Functional Foods, Ageing, and Degenerative Disease

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Author: C. Remacle | B Reusens

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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, UniversiteÂ 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

Contents

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

x

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

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 UniversiteÂ 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

xvi

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]


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 Àvenir' 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. FerreÂ Unit 465 INSERM, Centre de Recherches Biomedicales des Cordeliers UniversiteÂ 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]


xviii

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]

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


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


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


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.


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 èxtra 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


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 ìron 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


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 ìmproper 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


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


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


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


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 àny 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 àny substance or combination of substances which may be administered to human beings or animals with a view


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 ìf 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, UniversiteÂ 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

18


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

Diet and the prevention of degenerative disease

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

20


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


21

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

22


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


23

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

24


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


25

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


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


27

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


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

30


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


31

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


33

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

34


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


35

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).

36


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


37

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


39

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 èmerging 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).


41

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 òne-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

42


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). Òne-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.


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

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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

3.1.1 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.

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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).

New functional foods for age-related diseases

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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 òn' periods in patients with Parkinson's disease who have òn-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 òrganic' 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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3.7



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.


3.9

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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.

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

Diet and control of osteoporosis

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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 ìt 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 ìn 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/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


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

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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 à 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 èquol status' hypothesis suggests that the beneficial effects of soy proteins might be present only in èquol 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 èquol 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


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, Èffects 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 (ÈC-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/.


5.8

129

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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 65 y (n 185): 4.8

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

Norway

Norkost 1997 (Johansen and Solvoll, 1999)

Food frequency questionnaire

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

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

Sweden

Riksmaten 1997±98 (Becker and Pearson, 2002)

Seven±day record

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

Females 45±54 y (n 153): 5.8 55±64 y (n 81): 6.1 >65 y (n 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 271): 4.25 75±84 y (n 265): 3.81 >85 y (n 96): 3.18

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

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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: 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 SanteÂ 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.


153

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

154


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.


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

156


`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


157

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

158


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-


159

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 ( 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

Dietary lipids and immune function

355

Fig. 15.2 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


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 àverage' 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 ìnflammatory'.

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


381

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

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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.

Improving gut health in the elderly

16.2

395

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 10), adults (21±34 years, n 7), healthy elderly people (67±88 years, n 5) and elderly patients diagnosed with Clostridium

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difficile diarrhoea (68±73 years, n 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 DoreÂ (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


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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, especially broad spectrum antibiotics, target microorganisms within the gut in a nonspecific manner, acting against pathogenic and non-pathogenic bacteria alike. Disturbances in the species dynamic due to antibiotic therapy may then afford surviving pathogenic bacteria the opportunity to take advantage of the less competitive environment and cause diarrhoea, e.g., Clostridium difficile. Even low levels of antibiotics may disrupt the gut microbial dynamic or lead to the spread of antibiotic resistance determinants between bacteria and the emergence of pathogens with a wider spectrum of antibiotic resistances, e.g., methicillinresistant Staphylococcus aureus (MRSA) or vancomycin-resistant enterococci (Tuohy et al., 2002a). Clostridium difficile is the major cause of antibiotic-associated diarrhoea (AAD) and pseudomembraneous colitis (Bartlett, 1994). C. difficile associated diarrhoea (CDAD) has been associated with oral therapy with a range of antibiotics, but those of highest risk include clindamycin, penicillins (including ampicillin and amoxicillin) and oral cephalosporins. CDAD is ten times more common in patients aged 60±98 years compared to younger patients (Karlstrom et al., 1998). As well as antibiotics, enteral and tube feeding is also a risk factor


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of CDAD (Whelan et al., 2001; HeÂbuterne, 2003). Other risk factors include multiple comorbid conditions and associated high number of medical or surgical interventions as well as extended hospitalisation. C. difficile results in colitis via the action of two toxins. Toxin A is 308kDa (cdtA) and toxin B is 207kDa (cdtB), both toxins are responsible for the symptomatic intestinal inflammation found in the disease (Pothoulakis, 2001). Saccharomyces boulardii and Lactobacillus rhamnosus GG have both shown promise in the treatment of C. difficile associated diarrhoea (Gorbach et al., 1987; Surawicz et al., 2000; Tuohy et al., 2003). D'Souza et al. (2002) in a recent meta-analysis have shown the efficacy of probiotics in the treatment of AAD. When administered with antibiotic, both S. boulardii and lactobacilli significantly reduced the risk of developing AAD in nine controlled studies. In a retrospective study, regular lactulose therapy was shown to reduce the prevalence of urinary tract infections (often caused by the members of the gastrointestinal microflora such as E. coli) in elderly patients in long-term hospitalization (McCutcheon and Fulton, 1989). Lactulose intake also reduced antibiotic therapy in these patients compared to a control (no lactulose) group. Other pathogens linked to antibiotic associated diarrhoea include C. perfringens, Salmonella and Shigella species (Samuel et al., 1991; DuPont, 1991). Although carried out in young adults (mean age 28 years) Sullivan et al. (2003) showed that consumption of a yoghurt containing L. acidophilus NCFB 1748, B. lactis Bb12 and L. paracasei subsp. paracasei F19 limited ecological disturbances in Bacteroides sp. population levels upon treatment with clindamycin. However, probiotic supplementation appeared to have little effect on C. difficile infection, where one of the patients on the active probiotic treatment developed CDAD. Drugs, such as H2-antagonists, proton pump inhibitors and synthetic prostaglandin analogues, used to treat peptic ulcers, gastro-oesophageal reflux or Zollinger-Ellison syndrome, all conditions affecting the elderly, reduce gastric pH (Lovat, 1996; Ratnaike and Jones, 1998). This in turn leads to increased survival of ingested microorganisms and an increased risk of diarrhoea. In the elderly, H2-antogonists are reported to be a risk factor in developing C. difficile diarrhoea (Walker et al., 1993).

16.5 Immunosenescence and suscepibility to colon cancer in old age 16.5.1 Immunosenescence Changes in immune function with old age are associated with increased morbidity and mortality rates with infectious diseases. Immunosenescence is characterised by a decrease in mature CD3+ T cells, a reduced pool of naõÈve T cells, inhibited T cell proliferation and secretion of interleukin-2 (Hodes, 1997; Schmucker et al., 2001). The activity of natural killer cells and phagocytes is also reduced in old age (Butcher et al., 2000; Solana and Mariani, 2000). In the aged population the quality and proportion of T cells is reduced. This leads in

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turn to a reduction in secretory IgA which is the primary immune response of the GALT (Ratnaike and Jones, 1998). Immunosenescence is not fully understood as yet and at times seems contradictory in that there is a reduction in immune response to foreign antigens but an increase in auto-immune and auto-antibody production (Arranz and Ferguson, 1992). Lactobacilli and bifidobacteria have been shown to modulate the immune system in a number of animal studies and in a limited number of human feeding studies (Perdigon et al., 1988; Solis Pereyra and Lemmonier, 1993; Link-Amster et al., 1994; Schiffrin et al., 1995). They have been shown to enhance non-specific resistance to infectious agents or tumours (Fernandes and Shahani, 1990) or act as adjuvants to specific immune responses (Schiffrin et al., 1995; Maasen et al., 2000; Plant and Conway, 2002). Some aspects of cellular immunity may be modified using specific probiotic strains in the elderly (Matsuzaki and Chin, 2000). In thirty healthy elderly volunteers (63±84 years) dietary supplementation with a milk containing Bif. lactis HN019 over a three-week period, increased the proportion of total, CD4+ and CD25+ T lymphocytes and natural killer cells increased in blood samples compared to the control (low-fat milk) (Gill et al., 2001). Coupled with this were increased phagocyticity of polymorphonuclear and mononuclear cells and an increase in the tumouricidal activity of NK cells in ex-vivo samples. These authors also showed that L. rhamnosus HN001 and Bif. lactis HN019 increased ex-vivo tumouricidal activity and that this was significantly correlated with age, where those over 70 showed most improvement. Schiffrin et al. (1995) examined the immunomodulatory effect of milk fermented with either L. acidophilus La1 or B. bifidum/lactis Bb12 at 360 ml per day for three weeks (corresponding to 7 1010 CFU L. acidophilus La1 and 1 1010 CFU Bif. lactis Bb12 per day). Although no modifications in lymphocyte subpopulations were observed both probiotic drinks increased granulocyte and monocyte phagocytic activity against E. coli in peripheral blood. Stimulation of this antiinfective non-specific mechanism of defence may be best applied to sections of the population with defective immunocompetent blood cells, such as the elderly. Since such immonomodulatory effects are likely to be strains specific, efficacious probiotic strains should be chosen with care. A growing number of in vivo studies have shown that probiotics may impact on the incidence and/or duration of diarrhoeal infections including those caused by viruses and `winter' infections of the gut and respiratory tract. Turchet et al. (2003) conducted a pilot controlled study on the protective effects of a fermented milk product containing L. casei DN-114 001 (and yoghurt strains) on the incidence and duration of `winter' infections, both respiratory and gastrointestinal, in 360 healthy elderly subjects over the age of 60. Subjects in the treatment group consumed 2 100 ml probiotic fermented milk daily for three weeks and their health status was monitored. Although consumption of the probiotic milk did not affect the incidence of winter infections, it did significantly reduce the duration of illness by as much as 20% compared to subjects not receiving the probiotic. This reduction in duration of illness was significant for all infections monitored (i.e. total pathologies, influenza,


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gastrointestinal syndromes, respiratory diseases and ears/nose/throat pathologies). It is likely that such protection is mediated through interactions with the immune system. Probiotics may have both local and systemic effects on immune function including modulation of allergic response through stimulation of non-specific immunity or enhancing humoral or cellular immunity (Isolauri et al., 2001). There is currently limited information on the immunostimulatory effects of prebiotics and other dietary fibres, although it is likely that, as they stimulate indigenous probiotic growth within the gut, they may have an immunological influence. Evidence from animal feeding studies appears to support this with both fructooligosaccharides and lactulose (as well as less well defined dietary prebiotic fibres) showing some impact on immune parameters (Schley and Field, 2002). An increase in phagocytic activity of intraperitoneal macrophages was observed in rats fed lactulose (0.5% energy intake) compared to a control diet (infant formula) (Nagendra and Vankat Rao, 1994). Similarly, Gaskins et al. (1996) showed that mice fed fructooligosaccharides (30g/l drinking water) had increased numbers of caecal and colonic macrophages. Field et al. (1999) found that in dogs fed a mixture of fermentable fibres (including fructooligosaccharides), numbers of CD8+ cells increased in Peyers Patches, intraepithelial lymphocytes and lamina propria compared to a low fermentable fibre diet. Lactulose has also been shown to increase numbers of Ig-A positive cells in the caecum of rats compared to a cellulose containing control diet (Kudoh et al., 1999). Only on a few occasions has the effect of prebiotic consumption on immune parameters been determined in humans and there is limited information on how prebiotics interact with the elderly immune system. Guigoz et al. (2002) determined the bifidogenic effects of FOS in elderly subjects and investigated whether prebiotics improved non-specific immune function. Upon feeding 19 elderly subjects FOS at 8 g/day for three weeks, numbers of bifidobacteria and Bacteroides spp. increased significantly in faecal samples compared to pre-treatment levels. No changes were observed in other bacterial groups monitored (i.e. the Enterobacteriaceae, enterococci and lactobacilli). Conversely, changes were observed in non-specific immune function. The phagocytic activity of granulocytes and monocytes decreased, as did expression of interleukin-6 mRNA in peripheral blood monocytes. Such changes suggest a possible reduction in the inflammatory immune response in the elderly subjects. Bunout et al. (2002) investigated the effect of prebiotics on the immune response to influenza and pneumococcal vaccination in the elderly. These authors found that a mixture of fructooligosaccharides and inulin had no effect on serum proteins, albumin, immunoglobulins and secretory IgA or on interleukin-4 and interferon-aÄ secretion by cultured monocytes. Clearly, further studies in human subjects, especially the elderly, are needed to confirm some of the interactions between prebiotics and the immune system observed in animals.

