisation of the stomach, and acts as a crucial first line of defence in limiting the growth of invading microbial pathogens ingested from the external environment. Indeed, it is likely that many of the bacteria isolated from the stomach are allochthonous, originating in the oral cavity or being ingested with food (Gorbach, 1993). One important exception is Helicobacter pylori, evolved for gastric colonisation and occurring in approximately 40% of the adult population in developed countries. H. pylori possesses a number of unique ecological adaptations which allow it to colonise the stomach. This spiral shaped, flagellated, Gram negative organism, burrows into the gastric mucosa and adheres onto the gastric epithelium. Here, it may escape to some degree from the acidic environment of the stomach lumen. Specific physiological characteristics which enable H. pylori to cope with high acid environments include the ability to reduce cytoplastic H+ concentrations through hydrolysis of urea to ammonia, secretion of carbonate and production of outer membrane proteins with higher isoelectric points (Sachs et al., 2002). H. pylori has been shown to be the causative agent of gastric ulcers and is considered to be a major risk factor for the development of gastric cancer. Indeed, the organism was recognised by the International Agency for Cancer Research as a biological carcinogen in 1994, for its role in the inducing atrophic gastritis leading to the gastritis-metaplasia-carcinoma sequence (IARC Working Group, 1994; Baldini et al., 1999). Carriage of H. pylori can reach up to 80% of the population in developing countries and this has been attributed mainly to poor sanitation and spread via the faecal-oral route. In developed countries, where sanitation has been much improved over the last 50±60 years, the incidence of H. pylori infection is much higher in the elderly often reaching up to 80% of the over sixties. This cohort effect is probably due to infection of this population subset early in life, when sanitation was rudimentary and on a par with present-day developing countries, e.g., inner-city slums and under-developed rural areas. The small intestine is also relatively sparsely populated, with the rapid transit of digesta driven by peristalsis limiting microbial colonisation. The digestive capabilities of the small intestine and biliary secretions also inhibit microbial colonisation in this region of the gut. Indeed it is not until the distal ileum that microbial populations start to increase in number significantly. Here numbers of facultative lactobacilli, streptococci and enterobacteria, as well as some anaerobic bifidobacteria, Bacteroides spp. and clostridia reach population levels of about 104±108 CFU/ml. The ileocecal valve, in health, effectively limits the

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spread of a more diverse and dense microflora moving proximally from the colon (Gorbach, 1993; Conway, 1995). The colon is the major site of microbial colonisation within mono-gastric mammals with microbial numbers reaching up to 1011±1012 CFU/ml contents. This climax microbial population is also the most diverse within the human host, and a large proportion of species present are new to science (Suau et al., 1999; Blaut et al., 2002). Indeed many of the bacteria present defy cultivation by classical microbiological techniques and have only recently been recognised with the application of modern molecular microbiological tools to study this rich microbial habitat. The colonic microflora is dominated by the strict anaerobes such as Bacteroides spp., the clostridia and other families within the Clostridium mega-genus (including Ruminococcus spp., Butyrovibrio spp., Fusobacterium spp., Eubacterium spp., Peptostreptococcus), Bifidobacterium spp., Atopobium spp. and the peptococci. Facultative bacteria occur in much lower numbers, typically 1,000-fold lower, and include species of lactobacilli, enterococci, streptococci, the Enterobacteriaceae. In health, yeasts are also found in low numbers (about 102±104 CFU/ml), due to competitive exclusion by bacteria. This complex microflora, by its very nature, serves as an important barrier to the establishment of allochthonous microorganisms which may be pathogenic to the host. They effectively limit their colonisation potential through competition for nutrients, ecological niches, attachment sites on the gut wall and production of inhibitory metabolites such as short chain fatty acids (which also lower colonic pH) and bacteriocidal compounds such as bacteriocins. 16.2.2 The gut microflora in old age The composition of the gut microflora, both in relative numbers of different populations and in species present has been reported to be altered in old age. Indeed, it appears that microbial succession within the gastrointestinal tract does not stop with the relatively stable adult gut microflora, but may be thought of as entering senescence in the elderly. Gorbach et al. (1967) observed that elderly people had lower numbers of bifidobacteria and elevated population levels of yeasts and enterobacteria compared to adults. Mitsuoka and Hayakawa (Mitsuoka and Hayakawa, 1973; Mitsuoka, 1992) reported that numbers of bifidobacteria decreased in the aged, while numbers of clostridia, lactobacilli, streptococci and enterobacteria were present in high population levels. More recent studies have confirmed these observations. Hopkins et al. (2001) using a combination of traditional microbiological culture based techniques, a molecular technique which estimates relative abundance of 16S rRNA populations compared to total faecal 16S rRNA (dot-blot hybridisation), and community cellular fatty acid profiles, investigated the composition of the gut microflora in four different groups of individuals. The cohorts included children (16 months to seven years, n ˆ 10), adults (21±34 years, n ˆ 7), healthy elderly people (67±88 years, n ˆ 5) and elderly patients diagnosed with Clostridium

