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]

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

Chapter 10

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

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

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

xvii

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

Chapter 14 Chapter 11 Dr F. Foufelle and Professor P. 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]

Tel: +44 8059 4223 Fax: +44 8059 5489 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

Functional foods, ageing and degenerative disease

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

Regulatory context in the EU Table 1.1

3

Membership of the European Union

EU member states prior to May 2004

Additional EU member states from May 2004

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

Czech Republic Cyprus Estonia Hungary Latvia Lithuania Malta Poland Slovakia Slovenia

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

4

Functional foods, ageing and degenerative disease

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

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

Regulatory context in the EU

5

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

6

Functional foods, ageing and degenerative disease

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

Regulatory context in the EU

7

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

1.3

EU food law and regulation of food health claims

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

8

Functional foods, ageing and degenerative disease

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

Regulatory context in the EU

9

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

10

Functional foods, ageing and degenerative disease

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

1.4

National initiatives to regulate food health claims

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

Regulatory context in the EU

11

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

12

Functional foods, ageing and degenerative disease

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

Regulatory context in the EU

13

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

1.5

Approval and substantiation of health claims

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

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

14

3.

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

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

1.6

Medicinal products and EU legislation

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

Regulatory context in the EU

15

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

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

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

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

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

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

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

17. 18.

March 1998.

Agreement on health claims on foods of 20

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

2 Diet and the prevention of degenerative disease L. Kalbe, B. Reusens and C. Remacle, 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

Functional foods, ageing and degenerative disease

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

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19

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

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

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

2.2

Foetal and neonatal nutritional requirements

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

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

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

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

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

26

Functional foods, ageing and degenerative disease

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

Diet and the prevention of degenerative disease

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

28

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

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

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

2.3

The effects of supplement intake

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

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

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

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

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

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

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

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

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

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

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

2.5

Safety concerns of functional foods

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

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

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

2.6

Future trends

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

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

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2.7

43

Sources of further information and advice

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

2.8

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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.

59

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

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

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

3.2

Mediterranean foods and their functional properties

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

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

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

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

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

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

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

3.4

Diet and age-related diseases

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

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

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

3.5

Methods of identifying and analysing plant extracts

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

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

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

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

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

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Sources of further information and advice

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

3.8

Acknowledgement

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

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3.9

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

4.1

Introduction: definition and epidemiology of osteoporosis

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

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

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

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

4.2

Bone growth and factors affecting bone mass

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

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Table 4.1

Biochemical markers of bone turnover

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

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

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

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

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

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

4.3

Dietary strategies for preventing osteoporosis: minerals

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Diet and control of osteoporosis Table 4.2

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Candidate genes for osteoporosis

Candidate gene

Physiological function

Vitamin D receptor

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

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

Matrix component Regulation of calcium homeostasis Homocysteine metabolism Matrix component

Modified from Cusack and Cashman (2003).

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

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

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

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

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

4.6

Conclusions and future trends

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

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

4.7

Sources of further information and advice

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

4.8

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

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

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

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

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

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

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

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5.4

Phytoestrogen action on bone cells

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

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

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

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

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

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

5.6

Conclusions

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

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

5.7

Sources of further information and advice

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

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

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

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

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

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KILKINNEN A

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

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

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

6.1

Introduction

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

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

6.2

Vitamin D: sources, metabolism, function and deficiency

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

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

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

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

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

6.3

Vitamin D fortification and osteoporosis

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

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

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

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

6.4

Dietary intake of vitamin D

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

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

Survey

Method

Intake (g/day)

Denmark

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

Males 45±54 55±64 65±74 75±80

Finland

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

24±hour recall

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

France

INCA 1999 (Volatier, 2000)

Seven±day record

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

Germany

ErnaÈhrungsbericht 2000 (Deutsche Gesellschaft fuÈr ErnaÈhrung)