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16.5.2 Colon cancer The elderly are particularly susceptible to colon cancer and with the ageing population, this impacts greatly on medical expenditure (Gill and Rowland, 2002). Whether colon cancer can be seen truly as a disease of old age is debatable, since disease progression occurs over a long period of time often going un-diagnosed until later in life when more serious symptoms become apparent. Indeed, there may be as long as 20 years between initial DNA damage and the tumour development. The gut mucosa is continuously in contact with a vast array of potential carcinogens and genotoxins from dietary, environmental and microbial sources, and in the absence of underlying genetic susceptibility, disease occurs upon failure of host defences (DNA repair mechanisms and immune responses) (Gill and Rowland, 2002). Accumulation of genetic mutations over prolonged periods of time and alterations in the immune response with age may also explain the higher incidences of colon cancer in old age (Malaguarnera et al., 2001). However, the role played by immunosenescence in colon cancer is very much unclear at the moment (Bonafe et al., 2001; Tarazona et al., 2002). Evidence from a variety of sources is converging to support the view that the gut microbiota plays a role in the aetiology of colon cancer (Burns and Rowland, 2000). Conversely, there is convincing evidence that dietary modulation of the gut microbiota towards a more beneficial composition may play a protective role against colon cancer onset and progression (Burns and Rowland, 2000; Rafter, 2002). Probable modes of action behind this protection include microbial short chain fatty acid production, interactions with lipid metabolism (production of secondary bile acids), and direct interactions between beneficial microorganisms, such as bifidobacteria and lactobacilli, with cells of the gastrointestinal mucosa (e.g. immunomodulation, biological activators). Much of the supporting evidence on the protective role of probiotics, prebiotics and synbiotics has come from animal feeding studies (Rowland et al., 1998; PoolZobel et al., 1996, 2002). There is currently a need to conduct placebocontrolled blinded human feeding studies to examine the impact of dietary modulation on biomarkers of colon cancer, especially in high-risk populations such as the elderly.

16.6

Future trends

Changes in species diversity and relative bacterial numbers within the gut microflora, as well as immune dysregulation, suggest that the elderly are a population for which the application of probiotics, prebiotics and synbiotics may be particularly efficacious. However, there is a considerable lack of knowledge regarding interactions between the gut microflora (especially in old age) and host parameters such as the immune system and the mucosal surfaces within the gut. There is also a need to study interactions between the gut microflora and more chronic disease states, common in the elderly such as colon cancer. A number of key areas of research may be identified.

Improving gut health in the elderly 1.

2.

3.

4.

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Changes within the gut microflora with old age need further clarification, particularly using high-resolution molecular techniques to monitor changes in species diversity within the aged as well as monitoring changes in bacterial numbers in response to dietary interventions. Current EU-funded projects are providing the tools necessary for such an approach and are employing these tools to study the microbial ecology of the aged gut (Blaut et al., 2002; Mattila-Sandholm et al., 2002; Saunier and DoreÂ, 2002). Placebo-controlled, blinded human feeding studies are required to determine the efficacy of particular probiotic strains and prebiotics in modulating the course of specific gastrointestinal illnesses important in the elderly, e.g., constipation, gastroenteritis (of both viral and bacterial aetiology) and more chronic diseases such as colon cancer. Important to the design of such studies is controlling for nutritional status and concomitant medication within study groups. Interactions between the ageing gut microflora and the immune system of elderly people needs clarification. Currently, there is a lack of understanding regarding fundamental interactions between the role played by specific members of the gut microflora and immunosenescence. Such interactions may be important in determining the course of both infectious disease and cancer within the gut. Although probiotics, and to a much lesser extent prebiotics, have been shown to impact on specific immune parameters, it is not known how this impacts on gastrointestinal health and disease in the elderly. Choice of probiotic strain or prebiotic may be important for modulation of gut health in the elderly. Important selection criteria may be: ability to adhere to intestinal mucus (particularly elderly intestinal mucus); ability to inhibit important gastrointestinal pathogens of the elderly (e.g. C. difficile and food pathogens), stimulation of specific immune parameters (e.g. enhancement of phagocytic activities or improved response to vaccines); and selective stimulation of species of potentially beneficial members of the elderly gut microflora (e.g. species of bifidobacteria more commonly found in the elderly). Novel prebiotic oligosaccharides may also be designed to stimulate specific species of bacteria within the elderly gut microflora or to incorporate anti-adhesive moieties active against gastrointestinal pathogens or their toxins (Rastall and Maitin, 2002).

16.7

Conclusion

It is likely that a combination of immunosenescence, changes in gastrointestinal physiology (e.g. atrophic gastritis and impaired small bowel motility) and agerelated changes in the gut microflora all contribute to the predisposition of elderly people to severe and prolonged microbial infections. In the light of the importance of the gut microbiota to health, changes in the composition of the gut microbiota with age could be of major significance. In general, however, studies on the effect of age on microbiota have been confined almost exclusively to early life, babies,

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and infants. There is limited information concerning the composition of the gut microbiota in the aged or its contribution to healthy ageing. Confirmation of a decline in bifidobacteria and other lactic acid producing bacteria (LAB) in the gut of ageing subjects opens up the possibility of reversing such trends by administration of probiotics (bifidobacteria or lactobacilli), prebiotics that selectively encourage the growth of such bacteria within the elderly gut, or a combination of the two, synbiotics. Although currently few in number, feeding studies with probiotics and prebiotics in the elderly do show some health-promoting capabilities, e.g., relief from constipation, reduced duration of antibiotic-associated diarrhoea and some immunomodulation. Further studies are required to determine the interaction between immunosenescence and alterations within the gut microflora with old age. An important consideration in such studies however, is the nutritional status and general health of study groups and they should be matched for nutritional status and medication.

16.8

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and MARIANI E (2000), `NK and NK/T cells in human senescence', Vaccine, 18, 1613±1620. SOLIS PEREYRA B and LEMMONIER D (1993), Ìnduction of human cytokines by bacteria used in dairy foods', Nutr Res, 13, 1127±1140. SUAU A, BONNET R, SUTREN M, GODON J J, GIBSON G R, COLLINS M D and DOREÂ 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. Ê , BARKHOLT L and NORD C E (2003), `Lactobacillus acidophilus, BifidoSULLIVAN A bacterium lactis and Lactobacillus F19 prevent antibiotic-associated ecological disturbances of Bacteroides fragilis in the intestine', J Antimicrob Chemother, 52, 308±311. SURAWICZ C M, MCFARLAND L V, GREENBERG R N et al. (2000), `The search for a better treatment for recurrent Clostridium difficile disease: use of high-dose vancomycin combined with Saccharomyces boulardii', Clin Infect Dis, 31, 1012±1017. TARAZONA R, SOLANA R, OUYANG Q and PAWELEC G (2002), `Basic biology and clinical impact of immunosenescence', Exp Gerontol, 37(2±3), 183±189. TUOHY K M, KOLIDA S, LUSTENBERGER A M and GIBSON G R (2001), `The prebiotic effects of biscuits containing partially hydrolysed guar gum and fructo-oligosaccharides ± a human volunteer study', Br J Nutr, 86, 341±348. TUOHY K M, ROWLAND I R and RUMSBY P C (2002a), `Biosafety of marker genes the possibility of DNA transfer from novel foods to gut microorganisms', in Atherton K T, Genetically modified crops assessing safety, Taylor and Francis, London, 110±137. È BEL Y, POOL-ZOBEL B L and GIBSON G R (2002b), À TUOHY K M, ZIEMER C J, KLINDER A, KNO human volunteer study to determine the prebiotic effects of lactulose powder on human colonic microbiota', Microb Ecol Health Dis, 14, 165±173. TUOHY K M, PROBERT H M, SMEJKAL C W and GIBSON G R (2003), Ùsing probiotics and prebiotics to improve gut health', Drug Discov Today, 8, 692±700. TURCHET P, LAURENZANO M, AUBOIRON and ANTOINE J M (2003), Èffect of fermented milk containing the probiotic Lactobacillus casei DN-114 001 on winter infections in free-living elderly subjects : a randomised, controlled pilot study', J Nutr Health Aging, 7, 75±77. VAUGHAN E E, MOLLET B and DE VOS W M (1999), `Functionality of probiotics and intestinal lactobacilli: light in the intestinal tract tunnel', Curr Opin Biotechnol, 10, 505±510. WALKER, K J, GILLILAND S S, VANCE-BRYAN, K. et al. (1993), `Clostridium difficile colonization in residents of long-term care facilities: prevalence and risk factors', J Am Geriart Soc, 41, 940±946. WHELAN K, GIBSON G R, JUDD P A and TAYLOR M A (2001), `The role of probiotics and prebiotics in the management of diarrhoea associated with enteral tube feeding', J Hum Nutr Diet, 14(6), 423±433. SOLANA R

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(1996), `Health and ageing: a discussion paper', pp. 1±40. www.tt.mtas.es/imserso/healthy%/ 20Ageing%20booklet.pdf ZOETENDAL E G, AKKERMANS A D L and DE VOS W M (1998), `Temperature gradient gel electrophoresis analysis of 16S rRNA from human faecal samples reveals stable and host-specific communities of active bacteria', Appl Environ Microbiol, 64, 3854±3859. COMMUNICABLE DISEASE PREVENTION AND SURVEILLANCE)

17 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

Nowadays, consumers have become increasingly aware of the necessity to maintain their health through nutrition, and of the role of the gut flora in health and disease. This microflora includes more than 500 bacterial species and hence comprises about 95% of the total number of cells in the human body. It contributes significantly to the host's resistance to infectious diseases. Changes in the composition of the gut flora are often associated with disease and may, in some cases, be the cause of disease. Therefore, scientific research is focusing on the roles that diet, stress, reduced physical activity, environmental factors, and modern medical practices (e.g. the use of antibiotics or surgery) play in threatening human health. In particular, the shifting of the population towards older individuals is increasing the incidence of illnesses that may be caused by a deficient or compromised microflora, such as gastrointestinal tract (GIT) infections, constipation, irritable bowel syndrome, inflammatory bowel disease (Crohn's disease and ulcerative colitis), food hypersensitivity and allergies, antibiotic-induced diarrhoea, small bowel bacterial overgrowth, cardiovascular disease, and certain cancers (e.g. colorectal cancer). Also, the growing abundance of modern disorders such as neoplasms, hypertension, and HIV infection requires increasing interest. Furthermore, serious concern has been expressed as the degree of microbial resistance to indiscriminately prescribed and misused antibiotics increases. To combat these trends directly, the World Health Organisation (WHO) currently advocates the implementation of alternative disease control strategies. The exploitation of the prophylactic and therapeutic potential of probiotic microorganisms is very promising (Bengmark, 1998; Naidu et al., 1999).