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difficile diarrhoea (68±73 years, n ˆ 4). Using both traditional and 16S rRNA based dot-blot probing the authors found that numbers of enterobacteria were significantly higher in the children than in the adults. In the C. difficile group, numbers of bifidobacteria and Bacteroides spp. were much reduced while numbers of facultative anaerobes (enterobacteria, enterococci and lactobacilli) increased. Numbers of clostridia were also higher in this group than any other. Numbers of bifidobacteria were lower in both the aged groups compared to the children and the adults using the traditional culture based technique, while dotblot probing showed bifidobacteria to be lowest in the C. difficile patients. Using both techniques, bifidobacterial population levels were found to vary greatly within the elderly, with some individuals showing relatively high levels of bifidobacteria and others very low levels. In a further publication, the same authors looked in more detail at the species composition within the gut microflora of these subject groups (Hopkins and Macfarlane, 2002). Using traditional culture techniques, the authors examined microbial diversity within healthy adults, and in elderly subjects and elderly patients with C. difficile associated diarrhoea (CDAD). Bacteroides thetaiotaomicron, B. ovatus and Prevotella tannerae were common isolates from the adult subjects, while Bacteroides species diversity increased in the elderly. Diversity of the bifidobacteria however, decreased with age, with Bif. adolescentis and Bif. angulatum commonly isolated. In the CDAD patients, as observed in previous studies, numbers of Bacteroides spp., Prevotella spp. and Bifidobacterium spp. were low, while species diversity of facultative anaerobes (enterobacteria and enterococci), lactobacilli and clostridia increased. Although changes within the gut microflora of CDAD patients may be due to therapeutic metronidazole treatment, the authors suggested that Bacteroides spp. and Bifidobacterium spp. may play an important role in maintaining colonisation resistance towards C. difficile in health, as these groups were much reduced in the CDAD patients. A limitation of these studies was the low number of subjects examined in the different groups. Further studies involving a larger number of subjects are required to confirm these findings. An ongoing EU-funded project (CROWNALIFE) is investigating the impact of the gut microflora on health and assessing its amenability to dietary modification in larger numbers of elderly subjects at multiple centres throughout Europe. Using a much broader spectrum of 16S rRNA targeted oligonucleotide probes and fluorescent in situ hybridisation combined with cloning of faecal 16S rRNA species, CROWNALIFE aims to characterise microbial diversity and relative species composition within the elderly and monitor the effect of dietary interventions with synbiotics on the gut microflora itself and on important biomarkers of disease (Saunier and DoreÂ, 2002). Using traditional microbiological culture techniques Silvi et al. (2003) described the species composition of bifidobacterial and lactic acid microflora of faecal samples taken from 12 healthy elderly Italian volunteers. Although the authors recognised that each individual had their own collection of species making up these two moieties, L. fermentum and B. longum were the most

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commonly isolated species of lactic acid bacteria. Gavini et al. (2001) determined the differences within the bifidobacteria and enterobacteria moieties of the microflora from three different age groups, 3±15 years, 30±46 years and 69±89 years. Within the Enterobacteriaceae, E. coli was present in 93% of faecal samples and its occurrence was independent of age, while Enterobacter and Klebsiella species were isolated more frequently in children and the elderly than in adults. Species of Proteus and Providencia (increasingly accepted as a cause of diarrhoea) were common in the elderly. Overall, the enterobacterial microflora of the elderly and of children was more diverse than that of the adults. Many of the enterobacteria including Enterobacter, Klebsiella, Proteus and Providencia have been shown to cause diarrhoea or are frequently isolated from diarrhoeal stools (i.e. Proteus). On the other hand, Bif. longum was more common in children and the adults, while Bif. adolescentis was more common in the elderly. Characterisation of the gut microflora to the species level is important in that many changes within the gut microflora may be overlooked if relying on measurements at the genus level. Isolation of live bacteria is often critical for determining the particular ecological role of an organism or in isolation of potentially beneficial organisms which may then be screened for their use in probiotic functional foods (Dunne et al., 1999). Using dot-blot oligonucleotide probing Saunier and Dore (2002) in a recent review reported that Bacteroides, bifidobacteria and Clostridium leptum 16S rRNA species are decreased in the elderly, while proportions of Lactobacillus rRNA were elevated compared to adults. The authors also reported that as evidenced from the diversity coverage of the panel of probes used, only 50% of the elderly gut microflora was measured while the percentage coverage rose to 80% for adults. Results from the same laboratory using comparative sequencing of cloned 16S rRNA from faecal samples of different age groups have confirmed these findings, where statistically the number of species recovered by this technique rises from 15 for infants to 168 for the elderly. Moreover, only 8% of 16S rRNA sequences recovered from elderly faecal samples were related to bacterial isolates deposited in culture collections compared to 20% for adults (Blaut et al., 2002).