Three±day record

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

y y y y

(n (n (n (n

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

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

(n (n (n (n

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

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

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

Ireland

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

Seven±day record. Intake from supplements included

Males 51±64 y (n 173): 4.4

Females 51±64 y (n 162): 5.8

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

Two±day record

Males 50±65 y (n 454): 4.9 >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: <60 y: 5 g 61 y: 10 g

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

United Kingdom

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

Reference nutrient intake: >65 y: 10 g

Ten g for those confined indoors, irrespective of age

Germany, Austria, Switzerland

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

Netherlands

Gezondheidsraad (2000)

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

Higher recommendations with limited exposure to sunlight and dark skin colour

Belgium

MinisteÁre des Affairs Sociale, de la 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.

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

6.5

Strategies to improve vitamin D supply

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

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Functional foods, ageing and degenerative disease

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

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

*Vitamin D content in fatty fish shows large variations

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

Vitamin D fortification and bone health

6.6

155

Food fortification: reducing deficiency diseases

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

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Functional foods, ageing and degenerative disease

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

2. 3. 4. 5.

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

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

6.7

Issues in vitamin D fortification of food

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

Vitamin D fortification and bone health

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

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

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

Vitamin D fortification and bone health

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

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

Mean

10 percentile

Median

90 percentile

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

4.6 9.3 4.8 10.0 5.3 12.1 4.7

2.0 3.0 2.3 4.6 2.7 5.1 2.4

4.1 8.1 4.2 9.2 4.8 11.4 4.2

7.8 17.0 7.7 16.3 8.3 20.0 7.5

10.0

4.9

9.3

15.7

Food (fortification

Mean

10 percentile

Median

90 percentile

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

4.8 8.6 5.6 11.5 5.6 11.9 5.2

2.3 3.1 3.1 6.0 2.7 4.6 2.8

4.2 8.0 4.8 11.4 5.1 11.9 4.3

8.1 14.9 8.6 18.6 9.8 19.4 8.5

10.1

5.8

9.4

15.9

Women, 65±74 years (n 103)

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

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

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

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

6.8

Future trends

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

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6.9

Sources of further information

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

6.10

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

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

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

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

DOUGHERTLY C, GUNTER EW, BOWMAN BA

NIH CONSENSUS DEVELOPMENT PANEL ON OSTEOPOROSIS PREVENTION, DIAGNOSIS AND THERAPY

785±795.

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

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

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

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

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

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

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

STANDING COMMITTEE ON THE SCIENTIFIC EVALUATION OF DIETARY REFERENCE INTAKES

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

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

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

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

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

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

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

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

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

7.1

Introduction: bone formation and calcium fortification

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

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

Calcium citrate (TCC) and bone health Table 7.1

175

Optimal daily intake of calcium1

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

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

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

7.1

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

7.2

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

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

Calcium content of the different salts

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

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

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

7.2

Calcium citrate as a calcium supplement

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

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7.3

Measuring the effectiveness of TCC

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

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

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

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

7.4

TCC fortification

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

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

7.5

Future trends

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

7.6

Sources of further information

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

182 3.

4.

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

11. 12.

13.

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

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

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

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

Calcium citrate (TCC) and bone health

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

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

8.1

Introduction: key dietary factors in oral health

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

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

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

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The effects of ageing on oral health

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

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

8.3

Dietary strategies for oral health

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

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

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

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

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

8.4

Functional foods for promoting oral health

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

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

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

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

8.5

Future trends

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

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

8.6

Sources of further information

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

8.7

References

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

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

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

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

VIRTANEN JI, BLOIGU RS

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

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

Sweeteners and dental health Table 9.1

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Factors that affect the development of dental caries infection

Factor

Possible relationship to dietary sweeteners and caries

Diet Eating habits Oral clearance

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

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

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

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9.2

Xylitol and the prevention of dental caries

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

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

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

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

4.

5.

6.