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17.1.1 History of defining probiotics A beneficial association of microorganisms with the human host was probably first suggested by DoÈderlein in 1892 (DoÈderlein, 1892). He proposed that vaginal bacteria produce lactic acid from sugars to prevent or inhibit the growth of pathogenic bacteria. Such bacteria were also found in association with fermented milk products and were examined for their health benefits by Metchnikoff in 1908. In 1900, Moro first isolated a lactic acid bacterium (LAB), namely Lactobacillus acidophilus (first called Bacillus acidophilus), from infant faeces. Lactobacillus acidophilus is indeed found in the intestinal tract of humans and animals as well as infants having high milk, lactose, or dextran diets. In 1901, Beijerinck (1901) did early taxonomic studies on LAB and in the same year Cahn studied the gut (faecal) ecology from infant stool (Cahn, 1901). In 1908 in his work on The Prolongation of Life, Metchnikoff implicated a LAB found in Bulgarian yoghurts ± that he called the Bulgarian bacillus and later Bacillus bulgaricus, which is likely to be the organism later known as Lactobacillus bulgaricus and now called Lactobacillus delbrueckii subsp. bulgaricus ± as the agent responsible for deterring intestinal putrefaction and ageing. Hence, Metchnikoff hypothesised for the first time the importance of lactobacilli for human health and longevity. However, he considered the gut microbes in total as detrimental rather than beneficial to human health, and he suggested that desirable effects might be expected only from their substitution by yoghurt bacteria. In this context, Metchnikoff especially promoted LAB and their major metabolite of sugar fermentation, i.e., lactic acid. Around 1906 Cohendy administered milk soured with the Bulgarian bacillus to subjects exhibiting putrefactive-type fermentations on a mixed diet, and he found a decrease in the products of putrefaction (Cohendy, 1906a,b). In 1906, Tissier reported clinical benefits from modulating the flora in infants with intestinal infections through displacement of the pathogenic bacteria with bifidobacteria. Further, in the early 1920s Rettger and Cheplin documented that L. acidophilus milk has therapeutic effects. They believed that colonisation and growth in the gut were essential for efficacy, and therefore, advocated the use of intestinal isolates (Rettger and Cheplin, 1921). Finally, in 1930, Shirota was the first to culture a strain of `good' intestinal bacteria that was able to reach the intestines alive without being destroyed by the digestive system. It was first named L. acidophilus Shirota, after him, and later renamed Lactobacillus casei Shirota (Yakult, 1999). In just five years he was manufacturing and distributing a dairy drink containing this culture, named Yakult. Due to this and other keen experimental scientists and clinicians the key scientific selection criteria for probiotics were in place by the 1950s though the concept had not yet been defined at that time. The word `probiotic' stems from the Greek pqo bi*o| (pro bios, `for life') and has been used in several different ways over the past few decades. It was originally proposed to describe compounds produced by one protozoan that stimulated the growth of another (Lilly and Stillwell, 1965). In the early seventies of the twentieth century Sperti (1971) expanded the term to encompass

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tissue extracts that stimulated microbial growth. Although Metchnikoff had already suggested in 1907 that ingested bacteria could have a positive influence on the normal microbial flora of the GIT, the term probiotic was first used in that context by Parker (1974) to describe animal feed supplements that had a beneficial effect on the host by contributing to its intestinal microbial balance. Consequently, the word probiotic was applied to òrganisms and substances that contribute to the intestinal microbial balance'. This general definition was, however, not satisfactory, because such an imprecise word as `substances' might include a variety of supplements, including antibiotics that were also used to promote the growth of farm animals (Fuller, 1999). Fuller (1989, 1992) revised the early definition of a probiotic to stress the importance of living cells as an essential component of an effective probiotic, and thus defined a probiotic as à live microbial feed supplement, which beneficially affects the host animal by improving its intestinal microbial balance'. This modified version of the definition underlined the need for the supplement to be composed of viable microorganisms and excluded antibiotics, but was restricted to animals. On the other hand, this definition also included traditional yoghurts, which are produced by fermenting milk with L. delbrueckii subsp. bulgaricus and Streptococcus thermophilus. The definition of a probiotic has since been narrowed to focus on the human gut microflora that has a beneficial health effect (Havenaar et al., 1992; Fuller and Gibson, 1998; Klein et al., 1998; Rolfe, 2000), or broadened to encompass other microbial communities, man and animals, as well as cocktails of cultures (Havenaar and Huis in 't Veld, 1992; Guarner and Schaafsma, 1998; Schrezenmeir and de Vrese, 2001). A first adapted definition was suggested by Havenaar et al. (1992), according to whom probiotics are defined as `monoor mixed cultures of live microorganisms which, when applied to animal or man, beneficially affect the host by improving the properties of the indigenous microflora' (Huis in 't Veld and Havenaar, 1991; Elmer, 2001). A slightly modified definition and presently widely accepted is that of Havenaar and Huis in 't Veld (1992), where probiotics are defined as `viable microorganisms (LAB and other bacteria or yeasts, applied in a fermented product or as dried cells) that exhibit a beneficial effect on the health of the host upon ingestion by improving the properties of its indigenous microflora'. Still other definitions tried to encompass both the formulations and functionalities of probiotics, such as that of Guarner and Schaafsma (1998) stating that `probiotics are live microorganisms, which upon ingestion in certain numbers, exert health effects beyond inherent basic nutrition' and that of Salminen et al. (1998) `probiotics are live microbes of human origin, used as food supplements or pharmaceutical preparations, that survive passage through the upper GIT, transiently colonise the gut by adhesion to the intestinal mucosa, and are beneficial to health'. Finally, the FAO/WHO expert committee defined probiotics as `live microorganisms that, when consumed in an adequate amount as part of the food, confer a health benefit on the host' (FAO/WHO, 2001). It should be emphasised therefore that human origin and adhesion are no longer necessary to consider a strain as

Probiotics, prebiotics and gut health Table 17.1

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Microorganisms used or considered for use as probiotic in humans

Lactobacillus species

Bifidobacterium species

Other lactic acid bacteria

Other microorganisms

L. L. L. L. L. L. L. L. L. L. L. L.

B. B. B. B. B. B. B.

Enterococcus faecalis* Enterococcus faecium* Lactococcus lactis Leuconostoc mesenteroides Pediococcus acidilactici Sporolactobacillus inulinus* Streptococcus thermophilus

Bacillus cereus (e.g. toyoi)*§ Escherichia coli (e.g. Nissle, 1917)§ Propionibacterium freudenreichii*§ Saccharomyces cerevisiae§ Saccharomyces boulardii§

acidophilus amylovorus casei crispatus gallinarum* gasseri johnsonii paracasei plantarum reuteri rhamnosus salivarius

adolescentis animalis bifidum breve infantis lactis longum

* mainly applied in animals § mainly applied in pharmaceutical preparations

probiotic. It should be the ability to remain viable at the target site and to grow and/or be active in the human body that determine its effectiveness. The latter definitions have certain advantages compared to the original definition of a probiotic by Fuller in that: · A large number of microbial species and genera are considered as probiotics (Table 17.1). · They do not restrict `probiotic' activities to the colon microflora but also, to other intestinal microbial communities (stomach, small intestine) and to microbial communities at other sites of the body (oral cavity, urogenital tract, skin). · They do not restrict `probiotic' effects through mediation by the microflora, but also, for instance, on immune parameters. · An adequate dose of microorganisms has to be provided to exert a desirable effect. · The probiotic might consist of more than one microorganism. · The probiotic can be applied to both man and animals. Currently, delicate points of discussion relate to the viability of the probiotic strains (dead cells, alive upon ingestion, alive at the site of action, . . .), the site of activity (oral cavity, upper GIT, lower GIT, urogenital tract, skin, . . .), the amount of cells necessary to exert a specified probiotic effect, the format of intake and its carrier (mono- or mixed cultures, food (dairy) products, food supplements, pharmaceutical preparations, . . .), etc.. Furthermore, the lack of proper biomarkers and/or technologies to directly quantify the presence or efficacy of probiotic strains in healthy humans makes a reliable definition difficult (Mercenier et al., 2003).

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17.1.2 Defining prebiotics Prebiotics are non-digestible food ingredients that beneficially affect the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the colon (Gibson and Roberfroid, 1995). Thus, the prebiotic approach advocates the administration of non-viable entities, and therefore overcomes any viability problems of probiotics in the upper GIT. Further, a prebiotic can be considered as a growth substrate that fortifies the beneficial intestinal microflora. However, it differs from the classical dietary fibres in that it selectively stimulates the growth and/or activity of bifidobacterial species in particular. The general term `dietary fibres' refers to the remains of plant cells that are resistant to hydrolysis by human enzymes and that modulate the carbohydrate and lipid metabolism of the host (positive influence on constipation, hyperlipidemias, diabetes, obesity, and diverticular disease) (Trowell, 1972; Trowell et al., 1976; Schweizer and Wursch, 1991). Fibre components are not selectively fermented or are not fermented at all (Schweizer and Wursch, 1991). To be classified as a prebiotic, a food ingredient must (i) be neither hydrolysed nor absorbed in the upper part of the GIT, (ii) be able to alter the colonic flora in favour of a healthier composition through selective fermentation, and (iii) induce luminal or systemic effects that are beneficial to the hosts' health (Gibson and Roberfroid, 1995; Roberfroid, 1997; Van Loo, 1998; Cummings et al., 2001). 17.1.3 Defining synbiotics Synbiotics are defined as a mixture of probiotics and prebiotics that improve the survival and implantation of live microbial dietary supplements in the GIT, either by stimulating the growth or by metabolically activating the healthpromoting bacteria (Gibson and Roberfroid, 1995; Lewis and Freedman, 1998). The end result should be improved survival of the probiotic, which has a readily available (and specific) substrate for its fermentation, and hence increased numbers of bacteria reaching and residing in the colon, as well as the individual advantages that the pro- and prebiotic may offer.

17.2 Types of probiotics and prebiotics and their influence on gut health Different types of food products or food supplements containing viable microorganisms with probiotic properties are commercially available either as fermented or non-fermented food commodities or as specific preparations such as powders, tablets, or capsules. An overview of microorganisms used or considered for use as probiotics in humans is given in Table 17.1. As LAB are considered of importance with regard to human food and nutrition, only LAB strains that may be of prophylactic and therapeutic potential will be addressed in this chapter.


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Numerous studies report the generally unsubstantiated health-promoting properties of LAB, yeast and fermented dairy products in animals and humans. These properties include the beneficial influences probiotics apparently exert on the microbial ecology of the host, its food digestion, lactose intolerance, incidence of diarrhoea (rotavirus diarrhoea, travellers' diarrhoea, Clostridium difficile-associated diarrhoea, antibiotic-associated diarrhoea), mucosal immune response, intestinal inflammation, intestinal infections, Helicobacter pyloriassociated gastritis, vaginitis, allergic reactions, atopic dermatitis, blood cholesterol concentrations, intestinal microbial enzyme activity and faecal mutagenicity, metal detoxification, tumour development, and cancer (Dunne et al., 1999, Rolfe, 2000; Saavedra, 2000, 2001; Marteau et al., 2001, 2002; Vanderhoof, 2001; Dunne and Shanahan, 2002; Kaur et al., 2002; Mercenier et al., 2003). Currently, certain health effects of probiotic LAB are considered scientifically proved for specific strains (Salminen et al., 1998; Naidu et al., 1999; Ouwehand et al., 1999b; FAO/WHO, 2001). For the selection and assessment of probiotic LAB, the following criteria have been proposed (Havenaar and Huis in 't Veld, 1992; Huis in 't Veld and Shortt, 1996; Salminen et al., 1996b; Charteris et al., 1998; Collins et al., 1998; Guarner and Schaafsma, 1998; Holzapfel et al., 1998; MattillaSandholm et al., 1999; Ouwehand et al., 1999b, Dunne et al., 2001): human origin, non-pathogenic behaviour, safe, resistant to gastric acidity and bile toxicity, adhesion to or interaction with the gut epithelial tissue, ability to persist within the GIT (transient colonisation of the intestinal environment), production of antimicrobial substances and nutraceuticals, evidence of beneficial health effects (e.g. ability to modulate immune responses), ability to influence metabolic activities (e.g. lactase activity and vitamin production), and resistance to technological processes (i.e., viability and activity in delivery vehicles during the shelf-life period). Further, the demonstration of probiotic activity of a certain LAB strain requires welldesigned, randomised, double-blind, placebo-controlled human studies (Guarner and Schaafsma, 1998). These requirements were further outlined by several authors (Salminen et al., 1996a, 1998; Berg, 1998; HamiltonMiller and Gibson, 1999) as follows: each potential probiotic strain should be documented and assessed independently through in vitro, animal, clinical, and epidemiological studies; extrapolation of data from closely related strains is not acceptable; only well-defined strains, products, and study populations should be used in trials; all human studies should be with established end-points; results should be confirmed by independent research groups; and the study should be published in peer-reviewed journals. The joint FAO/WHO (2001) expert consultation further recommends a refinement of the current in vitro and in vivo tests to better predict the ability of probiotic microorganisms to function in humans. In this chapter we will focus on the antimicrobial activities of probiotic LAB and possible underlying mechanisms. The idea that specific foods provide protective functions has been a long-held belief of populations that eat