16.3 Modification of the gut microflora: probiotics, prebiotics and synbiotics Currently, there are three dietary rationales for modification of the gut microflora towards improved health probiotics, prebiotics and synbiotics. Probiotics, defined as live microbial food ingredients that are beneficial to health (Fuller, 1989) and are based on the concept of introducing a microorganism believed to improve host health into the intestinal environment often through a food vehicle. Probiotics with the strongest scientific support include species of lactobacilli and bifidobacteria (both lactic acid producing genera indigenous to

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the mammalian gastrointestinal tract), as well as the yeast Saccharomyces cereviseae, for which there is convincing evidence for its anti-diarrhoeal capabilities (Tuohy et al., 2003). Some of the proposed beneficial effects of probiotics include relief of lactose maldigestion, reducing the incidence and duration of diarrhoea (of bacterial and viral aetiology), protection against colon cancer, extension of remission in inflammatory bowel disease and relief from the symptoms of less well defined complaints such as irritable bowel syndrome, where both diarrhoea and constipation impact on patient quality of life (Saarela et al., 2002; Tuohy et al., 2003). Although questions remain about the mode of action of many probiotics against specific disease states, it is likely they include a combination of the following mechanisms of effect: · enhancement of the colonisation resistance to invading pathogens through increased competition for nutrients and mucosal adhesion sites · production of secondary metabolites inhibitory to pathogenic bacteria, e.g., short chain fatty acids and bacteriocin-like compounds · stimulation of the immune system in a non-inflammatory manner · fortification of mucosal integrity (Madsen et al., 2001). Existing evidence suggests that probiotics act in a strain specific manner, highlighting the need for rigorous scientific validation through initial determination of probiotic capability, e.g., inhibition of gastrointestinal pathogens, through to demonstration of efficacy in well controlled blinded human feeding studies (Vaughan et al., 1999). Prebiotics are non-viable food components that evade digestion in the upper gut, reach the colon intact and there they are selectively fermented by bacteria seen as beneficial to gastrointestinal health, namely the bifidobacteria and/or lactobacilli (Gibson and Roberfroid, 1995). The majority of prebiotics are oligosaccharides or short polysaccharides and amongst the most studied are the fructans (fructooligosaccharides and inulin), galactooligosaccharides and lactulose (Tuohy et al., 2001, 2002a,b; Ito et al., 1993). Other plant-derived oligosaccharides under investigation as prebiotics include pecticoligosaccharides, soyoligosaccharodes, isomaltooligosaccharides, various resistant starches, gentiooligosaccharides and chitooligosaccharides (Kolida et al., 2000). A characteristic feature of prebiotics, one which distinguishes them from other dietary fibres, is their selective fermentation by bifidobacteria and/or lactobacilli within the colonic microflora. Such specific modulation of the gut microflora has been repeatedly proved in human feeding studies and appears to be brought about at doses of prebiotic ranging from 4g to 15g per day (Roberfroid et al., 1998). Prebiotics have been investigated for their ability to bring about a number of specific health outcomes including: · modification of the gut microflora of formula-fed infants towards parity with that of breast-fed infants, i.e., increase in numbers of bifidobacteria · enhanced mineral absorption