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fermented foods such as yoghurt. Such gut protection as a functionality (gut health) is determined by living bacteria and their metabolic products. Organic acids, short-chain fatty acids, hydrogen peroxide, ethanol, carbon dioxide, and diacetyl, produced by LAB, display antimicrobial effects. In addition, bacteriocins or bacteriocin-like, proteinaceous substances display specific inhibitory activity against closely related species. They are extensively studied antimicrobials produced by LAB (De Vuyst and Vandamme, 1994; Jack et al., 1995; Nes et al., 1996; Cintas et al., 2001; Cleveland et al., 2001). The production of antimicrobial substances by probiotic LAB and/or as a result of growth of beneficial microorganisms on an appropriate prebiotic substrate may not only result in pathogen inactivation in the gut and balancing of the gut microflora, but also in good storage stability and shelf-life of such functional food products. 17.2.1 Probiotic lactic acid bacteria Currently, the most well studied probiotics are certain strains of LAB, particularly lactobacilli, and to a lesser extent, bifidobacteria (Charteris et al., 1997; Holzapfel et al., 1998, 2001; Gomes and Malcata, 1999). LAB are Grampositive, non-sporulating, catalase-negative, oxidase-negative microorganisms that are aerotolerant, nutritionally fastidious, acid tolerant, and strictly fermentative. Indeed, LAB represent a group of bacteria that are metabolically related by their ability to produce lactic acid during homo- or heterofermentation of carbohydrates. The acidification and enzymatic processes accompanying the growth of LAB impart to the preservative qualities, key flavour, and texture of a variety of fermented foods and beverages. Several members of the LAB occupy important niches in the GIT of humans and animals. Indeed, they are found as natural commensals of the GIT, the oral cavity, and the female urogenital tract of animals and humans. Due to the low pH in the stomach, microbial numbers are very low (103 colony forming units (CFU) gÿ1). The population of the stomach consists of LAB such as lactobacilli and streptococci, and yeasts. Increasing numbers of bifidobacteria, Gramnegative facultative aerobic bacteria such as Enterobacteriaceae, and the obligate anaerobic bacteria such as Bacteroides and Fusobacterium appear towards the more distal regions of the small intestine, in addition to LAB. Numbers increase from 104 CFU gÿ1 in the jejunum up to 108 CFU gÿ1 in the ileum. The colon contains around 1011±1012 CFU gÿ1 and harbours more than 300 to 500 species, among which strict anaerobes (Bacteroides, Eubacterium, Bifidobacterium) are the predominant group. Strict anaerobes such as Clostridium and facultative aerobes, including lactobacilli, are considered to be sub-dominant populations in the gut. Practically, all microorganisms used in probiotic foods or food supplements are representatives of the genera Lactobacillus, Bifidobacterium, and Enterococcus. Out of 91 species, strains of more than twelve species of lactobacilli are probiotic (Tannock, 1999; Holzapfel et al., 1998). Of the 31 Bifidobacterium


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species currently known, eleven have been detected in human faeces; strains belonging to five species are probiotic (Tannock, 1999). Unfortunately, most of the LAB cultures used in probiotic products do not have the appropriate species designation. This is particularly the case for strains belonging to the L. acidophilus complex, the L. casei complex, and the Bifidobacterium longum/ Bifidobacterium animalis complex (Klein et al., 1998). In addition, characterisation and study of the mechanisms of action of these microorganisms lag significantly behind characterisation and study of in vitro and clinical effects (Reddy, 1999; Ouwehand et al., 1999b; Reid et al., 2001). Lactobacilli Homofermentative lactobacilli that are typical of the human host are represented by three groups: (i) the Lactobacillus acidophilus complex (belonging to the Lactobacillus delbrueckii group), (ii) Lactobacillus salivarius (belonging to the L. salivarius group), and (iii) the Lactobacillus casei complex (belonging to the Lactobacillus plantarum group) (Klaenhammer et al., 2002). Lactobacillus acidophilus is considered to be the most likely species to fulfil the base criteria expected of a probiotic culture; survival through the GIT, acid tolerance, bile tolerance, and antimicrobial production. Although L. acidophilus is phenotypically difficult to assess, its heterogeneity has been confirmed by DNA-DNA hybridisation studies reported in 1980 (Johnson et al., 1980; Lauer et al., 1980). These authors suggested six different DNA homology groups. As a consequence, only strains belonging to the homology group that showed a high degree of DNA relatedness with L. acidophilus remained in this species, whereas members of the other DNA homology groups were classified in separate species, i.e., Lactobacillus amylovorus, Lactobacillus gallinarum, Lactobacillus crispatus, Lactobacillus gasseri, and Lactobacillus johnsonii. Although these are regarded as separate species, they are closely related and have been suggested as belonging to one phylogenetic group or branch, the so-called `L. acidophilus complex' (Klaenhammer and Russell, 2000). Of the six species in this complex, L. acidophilus continues to be the bacterium most often implicated in providing probiotic effects and remains to be the species most commonly found in foods or food supplements that contain probiotic cultures. This species was first described by Moro (1900) as Bacillus acidophilus, and was renewed by Hansen and Moquot (1970). In the early 1930s, preparations containing L. acidophilus were used to alleviate constipation (Rettger, 1935). Also, L. johnsonii strains have been mainly isolated from the faeces of humans and animals (Johnson et al., 1980; Fujisawa et al., 1992), suggesting that these bacteria constitute part of the natural intestinal microflora as well. Together with another species of this group relevant for probiotics, namely L. gasseri, they were described in the years 1980 to 1992. This may be the reason why the identification of strains such as L. acidophilus was quite common and not justified in most cases (Klein et al., 1998). Also L. crispatus, first described in 1953, is used as a probiotic.

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Lactobacillus johnsonii La1 (formerly L. acidophilus La1) is a NestleÂ proprietary strain that has been extensively studied for its probiotic properties. It is commercialised in the LC1 fermented milk product. It modulates the immune response (Link-Amster et al., 1994; Marteau et al., 1997; Pfeifer and Rosat, 1998; Donnet-Hughes et al., 1999, Haller et al., 2002). It adheres to human enterocyte-like Caco-2 cells in vitro (Granato et al., 1999), and it colonises the human GIT temporarily (Pfeifer and Rosat, 1998; Granato et al., 1999). It has been shown that lipoteichoic acids from the bacterial cell wall play a role in the adhesion of L. johnsonii La1 cells to Caco-2 cells (Granato et al., 1999), and the strain shares carbohydrate-binding specificity with enteropathogenic bacteria (Neeser et al., 2000). In addition, L. johnsonii La1 displays antimicrobial activity against both Gram-positive and Gram-negative pathogenic bacteria (Bernet et al., 1994; Bernet-Camard et al., 1997; PeÂrez et al., 2001), including H. pylori (Michetti et al., 1999; Felley et al., 2001; Gotteland and Cruchet, 2003). Finally, L. johnsonii La1 has also a potential activity against diarrhoeagenic protozoa like Cryptosporidum parvum and Giardia intestinalis (PeÂrez et al., 2001; Foster et al., 2003). Lactobacillus gasseri appears to represent the major homofermentative Lactobacillus species that occupies the human GIT. Lactobacillus gasseri demonstrates good survival in the GIT (Pedrosa et al., 1995; Fujiwara et al., 2001a), and has been associated with a variety of probiotic activities and roles including reduction of faecal mutagenic enzymes (Pedrosa et al., 1995; Sreekumar and Hosono, 1998). Also, inhibition of H. pylori (Sakamoto et al., 2001) and immunostimulation (Kitazawa et al., 2002) has been described for L. gasseri. Finally, several bacteriocins have been isolated from cultures of strains that were isolated from human faeces (Toba et al., 1991; Itoh et al., 1995; Kawai et al., 2001). Lactobacillus salivarius is another autochthonous member of the human GIT (Holzapfel et al., 1998). The best probiotic example is probably L. salivarius subsp. salivarius UCC118. This strain was selected from a range of bacteria isolated from resected human terminal ileum. Screening methods consisted of testing resistance to gastric acid, bile acid resistance, adherence to human gut epithelial cells, in vitro antimicrobial activity, survival through the GIT of mice and man, and the effect of feeding in a murine model of inflammatory bowel disease (Dunne et al., 1999). Furthermore, this strain produces the bacteriocin ABP-118 (Flynn et al., 2002). Members of the L. casei cluster may be isolated from the reproductive and intestinal tracts of humans and animals as well (Holzapfel et al., 1998; Reuter et al., 2002). This cluster also contains the species L. zeae and L. rhamnosus. The species L. casei was poorly defined and contained five subspecies based on phenotypic characteristics, namely L. casei subsp. alactosus, L. casei subsp. casei, L. casei subsp. pseudoplantarum, L. casei subsp. rhamnosus, and L. casei subsp. tolerans. Based on studies of DNA homology Collins et al. (1989) indicated that the majority of organisms designated L. casei subsp. casei, together with L. casei subsp. alactosus, L. casei subsp. pseudoplantarum, and L.


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casei subsp. tolerans, showed high levels of DNA relatedness but were distinct from the type strain of L. casei subsp. casei. They proposed to give three subspecies of this homology group the status L. paracasei subsp. paracasei, next to L. paracasei subsp. tolerans and L. rhamnosus. However, based on DNA homologies with other species and strains, the species name L. paracasei was questioned as the type strain of L. casei, and the species L. zeae, L. casei and L. rhamnosus were proposed (Dellaglio et al., 1991, 2002; Dicks et al., 1996). Both L. casei/paracasei and L. rhamnosus are autochthonous strains inhabiting the human GIT and that have been applied in probiotics in man and animals (Reuter et al., 2002). Examples are L. rhamnosus GG and L. casei Shirota. These strains are probably the best documented probiotic strains (for a review, see Goldin, 1998, and Yakult, 1999). Also, some strains of Lactobacillus plantarum are being marketed as probiotic as well. An example is L. plantarum 299v that confers various health benefits to the consumer (Adawi et al., 2001; Cunningham-Rundles et al., 2000; Goossens et al., 2003; Wultt et al., 2003). Furthermore, the genome of L. plantarum WFCS1, a strain that is able to survive passage through the stomach and is able to persist more than six days in the human GIT (Vesa et al., 2000), has been completely sequenced (Kleerebezem et al., 2003). Genome sequencing may facilitate future research on probiotic strains, for instance by microarray experiments. Finally, some heterofermentative lactobacilli as part of the normal microbial population of the human GIT have been identified as well, which include mainly Lactobacillus reuteri and, to a lesser extent, Lactobacillus fermentum, Lactobacillus oris, and Lactobacillus vaginalis (Holzapfel et al., 1998). Lactobacillus reuteri is of great interest as an antimicrobials (reuterin, reutericyclin, bacteriocins) producing probiotic and is used in animal nutrition as well as in yoghurt-type products and pharmaceutical preparations (Klein et al., 1998; Rodriguez et al. 2003; Rosenfeldt et al., 2003). Bifidobacteria The genus Bifidobacterium shares some phenotypic features with typical LAB but is phylogenetically distinct. Bifidobacteria exhibit a relatively high guanine plus cytosine (G + C) content of 55±67 mol% in the DNA, and form part of the so-called Actinomycetes branch of Eubacteria. The traditional LAB form part of the so-called Clostridium branch, which is characterised by a G + C content of 2 years at room temperature, increased the number of precancerous lesions (Gallaher et al. 1996). Davies et al. (1998) showed that ISP underwent browning during storage, and that this browning was parallel to genistein loss. Further evidence for bioactivity changes in isoflavones during processing was reported by Singletary et al. (2000) who showed that the extrusion of soy/corn mixtures (~120ëC) resulted in an average loss of 24% of the isoflavones. Interestingly, they also found a decrease in the antiproliferative activity of these mixtures after extrusion. Storage of daidzein at elevated temperatures reduced antioxidant potential of the solution (Ungar et al. 2003). The increase in consumption of soy-based and soy-containing foods is based on consumer perception of these products as health-promoting agents. It is therefore the responsibility of the food industry to ensure that these benefits are delivered to the consumer. It was shown that phytoestrogens content and composition changes from one product to another. Hence, the biological benefit depends on the raw material and processes being used. Therefore, favorable raw materials and processing conditions can be used only if we have a good understanding of the biological impact of each phytoestrogen; in this case, the prevention of cancer.

Phytoestrogens and the prevention of cancer 649

23.3 The role of phytoestrogens in the prevention of different cancers 23.3.1 Breast cancer Isoflavones, which are mainly consumed from soybeans and soybean products, are a part of the regular diet in Southeast Asian countries like China and Japan. Traditionally, these countries have a very low incidence of cancer and coronary heart disease, however, the incidence is increasing, particularly in Japan due to change in dietary habits and life style (Adlercreutz 1998a). It is becoming apparent that the effect of an isoflavone-rich diet on breast cancer risk might be significant only if consumption occurs before puberty or during adolescence (Lamartiniere 2000). An epidemiological study conducted in China showed that adolescent soy food intake is inversely associated with adult breast cancer risk (Shu et al. 2001). Consistent with these observations, studies of Japanese and white people who emigrated to the United states showed that when emigration occurs later in life, breast cancer risk is substantially less compared with those who emigrated while young (Lamartiniere et al. 1995). In a study in Hawaii, Japanese women excreted more isoflavones than Caucasian women in the urine (Maskarinec et al. 1998). In addition, using urinary phytoestrogen excretion as a marker of intake and exposure, three case control studies reported inverse associations between urinary phytoestrogen and breast cancer risk in Australia (Ingram et al. 1997; Murkies et al. 2000) and China (Zheng et al. 1999). Australian Chinese populations excreted two to five times more genistein and daidzein, compared to the Anglo-Celtic population (Dalais et al. 1998). It should be noted, however, that one prospective cohort study, performed in the Netherlands, reported no association (den Tonkelaar et al. 2001). Study conducted in the United States on non-Asian US women aged 35±79, found that phytoestrogens appear to have little effect on breast cancer risk at the levels commonly consumed by these women (an average intake equivalent to less than one serving of tofu per week) (Horn-Ross et al. 2001). In a study conducted by Zheng et al. (1999) urinary excretion of isoflavones, particulary glycitin, was lower in breast cancer patients than in controls. In Japan Key et al. (1999) did not find any effect of soy consumption on breast cancer risks. Premenopausal patients with various breast diseases including cancer were given a course of 60 g of soy protein containing about 45 mg of isoflavones per day, over 14 days. No differences in thymidine labeling index estrogen and progesterone receptor labeling indices, apoptotic and mitotic indices were observed compared with age and menstrual cycle matched control. These studies might indicate that the assumption that `consumption of phytoestrogens at an early age is an important factor in the ability of the isoflavones in reducing cancer risk' is not conclusive. Epidemiological studies tried to correlate phytoestrogen intake with breast cancer risk. In several studies conducted in Singapore (Lee et al. 1991), Japan (Hirose et al. 1995), and with Chinese, Japanese and Filipino origin women in the United States (Wu et al. 1996), premenopausal women consuming large quantities

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of soy products had a considerably lower risk of breast cancer compared to women with low consumption. In an American study (Wu et al. 1996), a lower breast cancer risk was also noted for postmenopausal women consuming higher levels of soy products. However, no evidence for a protective effect of higher phytoestrogen consumption was found among a mixed pre- and postmenopausal Chinese population (Wu et al. 1996). A case control study conducted in Shanghai reported that regular soy food intake was found to be related to reduced risk of breast cancer, particularly for cancer positive for estrogen and progesterone receptors (Dai et al. 2001). In addition, it was reported that excretion of urinary isoflavones was associated with reduced risk of breast cancer (Zheng et al. 1999; Dai et al. 2002). From a recent study conducted by Dai et al. (2003) in Shanghai, it was suggested that the potential protective association of phytoestrogens may be modified by body mass index, waist:hip ratio, and blood levels of SHBG- sex hormone-binding globulin, and steroid hormones. Reducing breast cancer risk is also associated with another important group of phytoestrogens ± lignans, which are considered to be mammalian phytoestrogens. Lignans differ from the isoflavones because they are mostly produced in the colon from plant precursors and bacteria. A study from the early 1980s on lignan intake and breast cancer showed that the lignan concentration excreted from the urine in breast cancer patients was higher in vegetarians (Adlercreutz 1998b, Adlercreutz and Mazur 1997). In addition, low urinary excretion of enterlactone in patients with breast cancer was shown in a long-term Australian study of 12 women (Ingram et al. 1997). It is thought that the presence of lignan precursors in fiber-rich diets is connected with the low rate of breast cancer in women. Ingram et al. (1997) measured urinary isoflavone and lignan excretion in women newly diagnosed with breast cancer and community controls. After adjustment for age, total fat intake, and alcohol intake, a high excretion of both equol and enterolactone was associated with a substantial reduction in breast cancer risk. Low breast cancer risk was associated with intake of rye products, fiber, tea (known to be rich in lignans), and vitamin E in a recent study conducted in Finland (Pietinen et al. 2001). Flaxseed or purified lignans, administered neonatally or before puberty, may have the same effect on the mammary gland as isoflavones (Tou and Thompson 1999). A study (Kilkkinen et al. 2001) conducted on 12 women who were put on a diet containing at least 200 g of white wheat low-fiber bread per day and then were given a diet with equivalent amount of whole grain rye bread concluded that obesity is negatively associated with plasma enterolactone in women. Their plasma enterolactone concentration decreased from 28 nmol/l to a mean of 15.4 nmol/l during the first week and increased to 41.4 nmol/l on the rye bread diet (Adlercreutz unpublished data). In addition, intake of whole grain rye stimulates the formation of butyrate (a short-chain fatty acid with anticancer activity) at the time as enterolactone production is increased (Bach Knudsen et al. 2001, Avivi-Green et al. 2000). It is very important to note, that there is also evidence that high, prenatal, endogenous estrogen concentrations may increase breast cancer risk in women

Phytoestrogens and the prevention of cancer 651 (Ekbom et al. 1992; Hilakivi-Clarke et al. 1999). In addition, to date, the data is not conclusive regarding the beneficial effects of phytoestrogens in reducing breast cancer risk. Anti-breast-cancer activity in cell culture and animals Animal and culture studies provide more compelling evidence of the cancer preventing effects of phytoestrogens consumption. Messina and Loprinzi (2001) concluded that animals consuming soy in place of other proteins develop 25± 50% fewer tumors. Studies using purified isoflavones generally show protective effects against breast cancer (Ohta et al. 2000; Gotoh et al. 1998), although one study showed increasing tumorigenesis (Day et al. 2001). Genistein and daidzein have been studied extensively for anti-breast-cancer activity because of their estrogen receptor anatagonist and agonist activities. Studies by Constantinou et al. (1996) assessed the ability of genistein and daidzein injection (0.8 mg/day for 180 days) against N-methyl-N-nitrozoureainduced mammary tumors in Sprague-Dawley rats. Genistein and daidzein moderately reduced the number of tumors, but only marginally reduced the tumor incidence. Further research by this group with cultured human breast cancer cells demonstrated the inhibition of growth of estrogen receptor positive (MCF-7) or estrogen-receptor negative (MDA-MB-468) cells by 30±150 M genistein (Constantinou et al. 1998). In addition, treatment of these cells with genistein for six days with 30 M before implantation into nude mice decreased the growth of these cells in the animal. These studies suggested that the inhibition of human cancer cell growth by genistein was unrelated to the estrogenic activity of this compound. In a study conducted by Lamartiniere et al. (1995) the role of soy-containing diet in human breast cancer in early life was investigated. These researches treated neonatal rats with 5 mg genistein on days 2, 4, and 6 postpartum. They then induced mammary tumors with DMBA on day 50, and observed the delay in the development of tumors and reduction in the number of tumors in the rats that were pretreated with genistein (Lamartiniere et al. 1995). Fritz et al. (1998) concluded that the inhibition of mammary tumors by genistein is dose responsive. So et al. (1996) determined the impact of genistein on MDAMB-435, a human breast cancinoma cell line, and assessed the inhibition of cell proliferation in culture. The IC50 was highest for genistein (140 g/ml). Genistein showed inhibitory effect on breast cancer cells (Constantinou et al. 1998) and also inhibits the metastatic activity of breast cancer cells independent of its effect on cell growth. Scholar and Toews (1994) showed that daidzein also exhibits anticancer properties. Biochanin A produced greater growth inhibition than genistein in MCF-7 breast cancer cells and has potent antimutagenic activity (Peterson et al. 1996). While most studies indicate a positive effect, it is important to point out that isoflavones can also become precancerous as was found in numerous studies. Research conducted on laboratory animals showed a negative effect of phytoestrogens during pregnancy. Genistein, like estrogen, when administered

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during tumor development, enhances tumor growth of estrogen responsive tumors (Hsieh et al. 1998). It was found to enhance the proliferation of MCF-7 human breast cancer cells, and in ovariectomized athymic mice. Genistein acted as an estrogen agonist for inducing pS2, estrogen-responsive gene expression. These results suggest that caution should be used in considering cancer prevention by soybean isoflavones in humans. As previously noted, this is a particular concern because of the growing numbers of different soy and soycontaining products available to consumers as dietary supplements. The mammalian phytoestrogen lignans were also shown to affect breast cancer. Two studies by Serraino and Thompson (1991, 1992) suggested that the protective effect of flaxseed is at the initial stage of carcinogenesis. This was found by Ingram et al. (1997) when flaxseed supplementation in a basal high-fat diet reduced the epithelial cell proliferation in the female rat mammary gland, and by using the dimethlbenzanthracene rat model (Serraino and Thompson 1992). In addition, a number of studies showed that dietary treatment with flaxseed or SDG (secoisolariciresinol diglucoside) during early (Thompson et al. 1996a; Rickard et al. 1999) and late (Thompson et al. 1996b) promotion stage of mammary carcinogenesis can inhibit tumor growth (size, number or multiplicity) in rats. An interesting study was conducted on the effect of flaxseed supplementation on plasma IGF-I levels in rats treated with the carcinogen, N-methyl-Nnitrosourea (MNU) (Rickard et al. 2000). This insulin like growth factor-I (IGF-I) is important in the development of terminal end buds in the mammary glands (Kleinberg 1998). Many studies have shown an increase in the breast cancer risk in women with high level of IGF-I (Pollak et al. 1992, Peyrat et al. 1993, Hankinson et al. 1998). In N-methyl-N-nitrosourea (MNU) treated rats, flaxseed significantly reduced plasma IGF-I concentration suggesting an inverse relationship between urinary lignan levels and plasma IGF-I (Rickard et al. 2000). Flaxseed and the purified SDG seem to inhibit the growth of mammary tumors in experimental rat studies both in the initiation and promotional phase of the disease. Both tumor size and multiplicity were influenced. In addition, the oil components of flaxseed containing unsaturated fatty acids contributed to the effect (Avivi-Green et al. 2000, Knekt et al. 2000). Recently, the lignan Arctiin, which is the glycoside of arctigenin and is found in burdock seeds, was shown to inhibit chemically induced rat mammary carcinogenesis (Hirose et al. 2000). 23.3.2 Prostate cancer Prostate cancer mortality is high in the western world compared with Asian countries. It was postulated by Adlercreutz (1990) that diets in countries with low prostate cancer risk may contain high amounts of hormonally active, cancer protective compounds such as isoflavones. Recent studies support the hypothesis that high soy intake prevents prostate cancer. It was shown that a reduced cancer risk is related to phytoestrogen intake from soy foods (Adlercreutz et al. 2000). Indeed, a study showed that consuming soy milk more than once a day is

Phytoestrogens and the prevention of cancer 653 protective against prostate cancer (Jacobsen et al. 1998). In addition, Japanese men who consumed large amounts of soy products have lower prostate weights than western men of similar age (Oesterling et al. 1995). These observations are supported by the data from a study including 617 prostate cancer cases from Canada (Jain et al. 1999). Similar results were obtained in two cohort studies from the U.S., both showing significant beneficial effects of soybean product consumption on prostate cancer risk (Severson et al. 1989, Jacobsen et al. 1998). In lignans, studies conducted on population groups showed that Portuguese men have higher levels of enterolactone (162 ng/ml) in prostatic fluid compared with British men (20.3 ng/ml) and Hong Kong men (31.0 ng/ml) (Morton et al. 1997). However, the mean plasma concentrations of enterolactone from the three centers were similar, at 6.2, 3.9 and 3.9 ng/ml in samples from Hong Kong, Portugal and Britain, respectively. The incidence of prostate cancer in Portugal is higher than in Hong Kong, but is half that of Britain. The effect of the lignans is then unclear. Recently, 25 patients with prostate cancer who were awaiting prostatectomy were fed a low-fat, flaxseed-supplemented diet (DemarkWahnefried et al. 2001). Nutrition is apparently a major factor in the development and progression of prostate cancer. Based on experimental studies and epidemiologic data mainly from case control studies or cohort studies, it may be suggested that intake of phytoestrogen could yield a decrease in prostate cancer incidence. Inhibition of prostate cancer in cell culture and animals The inhibition of prostate cancer growth and development was investigated in several animal and cell culture studies. Studies conducted on inhibition of tumor growth by diets containing soy flour were reported by Zhang et al. (1997). Feeding 33% of the diet (by weight) as soy flour resulted in a 30±40% reduction in the growth of transplanted Dunning R3327 prostatic adenocarcinoma in rats. In addition, studies with cultured prostate cancer cell lines suggested that genistein was cytotoxic to the rat prostate cancer cell line MAT-lylu and the human prostate cancer cell line PC-3. However, genistein added to the drinking water (0.07±0.285 mg/kg/day) failed to inhibit the growth of MAT-lylu cells implanted into rats (Naik et al. 1994). Soybean isoflavones inhibited methylnitrosourea (MNU)-induced prostate seminal vesicle adenocarcinomas in rats by feeding high isoflavones (1.69 mg/g) supplemented soy diet before the initiation, and was compared with the same diet low in isoflavones (Pollard and Luckert 1997). Studies using the 3,20 -dimethyl-4-aminobiphenyl and testosterone propionate model in rats indicated that feeding a soybean isoflavone mixture containing 74% genistein and 21% daidzein, at total doses of 100 and 400 ppm, reduced the incidence of adenocarcinoma in the prostate by 50% compared with rats fed control diet (Onozawa et al. 1999). The extent of the anti-prostate-cancer effect of phytoestrogens depends on the nature of the specific phytoestrogen being tested. A study on the effect of isoflavones and lignans on human prostate cancer cells in culture (LNCaP, DU145, and PC cell lines) (Aldercreutz et al. 2000) concluded that the most