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· protection against the onset and development of colon cancer (evidence from animal models) · lowering of serum triglycerides and plasma cholesterol levels · normalisation of bowel habit (relief from constipation and diarrhoea). The prebiotic mode of action is likely to be similar to that of probiotics, in that prebiotics stimulate the growth and activity of probiotic organisms (bifidobacteria and lactobacilli) already present within the hosts gut microflora (Tuohy et al., 2003). Synbiotics combine probiotics and prebiotics into a single food product. The aim is to enhance the survivability or colonisation potential of particularly efficacious probiotic strains within the gut microflora by providing in parallel a selective substrate for their growth (Collins and Gibson, 1999). Synbiotics may also stimulate the growth of indigenous bifidobacteria and lactobacilli within the host gut microflora. Although few synbiotics are currently on the market, the scientific support for certain synbiotic combinations is encouraging. For example, the synbiotic combination of Bif. longum and inulin has been shown in animal feeding studies to be protective against colon cancer. Indeed, the synbiotic mix was shown to be more effective than either its probiotic or prebiotic moieties when fed separately (Rowland et al., 1998). The elderly may form a subset of the population for which microflora modulation through dietary probiotics, prebiotics and synbiotics may be especially relevant. Changes within the gut microflora, as detailed above, suggest a reduction in numbers of bifidobacteria and a concomitant increase in species diversity and relative numbers of the Enterobacteriaceae. Probiotics, prebiotics and synbiotics are effective at increasing numbers of probiotic bacteria within the gut microflora and have on occasions been shown to reduce numbers of potentially disease-causing Enterobacteriaceae and offering some protection against the onset or clinical course of diarrhoeal infections (Roberfroid et al., 1998; Asahara et al., 2001; Tuohy et al., 2003). Prebiotics may be particularly efficacious in this regard, as in healthy adult populations, they have been shown to be most effective in increasing numbers of intestinal bifidobacteria in subjects with low initial levels of bifidobacteria (Roberfroid et al., 1998; Tuohy et al., 2001). Although lower numbers of bifidobacteria identify the elderly as a population group likely to benefit from prebiotic dietary supplementation, the limited species diversity (one apparently dominated by Bif. adolescentis) suggests that prebiotics for the elderly should be chosen for their ability to selectively stimulate the growth of this species or the complementary application of synbiotics providing a bifidogenic oligosaccharide plus a proved probiotic bifidobacterial strain. Probiotics, and to a lesser extent prebiotics, have also been demonstrated to impact on the immune function (described below) and such approaches may be useful in stimulation of the ageing, dysregulated immune system bringing about reduced inflammatory responses, enhanced response to bacterial antigens and response to vaccines. Extended gastrointestinal transit time and constipation can

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impact greatly on quality of life in old age. Probiotics have been shown to increase transit times in human feeding studies (Marteau et al., 2002). Similarly, intervention with prebiotics has also been shown to relieve constipation in elderly men (Chen et al., 2001). Probiotics and prebiotics may also improve mineral absorption (e.g. Ca2+) and provide vitamins, often deficient in the elderly such as folate (Crittenden et al., 2002).

16.4

Factors affecting gut microflora in old age

A number of physiological changes within the gastrointestinal tract are commonly observed in the elderly. The mucosal surface changes, with reduced surface area (although this is not associated with malabsorption), alteration in the structure of intestinal mucus and greater permeability of mucosal membranes (which has been linked to increases in circulating antibodies to commensal gut bacteria) (Ouwehand et al., 1999; Hopkins et al., 2002; HeÂbuterne, 2003). Atrophic gastritis is common in the elderly and associated with hypochlorhydria. This may lead to a diminution of the acid barrier of the stomach allowing higher numbers of ingested bacteria to reach the small intestine in a viable form. Changes in small intestinal motor patterns may be associated with dyspepsia, irritable bowel syndrome and bacterial overgrowth in this region of the gut. Bacterial overgrowth in the small bowel may then affect nutrient absorption. Although colonic motor function remains intact in old age, changes occur in anorectal function leading to constipation, faecal incontinence and faecal impaction, all of which impact greatly on quality of life in old age (HeÂbuterne, 2003). It is likely that members of the gut microflora, or ingested bacteria, may take advantage of such changes in gastrointestinal physiology and occupy niches otherwise inaccessible to them. It is difficult to determine whether changes in the gut microflora in the elderly are the cause of physiological deterioration or functional alterations in the gut or the effect of such changes. Gastric acid output (basal and total) decrease with old age (Baron, 1963) and is thought to be due to increased gastric atrophy. Healthy elderly subjects are likely to maintain gastric acid secretion in the absence of atrophy (Katelaris et al., 1993; Aryeh et al., 1997). H. pylori plays an important role in the pathogenesis of gastric atrophy and hypochlorhydria (Lovat, 1996). It may be that changes in gastric histology and function, previously assigned to ageing, may be in part due to H. pylori infection, particularly since H. pylori infection (or previous infection) is found in most patients with gastric atrophy. In addition, elderly patients (aged 60 or over) with normal gastric mucosa were shown to be unlikely to develop atrophy during a ten-year study into the development of gastritis (Ihamaki et al., 1985). Taken with the cohort distribution of H. pylori infection, with higher prevalence of H. pylori in the elderly being ascribed to colonisation during the early years of life, under the less sanitary conditions of the early 20th century, it is likely that ageing per se is not the only contributory factor towards gastric atrophy and