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effective phytoestrogen was 40 -methylequol. When daidzein metabolite, equol, was administered to human prostate cancer cell line (LNCaP) it was observed that 80 M of equol decreased the proliferation by 50% (Aldercreutz et al. 2000). Studies of the tumors revealed that apoptosis was increased and angiogenesis was inhibited. Another important study showed that genistein inhibits the expression of the epidermal growth factor receptor in rat prostate (Dalu et al. 1998). A variety of other phytoestrogenic compounds were examined in relation to prostate cancer. Diets rich in lycopene from tomato products have been associated with a decreased risk of prostate cancer (Boileau et al. 2000). The consumption of green and black tea in large amounts in Asia may explain lower risk for prostate cancer. In green and black tea there is a large concentration of polyphenols, e.g., quercetin and epigallocatechin gallate, which are considered to be phytoestrogens and may have an inhibitory effect on prostate cancer. Epidemiological studies showed that high consumption of polyphenols in green tea resulted in a low risk of prostate cancer (Conney et al. 1995). In addition, green tea polyphenols and epigallocatechins inhibit the growth of prostate cancer call lines including DU145 (Ahmad et al. 1997), CWR22 (Carlin et al. 1996) and the mouse model including PC-3 (Liao et al. 1995) and LNCaP 194-R (Liao et al. 1995). Cumulative lab experiments seem to provide substantial evidence of phytoestrogens being a factor in reduction of prostate cancer risks in animal and cell culture studies. These are in agreement with most of the epidemiological and case studies reported in the literature, thus suggesting that the consumption of dietary phytoestrogens may be helpful in reducing the risk of prostate cancer. 23.3.3 Colon and gastric cancer The available data on the effect of phytoestrogens on colon and gastric cancers are conflicting and the evidence for the protective effect of the phytoestrogens is not clear. Interestingly, a case control study of gastric cancer in China (Hu et al. 1998) found that consumption of fermented and salted soy paste was associated with increased occurrence of gastric cancer. On the other hand, genistein was found to inhibit AOM-induced colonic ACF at doses of 75 and 150 mg/kg (Pereira et al. 1994). Studies with soy flakes, soy flour, genistein, and Ca++ showed that soy flakes, soy flour, and genistein reduced ACF, and that genistein (0.015%) caused the greatest reduction (Thiagarajan et al. 1998). In addition, rye bran, flaxseed, and lignans have an inhibitory effect on colon cancer or formation of colon polyps (Davies et al. 1999, Mutanen et al. 2000). In ApcMic mice, inhibition of colon and caecum tumor incidence was 33% with rye bran, 75% with wheat bran, and 88% with oat bran (Mutanen et al. 2000). Davies et al. (1999), who examined the effect of soy and rye diet supplementation to rats on colon tumor incidence, suggested that soy isoflavones have no effect on the colonic tumors while rye bran supplementation decreased the frequency of colon

Phytoestrogens and the prevention of cancer 655 cancer. However, it was demonstrated that 20% by weight of dietary soy protein significantly reduced rat intestinal mucosa levels of polyamine, a biomarker of cellular proliferation for colorectal cancer risk (Fournier et al. 1998). Genistein and daidzein affected polyamine levels in a study conducted by Wang and Higuchi (2000). In conclusion, isoflavones and lignans may have some protective properties against colon cancer, and there is some evidence of their ability to inhibit colon cancer development in animal models. 23.3.4 Lung cancer and melanoma A study that was conducted in Finland between 1967 and 1991 investigated the association between flavonoid intake and human cancer (Knekt et al. 1997). The incidence of cancer at all sites was inversely associated with flavonoid intake, and this association was primarily due to the lower rates of lung cancer in the groups with the highest flavonoid intake. The protection was greatest in individuals who were under 50 years of age and in non-smokers (Knekt et al. 1997). Garcia-Closas et al. (1998) assessed the dietary intake of four flavonoids (quercetin, kampherol, myricetin, and luteolin) in relation to lung cancer in Spanish women. Phytoestrogens and lung cancer showed only weak association. Lesser rates of lung cancer were found in Chinese women in Hong Kong with greater consumption of leafy green vegetables, carrots, tofu, fresh fruit, and fresh fish (Koo 1988). Skin tumorigenesis was inhibited by genistein (Wei et al. 1998), which reduced tumor incidence and multiplicity. A long-term study by Li et al. (1999) examined the antimetastatic potential of genistein and daidzein using murine melanoma cells injected to mice. In mice fed diets containing isoflavones for two years before and after intravenous injection of melanoma cells, the isoflavone diet inhibited the number of lung metastases in a dose-dependent manner.

23.4

Mechanisms of action of phytoestrogens

The role of isoflavonoid phytoestrogens in cancer prevention is often categorized as estrogenic and antiestrogenic activity, antiproliferative activity, and the inhibition of various enzymes at different levels. Another plausible mechanism for the anticancer activity of various phytoestrogens is their antioxidant activity. This mechanism, however, is not specific to phytoestrogens and will not be discussed in this chapter. 23.4.1 Estrogenic activity Some of the therapeutical functions of isoflavones may involve an estrogenicrelated mechanism. This claim is supported mostly by their recorded physiological effects. These include preventing osteoporosis, improving

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postmenopausal symptoms, and lowering cholesterol (Clarkson et al. 1995, Adlercreutz et al. 1995, Kurzer and Xu 1997, Anderson and Garner 1997, Setchell 1998, Brzezinski et al. 1997, Anderson 1999, Lichtenstein 1998). Despite the fact that their estrogenic potency is extremely weak (10ÿ3 to 10ÿ5 fold less that estradiol (Davis et al. 1998)), isoflavones showed estrogenic agonist activity in animal models (Moule et al. 1963, Farnsworth et al. 1975). This estrogenic potency may be explained by their high circulating concentrations, which is up to 100-fold greater than estradiol. The estrogenic activity of some flavonoids and isoflavones was ordered by Miksicek (1995) as genistein > kampherol > naringenin > apigenin > daidzein > biochanin A > formononetin > luteolin > fisetin > catechin > hesperatin. One possible route for the interference of these compounds with estrogen activity is through competitive binding to the estrogen receptors. As previously mentioned, the affinity of isoflavones and flavonoids is up to 1% of the binding affinity of estradiol (Shutt and Cox 1972). However, an indication for their binding to estrogen receptors was demonstrated by the finding that the inhibition of cell proliferation by genistein in MCF-7 cells was reversed by 17 -estradiol (So et al. 1997). Among estrogen receptors, apparently, phytoestrogens prefer the ER receptor subtype (Kuiper et al. 1997). This observation was strengthened by the good fit of genistein to the hydrophobic core of the ligand-binding domain of ER (Pike et al. 1999). It can be concluded that direct competition of dietary phytoestrogens with estrogen is likely only when their circulating concentrations are high. 23.4.2 Antiproliferative activity In many studies, phytoestrogens such as soy isoflavones exhibit inhibitory effect on cell proliferation. This may be a plausible mechanism for their anticancer properties, as cancer prevention is all about inhibition, prevention and cessation of hyperproliferation. Evidence on such activity was reported in cell lines meningioma (Piantelli et al. 1993), colon cancer cells (Kuo 1996), breast cancer (Le Bail et al. 1998), and lung cancer (Kawaii et al. 1999) cells. Interestingly, regardless of the specific phytoestrogen being tested, the concentration at which these compounds were active was at the tens of M. It should also be noted that the data on inhibition of cell proliferation in vivo is scarce. The antiproliferative activity indicates an interference of these compounds with the cell cycle, and probably also induction of apoptosis. Genistein induced cell cycle arrest in human myelogenous leukemia and lymophocytic leukemia (HL-60 and MOLT-4) cell lines (Traganos et al. 1992). Such activity at the G2/ M was also reported in gastric cancer cells (Matsukawa et al. 1993). Induction of apoptosis was reported for genistein and other isoflavones in lung cancer, bladder cancer, prostatic cancer, and leukemia cell lines (Zhou et al. 1998, Kyle et al. 1997, Spinozzi et al. 1994). The cell cycle arrest in non-small-cell lung cancer cell line was induced by up-regulation of p21 and apoptosis induction (Lian et al. 1998).

Phytoestrogens and the prevention of cancer 657 It should be noted, that the antiproliferative activity of phytoestrogens is concentration dependent. At low concentrations up to 10 M genistein stimulates cell growth, whereas at higher concentrations of 10±100 M it inhibits cell growth (Zava et al. 1997, Wang et al. 1996, Peterson and Barnes 1991). Since the proliferative effect of genistein was not expressed in ER negative cells, it can be suggested that this activity of genistein is mediated by the ER receptor. Induction of cell proliferation, and of the expression of the estrogen responsive gene pS2 were detected in response to treatment of MCF-7 cells with genistein (1 M) (Hsieh et al. 1998). All these observations and many others, support the notion that at low concentrations the proliferative effects of genistein are ER-mediated. Similar observations were also made for daidzein, coumestrol, and biochanin A (Verna and Goldin 1998, Verna et al. 1997). Growth inhibition was observed also in ER-negative cells (Wang and Kurzer 1997). It may be suggested that while the proliferative effect is ER mediated, the antiproliferative activity of genistein, daidzein, coumestrol and biochanin A on MCF-7 cells is not mediated by the ER receptor. 23.4.3 Inhibition of enzymes The interference of phytoestrogens with the development of cancer may also be attributed to the inhibition of enzymes involved in the development of the carcinogens, and enzymes that contribute to the development of cancer. Often the carcinogenic form of a carcinogen depends on its metabolism. Partial activation of carcinogens is being performed via oxidative metabolism by enzymes such as cytochromes P450 (Talalay 1989). The route for the detoxification of the oxidized carcinogens is detoxification by additional enzymatic system. These enzymes convert the carcinogen into an inert form, or one which is easily excreted (Talalay et al. 1995). Dietary flavonoids and isoflavonoids induce detoxification enzymes in cells. NAD(P)H:quinone reductase (QR) protects cells against mutagenicity and carcinogenicity resulting from free radicals and toxic oxygen metabolites (Ernster 1967), and GST detoxifies a number of carcinogenic electrophiles (Chasseaud 1979). Dietary antioxidant flavonoids, such as quercetin (50±100 M in cultured cells or 1% in murine diet) and epicatechin (25±100 M) induce these enzymes (Gordon et al. 1991, Benson et al. 1980, Rodgers and Grant 1998, Nijhoff et al. 1993). A dosedependent induction in QR enzyme activity up to 6- to 8-fold after addition of genistein and up to 2- to 3-fold induction with biochanin A was reported in a human colonic Colo205 cell line (Wang et al. 1998b). Other phytoestrogens affect cancer development by modulating the activity of enzymes involved in estrogen synthesis. The two mammalian lignans, enterodiol (END) and enterolactone (ENL), are inhibitors of several steroid metabolizing enzymes, such as aromatase, 5-reductase, 7-hydroxylase and 17hydroxysteroid dehydrogenase. These are produced by intestinal bacteria from plant lignans. It is also known that some flavones, flavanones, isoflavones, isoflavanones and -naphthoflavones inhibit human estrogen synthetase

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(aromatase) (Kellis et al. 1984, 1986; Ibrahim and Abul-Hajj 1990). This aromatase catalyzes the conversion of androgens to estrogens in many tissues (Kellis and Vickey 1987). Its role in estrogen synthesis makes this enzyme important in hormone dependent cancer such as breast cancer. Initially, Adlercreutz et al. (1993) and later also Wang et al. (1994) reported that enterolactone (ENL) is a moderate inhibitor of aromatase, while enterodiol (END) is a somewhat weaker aromatase inhibitor. Lignans and isoflavonoid phytoestrogens inhibited 5-reductase in human genital skin fibroblast monolayers, and in benign prostatic hyperplasia tissue homogenates (Evans et al. 1995). Of all known phytoestrogens, genistein is probably the most investigated enzyme inhibitor. It is mostly known as an inhibitor of various kinases. The activity of these kinases is enhanced in breast cancer. Genistein, and also daidzein, inhibit protein kinase C (PKC). This may lead to antiproliferative effect (Hilakivi-Clarke et al. 1999). Genistein also affects cell survival in adhesion-dependent cells by the inhibition of tyrosin kinase (Fu et al. 1999; Yokoshiki et al. 1996), and interferes with breast cancer development by the inhibition of src-family kinases (Clark et al. 1996).

23.5

Future trends

The role of phytoestrogens in the food market is bound to increase with the continuous rise of the food-for-health market. One can only expect a parallel increase in knowledge-based foods and food ingredients, that will meet the ever more knowledgable consumer. To meet these requirements, more in-vivo evidence should be gathered on the health benefits related to phytoestrogens. Such studies should deal not only with the food, but also with the isolated bioactive compounds where data is still needed. Establishing databases and standards for phytoestrogen content in raw materials and final products is also essential. These should be supported by a unified approach for analysis and quantification of active ingredients. Being a key family of ingredients of the functional foods and nutraceuticals products, phytoestrogen delivers a promise of health to the consumer. Such chemicals often have a `medicine' image. It is therefore extremely important to ensure that the health attributes of phytoestrogens are being delivered and are not affected by the processing and storage conditions of the food. Changes such as degradation or chemical modification of phytoestrogens may render their biological activity. Further study on the stability of phytoestrogens and how it is related to their biological impact is therefore greatly needed.

Phytoestrogens and the prevention of cancer 659

23.6

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24 Food phenolics and cancer chemoprevention F. Shahidi, Memorial University of Newfoundland, Canada

24.1

Introduction

A number of epidemiological studies have demonstrated the relationship between diet and cancer and have provided evidence that consumption of plant foods protects against various types of cancer (Wattenberg, 1992). The protective effects were first attributed to the antioxidative effects of vitamins C and E and -carotene. In this connection, it was found that -carotene, as such, had the opposite effect in long cancer incidences in smokers (Omenn et al., 1996). Hence, it was later recognized that the observed effects were due to the presence of a cocktail or soup of phytochemicals present and in that phenolic compounds played a major role. The intervention of phenolics is generally with a specific stage or several stages of the carcinogenic process. The phenolics involved may exert their effects by ameliorating oxidative stress, while some may target intracellular signaling molecules or events while others act as antitumor agents. Inhibition of certain enzymes or their upregulation may be involved as well as subsequent suppression of activation, such as that of NF-B. Induction of apoptosis of cancer cells, inhibition of angiogenesis and proliferation of cancer cells, among others, might also be responsible for the chemopreventive effects of various phenolics. Many of these effects inhibit overexpression of cyclooxygenase-2 (Cox-2) and hence cancer.

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24.2 Functional properties of plant phenolics and polyphenolics Phenolic compounds are the most widely distributed secondary metabolites in plants and constitute several thousands of compounds. They are involved in the defence operations that help protect plants against herbivores, pathogens and insects. They also serve as attractants for pollinators and animals and protect the plants from UV light and free radical stress as well as serving as wound-healing agents. They also contribute to the variety of color and taste of foods. Structurally, phenolics are derived primarily from phenylalanine, and in a small number of plants from tyrosine, via the action of ammonia lyase. These compounds generally contain at least one aromatic ring and one (phenols) or more (polyphenols) hydroxyl groups. The first products from phenylalanine and tyrosine are trans-cinnamic and p-coumaric acids, respectively, and these are known as phenylpropanoids (C6±C3 compounds). Further hydroxylation, via the action of hydroxylase, or methylation, via the action of O-methyl transferase, produces other C6±C3 compounds such as caffeic acid, ferulic acid and sinapic acid, among others (Fig. 24.1). Meanwhile, loss of a two-carbon moiety from phenylpropanoids leads to the formation of benzoic acid (C6±C1) derivatives such as p-hydroxybenzoic acid, protocatechuic acid, vannilic acid, syringic acid and gallic acid (Fig. 24.1). Condensation of C6±C3 compounds with malonyl coenzyme A affords chalcones or stilbenes, such as resveratrol, which may subsequently cyclize under acidic conditions to produce flavonoids, isoflavonoids and related compounds (Fig. 24.2). There are over 2,000 naturally occurring flavonoids, amongst which quercetin and rutin are most widely distributed and are present in tea, coffee, cereal grains and a variety of fruits and vegetables. Among flavonoids, anthocyanins and catechins, known collectively as flavans because of lack of the carbonyl group on C-3, are important; flavan-3 ols and flavans-3,4-diols belong to this category. Anthocyanins are glycosidicaly bound anthocyanidins. Anthocyanins are water-soluble pigments responsible for the bright red, blue and violet colors of fruits, flowers and foods (Mazza and Miniati, 1994). Thus, the bright red skins of radishes, red skins of potatoes, the dark skin of egg plants and the color varieties in different berries, cherries, grapes, pomegranate and plums are due to the presence of anthocyanins. Condensation of flavonoids, especially flavan-3-ols, leads to the formation of condensed tannins or proanthocyanidins. Over 50 procyanidins ranging from dimer to hexamer have been identified (Hemingway, 1989). The consecutive units of condensed tannins are linked through the interflavonoid bond between C-4 and C-8 or C-6. Their molecular weight is often in the range of 2000±4000 Da (Macheix et al., 1990). Hydrolyzable tannins are another group of high-molecular-weight phenolics that are glycosylated gallic or ellagic acid. Based on their hydrolysis products, hydrolyzable tannins may be referred to as gallotannins or elagitannins. Several phenolic acids are attached to the same sugar molecule and the molecular weight of the resultant product varies between 500 and 2800 Da (Haddock et al., 1982).

Food phenolics and cancer chemoprevention

671

Fig. 24.1 Biosynthesis of phenylpropanoids (C6±C3) and benzoic acid derivatives from phenylalanine and tyrosine. PAL, phenylalanine ammonia lyase; TAL, tyrosine ammonia lyase.

Phenolic compounds in foods occur mainly in their conjugate form, that is esterified or etherified, and only partially in the free form. Thus, phenylpropanoids which occur predominantly in grains and cereals are often esterified while flavonoids which are dominant in fruits occur as glycosides. In this relation, biological activity of compounds involved might be different from those examined in in-vitro systems. Hence it might be necessary to subject

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Fig. 24.2 Biosynthesis of flavonoids (C6±C3±C6) and condensed tannins.

phenolics to hydrolysis prior to their evaluation. Metabolites of phenolics in the body might also have better or compromised activity compared to those consumed. We consume some 3.5 kg of oxygen on a daily basis. A small portion of this oxygen leads to the formation of oxidation products and reactive oxygen species (ROS) in the body that remain un-neutralized. ROS, if not neutralized, may be the culprit in a number of diseases and tissue injuries such as those of the lungs, heart, kidneys, liver, gastrointestinal tract, blood, eyes, skin, muscles and brain as well as the aging process (Fig. 24.3). While antioxidants and antioxidant enzymes in the body are responsible for preventing damage from ROS, upon illness and during infancy or due to aging, the neutralization process may not be adequately addressed. Thus, augmentation of endogenous antioxidants through dietary means might prove beneficial in preventing lipid oxidation, protein cross-linking and DNA mutation, among others (Shahidi, 1997). 24.2.1 Carcinogens and anticarcinogens Carcinogenicity is a multistep process in which molecular and cellular alterations occur. These include tumor initiation, promotion and progression. Similarly, each type of cancer is a multi-factorial disease which requires


Fig. 24.3

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Diseases and damages caused by reactive oxygen species.

identification of factors responsible for the initiation of the process, presumably genotoxic agents, and those that enhance the process, namely cocarcinogens or promoting agents. This may also be achieved by preventing the formation of carcinogens, preventing access of carcinogens to critical tissue target or acting after exposure to carcinogens. Initiation is a rapid and irreversible process that includes a series of extra- and intracellular events. In this, exposure to or uptake of a carcinogen, its distribution or transport to target tissues or organs where metabolic activation and detoxification can occur and the subsequent interaction/reaction with target cell DNA are necessary in order to lead to genotoxic damage. On the other hand, tumor promotion is a lengthy, but reversible process in which proliferating preneoplastic cells accumulate. Finally, progression is the final stage in which neoplastic transformation leads to the growth of a tumor with invasive and metastatic potential. Wattenberg divides chemopreventive agents to blocking agents and suppressing agents (Wattenberg, 1983, 1985, 1992). While blocking agents prevent carcinogens from reaching the target site and from undergoing metabolic activation with subsequent interaction with DNA and other biomolecules, suppressing agents inhibit promotion and progression of malignant transformation of initiated cells (Surh, 2003). Phytochemicals, including phenolics, generally block or reverse the initiation and promotion of carcinogensis. However, recent advances have clearly shown that numerous cellular molecules and events could be potential targets for chemopreventive agents in a more complex manner than that proposed by Wattenberg (1985); the effects might indeed be the outcome of a combination of several sets of intracellular effects than a single biological response (Surh, 2003).

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24.3 The role of phenolic compounds in the prevention of cancer Phenolic compounds are well known to protect cells and their components against cancer development. Phenolic compounds are generally good antioxidants, thus they afford protection against tumor promotion by inhibiting oxidative stress induced by tumor promoters (Agarwal and Mukhtar, 1993; Perchellet and Perchellet, 1989; Gali et al., 1991). Simple phenolic acids and tocopherols are known to act as potent inhibitors of formation of carcinogens such as N-nitroso compounds (Kuenzig et al., 1984). Meanwhile, inhibition of benzo [a] pyrene-induced neoplasia in the forestomach of mice fed various plant phenolics was reported by Wattenberg et al. (1980) and Wattenberg (1992). Chromosomal aberrations induced by polycyclic aromatic hydrocarbons were inhibited by caffeic acid, while chlorogenic acid and other phenolics blocked chemically-induced carcinogens in the large intestine, stomach and colon of hamsters and/or rats (Tanaka et al., 1990; Morishita et al., 1997; Shimizu et al., 1999). Antitumor-promoting activity of ellagic acid and quercetin was also reported (Chang et al., 1985). In addition, several phenolics such as salicylic acid and quercetin were found to inhibit the cydooxygenase pathway to prostaglandins and other prostanoids (Dehirst, 1980). Arachidonic acid metabolism modulation and inhibition of cyclooxygenase appear to affect promotion rather than initiation of carcinogenesis. Thus, plant phenolics act as modulators of arachidonic metabolism by inhibiting lipoxygenase and hence as inhibitors of cancer promotion. Table 24.1 summarizes difficult food phenolics with chemopreventive effects and their source. Inflammation and ROS also appear to play important roles in tumor promotion. Phenolics, such as quercetin, have been found to inhibit 12-0tetradecanoyl phorbol-13-acetate (TPA)-induced mouse skin inflammation (Wang et al., 1991). Quercetin, rutin and other flavonoids are known to inhibit the generation of superoxide anion by neutrophils (Hoeman, 1989; Walaszek, 1990). Tea polyphenols were reported to inhibit over 90% of mutagenicity of Table 24.1 Selected phenolic compounds with chemopreventive effect Phenolics

Source

Benzoic acid derivatives Catechins Capsaicinoids (capsaicin) Curcuminoids (curcumin) Ellagic acid Flavonoids Hesperidin Isoflavones (Genistein) Phenylpropanoids Stilbenes (Resveratrol)

cereals, blueberries, cranberries, oilseeds, etc. teas, berries, etc. pepper fruits, etc. turmeric, curry, etc. grapes, strawberries, raspberries, walnuts, etc. fruits, vegetables, etc. citrus fruits, etc. soybeans, legumes, etc. cereals, apricots, berries, carrots, cherries, etc. red grapes skin, red wine, etc.