Improving gut health in the elderly

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associated hypochlorhidria, and a pathological effect of H. pylori infection may be involved. The ability to adhere to the gastrointestinal mucosa has been identified as a prerequisite for intestinal colonisation by a bacterial strain and has been identified as a desirable characteristic in the selection of probiotics. Little is known about differential adherence of bifidobacterial strains to intestinal mucosa. He et al. (2001) compared the ability of 51 different bifidobacterial faecal isolates from adult and healthy elderly subjects for their ability to adhere to human intestinal mucus in vitro. Although percentage adhesion of the faecal isolates were all lower than the control strains (probiotics L. rhamnosus GG and Bif. lactis Bb12) bifidobacterial isolates from the adults showed significantly higher adhesion than did those isolated from healthy elderly subjects. This difference in adhesion rates was attributed to lower adhesion of Bif. adolescentis from elderly subjects compared to those from adult subjects. No difference in adhesion of Bif. longum isolates from the two populations was observed. Bif. longum from elderly subjects adhered to a greater extent than did Bif. adolescentis strains from the same individuals. Ouwehand et al. (1999) determined the ability of four probiotic bifidobacterial strains (Bif. lactis Bb12, Bifidobacterium 913, Bifidobacterium 420, and Bifidobacterium BF1100) to adhere to intestinal mucus isolated from different age groups. All probiotic bifidobacterial strains adhered less to mucus isolated from elderly individuals than to that from infants or adults. The mucus preparation from the elderly, although produced in equal amount to the adults, contained less protein and more carbohydrate. However, the mucus was not analysed in enough detail to pick out specific differences in glycosylation between the different mucus groups. These observations suggest that changes in mucus composition within the elderly gut as well as changes in species composition of the elderly bifidobacterial microflora (with less adherent B. adolescentis strains being more commonly isolated) may account for the lower numbers of bifidobacteria reported in the elderly gut microflora. Diet is likely to have an impact on metabolic activity and maybe population dynamics of the gut microflora in old age. More recent studies into malnutrition in the elderly have concluded that in general, despite some reduced absorptive capacity within the intestinal tract, healthy free-living elderly subjects do not differ significantly from younger adults in overall nutritional status. The elderly do, however, consume reduced amounts of dietary fibre, fruit and vegetables and are often deficient in a number of important nutrients including calcium, folate, and vitamin B12 (Russell et al., 1986; Lovat, 1996; Baik and Russell, 1999). One contributory factor may be reduced mastication due to tooth loss or altered taste/smell sensation which may affect the type of food eaten. Reduced intake of complex carbohydrates such as resistant starches or non-starch plant polysaccharides is likely to impact on microbial metabolism and relative population levels within the gut. Many of these compounds resist degradation in the small intestine, reach the colon intact and drive microbial carbohydrate fermentation.

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Benno et al. (1989) studied differences in faecal microflora in elderly individuals from rural areas (associated with long-life and high fibre intake) and urban areas (low fibre intake) of Japan. Fifteen elderly people from the rural areas were aged from 75 to 90 (median 84) and 15 from the urban area aged 65 to 76 (median 68). Intake of dietary fibre was significantly higher in the rural area (28.8 g/day) compared with the urban area (13.1 g/day). Numbers of total anaerobes were significantly higher in elderly people from the urban areas, whereas numbers of bifidobacteria were greater in individuals from the rural areas characterised by high fibre intake. Clostridia lecithinase positive (mainly C. perfringens) and negative (mainly C. coccoides and C. paraputrificum) were higher and isolated more frequently from individuals in the urban areas. Bacilli (mainly B. subtilis) were also more common in the elderly from the urban area and also occurred in significantly higher numbers. Although numbers of faecal bifidobacteria in the rural elderly population were lower than those found in healthy adults, they were higher than those observed in the urban elderly. Mitsuoka et al. (1974) demonstrated that a higher incidence of Bif. adolescentis and a lower incidence of Bif. longum were characteristic of the elderly microflora. Here the number and incidence of Bif. adolescentis was higher in the elderly from the rural area compared to that of the urban area. The authors suggested that diet, particularly the higher intake of dietary fibre, may be responsible for the observed differences between the populations. 16.4.1 Effect of drugs on the gastrointestinal microflora in old age Due to the increased incidence and severity of infectious and chronic disease, the elderly undergo a disproportionately high level of medical intervention compared to younger adults (Ratnaike and Jones, 1998). Oral antibiotics, in particular, have a significant impact on gut microflora. Antibiotics, especiall