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heterocyclic amines, benzo [a] pyrene and aflatoxin B1 (Yen and Chen, 1994). Imai et al. (1997) and Nakachi et al. (1998) reported that increased consumption of green tea reduced the risk of breast cancer metastasis and recurrence in Japanese women. The (-)-epigallocatechin-3-gallate (EGCG) from green tea reduced the incidence of tumors of liver, stomach, skin, lungs and esophagus in experimental animals (Huang et al., 1992). EGCG caused cell cycle dysregulation and apoptosis of cancer cells mediated through inhibition of NF-B (Ahmad et al., 2000a,b). Pan et al. (2000) reported that theasinensin A and mixtures of theaflavin-3-gallate and theaflavin-30 -gallate exhibited strong growth inhibitory effects against human hystolytic lymphoma U937. Yang and Chung (2000) demonstrated that effective levels of tea polyphenols needed for imparting signal transduction, inhibiting cell proliferation and inducing apoptosis of cancer cells are higher than those detected in the blood and tissues. It was also reported that epicatechin gallate inhibits invasion of highly metastatic human fibrosarcoma HT1080 cells in the absence of cytotoxicity (Maeda-Yamamoto et al., 1999). Green tea polyphenols also reduced the incidence and average tumor yield in rats and inhibited promotion stage of azoxymethane-induced (AOM) colon carcinogenesis (Kim et al., 1994). The effects of citrus flavonoids on poliferation, growth and viability of human breast cancer cells were investigated (So et al., 1997). Inhibition of tumorigenesis was more effective for orange juice as compared to grapefruit juice. The existing differences were attributed to the type of flavonoids present. Auraptene found in citrus fruit peel was found to exhibit chemopreventive activity in mouse skin (Murakami et al., 1997), rat colon (Tanaka et al., 1997), rat tongue (Tanaka et al., 1998) carcinogensis models. Flavonones displayed protective effects against cancer (Koyuncu et al., 1999). Isoflavones exert a broad spectrum of biological activity with genistein being the most potent inhibitor of cancer cell growth and daidzein and biochanin A displaying weaker inhibitory effects (Peterson and Barnes, 1991, 1993). However, isoflavone glucosides, namely genistin and daidzin, had little effect on the growth of cancer cells. Genistein was also found to inhibit human prostate and mammary cancer cell lines by reducing phosphorylation and downregulation processes, respectively (Davis et al., 1999, Gong et al., 2003; and Li and Sarkar, 2002). Curcuminoids, especially curcumin, were found to display anticancer, antimutagenic and anti-inflammatory activities (Brouet and Ohshima, 1995; Chang and Fong, 1994; Lin et al., 1994). Simon et al. (1998) evaluated curcumin, demethylcurcumin and bisdemethylcurcumin against human breast cancer cells and found that demethylcurcumin displayed the best inhibitory effect, followed by curcumin and bidemethylcurcumin. Curcumin was also an effective inhibitor of chemically induced tumor promotion (Lin et al., 1994) and induces apoptosis (Anto et al., 2002; Bharti et al., 2003). Resveratrol was found to prevent proliferation in tumor cell lines in vitro (Jang et al., 1997; Hsieh and Wu, 1999) and also decreased tumor growth in a rat tumor model (Carbo et al., 1999). Resveratrol was also found to induce

676


apoptosis in fibroblasts after induced expression of oncogens (Holmes-McNary and Baldwin, 2000). However, the first report of an inhibitory effect of resveratrol on transcription and activity of Cox-2 was reported by Subbaramaiah (1998). Suppression of dimethyl benzanthracene (DMBA)-induced mammary tumorigenesis via suppression of activation of NF-B has also been demonstrated (Banerjee et al., 2002). Capsaicinoids, namely capsaicin and its related products, are known for treatment of inflammation and several studies have shown that they may inhibit the metabolism and carcinogenicity of chemical carcinogens (Surh et al., 1998). In addition, their induction of apoptosis and removal of carcinogen has been well documented (Moore et al., 1995; Surh 2002). Ellagic, protocatechuic and chlorogenic acids, found in a wide range of fruits and vegetables, have been shown to serve as potential chemopreventers against several carcinogens (Tanaka et al., 1992). Repeated oral administration of protocatechuic acid was found to inhibit the growth of colon and oral cancers in rats (Tanaka et al., 1993; Ueda et al., 1996). Meanwhile topical application of protocatechuic acid effectively inhibited growth of tumor in mouse skin (Nakamura, 2000).

24.4

Future trends

Chemoprevention through consumption of dietary phytochemicals is becoming readily acceptable. This is an economical means to control cancer. However, little is known about the mechanism(s) of action of phytochemicals in cancer prevention. While the cocktail or soup of phytochemicals is expected to be responsible for their health benefits, their effects may be exerted at several stages during carcinogensis and via a combination of mechanisms. The absorption and bioavailability of food phenolics, their gastrointestinal chemical alterations and formation of metabolites deserve attention. The metabolites of phenolics might exert superior or compromised effects compared to those, as such, from dietary sources. Therefore the effects of metabolites and their rate of formation/disappearance need to be investigated. In relation to nutragenomics, i.e., the effects of dietary phenolics on gene expression, special attention might also prove to be important in future studies.

24.5


American Institute for Cancer Research. 1996. Dietary Phytochemicals in Cancer Prevention and Treatment. Advances in Experimental Medicine and Biology. Plenum Press, New York, NY. Naczk, M. and Shahidi, F. 2003. Phenolic compounds in plant food: chemistry and health benefits. Nutraceuticals & Food. 8: 200±218. Surh, Y.-J. 2003. Cancer chemoprevention with dietary phytochemicals. Nature Rev. 3: 768±780.


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24.6

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25 Vitamins and the prevention of cancer C. A. Northrop-Clewes and D. I. Thurnham, University of Ulster, UK

25.1

Introduction

There are thirteen vitamins and all play a role in intermediary metabolism. Four of them are fat-soluble vitamins and the rest are water-soluble, and while they can be categorised by simple physical characteristics, their functions are very different. Two of the fat-soluble vitamins are described as hormones, vitamins A (Ross and Ternus 1993) and D (Stumpf 1988), since both react directly with response elements at the level of the gene, stimulating transcription of specific mRNAs to promote a variety of functions depending on the genes which are stimulated. In contrast the majority of the water-soluble vitamins are involved in enzyme activity, facilitating enzymic action by acting as coenzymes or redox stabilisers to enable optimal enzyme activity. In order for normal metabolism to be maintained and for the organism to remain free from the specific deficiency diseases, such as beriberi or scurvy, there are minimum dietary requirements for all the vitamins. However, dietary requirements vary between different individuals and the concept of minimum requirement is not very useful for health maintenance. A more useful standard is the recommended dietary allowance (RDA) and RDAs have been calculated for most of the vitamins by many national and international health bodies. In general the RDA is calculated such that it represents an amount of nutrient that should be sufficient to meet the metabolic needs of 95% or more of the population. Thus somebody who consumes an RDA of a specific vitamin can be said to have a very low risk of being unable to meet their metabolic needs for that vitamin. The further an intake deviates below the RDA, the greater is the risk that the person may not meet their dietary requirements and they may show evidence of deficiency.

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Specific circumstances may make somebody more vulnerable to deficiency through an increase in their requirements. During sickness, increased metabolic demands will increase requirements for those nutrients involved in energy generation or redox protection, there may be increased losses of nutrients in urine and of course in severe illness, appetite is decreased so intake reduces. The increased intake of certain drugs may impair absorption of nutrients in a variety of ways as well as increasing urinary losses. Also individual lifestyles can influence nutrient requirements. Excessive intake of alcohol can both displace the normal consumption of a mixed diet as well as specifically block the absorption of thiamin. Smoking too interferes with normal dietary intake and alters metabolism of nutrients. Functional foods in the case of vitamins would be foods that are especially rich in one or more vitamins, either naturally through specific breeding of new varieties or via fortification. The enhanced nutrient content may make a food specifically suitable for people with increased requirements to ensure that deficiency does not arise. In general with nutrient supplements, the larger the nutrient content, the lesser amount of the supplement that is absorbed. However, foods rarely contain amounts of nutrients in such large amounts that they might seriously impede the absorption process. In fact, the combined administration of food and nutrient slows the absorption process but often makes it more efficient and manageable by the body and reduces the risk of toxicity. The other prospective use of functional foods is to increase dietary intake above the RDA to try to optimise nutrient availability. Optimal nutrition is a difficult concept to define. Proponents of optimal nutrition suggest that high or super-adequate intakes of nutrients will enable the body to better cope with diseases like cancer and cardiovascular disease. These ideas originate from the many epidemiological studies that have shown people who consume fruits and vegetables on a regular basis have lower risks of chronic diseases. Fruits and vegetables are rich sources of vitamins like folate, vitamins C and E, provitamin carotenoids, minerals and many hundreds of polyphenolic compounds that have important antioxidant properties. That is, somebody whose nutrient requirements are always 100% saturated will have a lower risk of chronic diseases. The evidence from supplementation studies in support of these ideas is not very encouraging but proponents argue that such studies are usually begun too late in life, when recipients may already have sub-clinical chronic disease. Furthermore, supplements used in such studies are restricted to one or two components or a mixture of the main vitamins and minerals. Therefore, the supplementation studies that have been done to date are flawed as they start too late and frequently only òptimise' a restricted number of micronutrients. Vitamins have unique functions but inadequacies of one will affect the metabolism of others and optimal status requires adequacy in all nutrients. Evidence for the preventive effects of nutrients against cancer is obtained from epidemiological studies and includes the following types: case-control, cohort and nested case-control studies. In case-control studies, the diet of individuals with cancer is compared with that of matched persons from the

Vitamins and the prevention of cancer 683 general population. There are many problems with this type of study not the least of which is the difficulty in assessing dietary habits many years in the past. In a cohort study, a group of persons whose diet has been ascertained (and sometimes blood collected as well) is followed up over a number of years with respect to disease incidence. Dietary characteristics of subjects who develop cancer is later compared with that of those who did not develop cancer. Or in the case of the nested cohort studies, with a smaller group of matched subjects within the cohort who did not develop cancer. A major problem with these studies is that they are expensive to undertake, as tens of thousands of subjects must be followed for many years to generate enough cancer cases to provide statistical power. Additionally, evidence for cancer-preventive effects of specific nutrients is sometimes obtained by intervention studies. Usually large amounts of potentially beneficial nutrients or placebo are administered preferably in a blinded fashion so that neither operator nor subject know their treatment. These studies very often involve thousands of participants who may be treated from a few months to several years. Participants in such studies very often have a higher risk of specific cancers as indicated by premalignant lesions or the previous removal of a benign cancer. Finally, studies have sometimes been done on self-selected persons, for example, persons regularly consuming elevated amounts of micronutrients and risks of cancer occurrences compared with those of the general population. Both the last two types of study might provide evidence of cancer-preventive benefits from elevated intakes of nutrients, i.e., the òptimal' concept but there are difficulties in relating such results to the general population. This chapter will examine the role of vitamins in the prevention of cancer. There are an enormous number of studies reporting associations between cancers and nutrient status. Ames (2001) has argued that a deficiency of any of the micronutrients ± folic acid, vitamin B12, niacin, vitamin C, vitamin E, iron or zinc ± mimics radiation in damaging DNA by causing single- and doublestrand breaks, oxidative lesions, or both. He has suggested that somewhere between 2 and 20% of the US population have low intakes (13 nmol/L) and red cell folate concentrations of 0.16 mol/L (normal >0.35 mol/L). Following folate supplementation, minimum spontaneous micronucleus frequencies were obtained only when serum folate reached concentrations between 34±45 nmol/L; three times the usual threshold for normality. In another study, elevated micronucleus frequencies in 122 splenectomised subjects were associated with serum folate concentrations

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