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

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

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

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

Remarks

1. Chewing gum

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

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

4. Mouthwashes, sprays 5. Pacifier

6. Cotton swabs, toothbrushes

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

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

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

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

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

Comments

Glucose and sucrose

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

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

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

Sweeteners tested (reference)

Duration (years)

Dose (g/day)

Caries reduction

Sweden (Vipeholm study)

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

4

Up to 300

Sucrose was highly cariogenic.

Denmark

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

2

3.6

10%

Sweden (Roslagen study)

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

Up to 2.5

Not given

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

England

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

2

Not given

`Small but significant benefit' (quote).

Island of Crete (Greece)

Sucrose chewing gum (Slack et al., 1972)

3

Five sticks daily No significant effect

Finland (Turku study)

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

2

67

>85% (with xylitol)

Finland (Turku study)

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

1

6.7

>82%

Soviet Union (Kazan study)

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

2

30

Up to 73%

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

2.3±2.7

20

Preventive effect noted.

WHO studies Thailand

French Polynesia

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

3

Up to 20

58±62%

Hungary

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

2±3

14±20

37±45%

Canada (Montreal study)

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

1±2

1.0±3.9

52%

Sweden (Gustavsberg)

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

2

Not given

35%

Finland (Ylivieska)

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

7±10

30±57%

7±10

59±84%

USA (Boston)

Sorbitol gum vs. normal diet (Glass, 1983)

2

Not given (2 sticks)

Not significant.

Costa Rica

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

3

Twice daily

Up to 12.3%.

3

Twice daily

Up to 10%.

3.3

Up to 10.7

Xylitol reduced by up to 73%d.

2

Up to 10.7

Xylitol reduced by up to 63%e .

Belize

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

Table 9.4

Continued

Study location

Sweeteners tested (reference)

Duration

Dose

Caries reduction

USA (Dayton, OH)

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

1.8

Up to 8.5

80%

Estonia

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

2±3

5

50±60%

Finland (Ylivieska)

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

6±7

70% (in children).

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

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

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

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

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215

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

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

9.4

Future trends

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

9.5

References

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

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

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

LARJE O, NORD CE

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

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

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

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

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

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

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

10.1

Introduction

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

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

10.2

Genetic influences on obesity

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

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

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

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

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

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

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

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

Endocrine function

Energy intake Central Peripheral Food preferences

Energy expenditure Central

Physiological mechanism

Candidate genes

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

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

Mediator

Hypothalamic neurotransmitters or receptors Symphatoadrenergic system

Effector

EE as such, fat oxidation

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

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

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

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

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

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

10.3

Nutrient-sensitive genes

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

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

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

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

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

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

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

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

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

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

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

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

10.5

Managing obesity: dietary and other strategies

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

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

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

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

10.6

Future trends

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

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

10.7

Sources of further information

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

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

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

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

10.8

References

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

11.1

Introduction: fat synthesis and nutrition

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

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Fig. 11.1

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The glycolytic and lipogenic pathways in the liver.

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

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

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

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

11.2

Regulation of glycolytic/lipogenic enzymes

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

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

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

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

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

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

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

Fig. 11.2 Integration of insulin/glucose regulation of glycolytic and lipogenic genes.

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

11.4

Improving lipogenesis using functional foods

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

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

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

11.5

Future trends

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

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

11.6

Sources of further information

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

11.7

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Abbreviations

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

and

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

12.1

Introduction: satiety and obesity

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

12.2

Factors influencing satiety and satiation

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

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

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

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

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12.3

The impact of different food components on satiety

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

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

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

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

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

12.4

Developing biomarkers of satiety

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

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

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

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

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

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Sources of further information and advice

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

12.7

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

13.1

Introduction: the human gut

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

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Fig. 13.1 The human gut.

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

13.2

The structure of the gut and its immune system

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

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Fig. 13.2 The basic structure of the gut wall.

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

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

3. 4. 5.

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

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

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

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

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

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

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

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

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

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

304 2. 3. 4. 5.

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

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

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

13.3

Nutrients and gut function

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

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

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

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

13.4

Nutrients and the gut immune system

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

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

13.5

Nutrition and gut health

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

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

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

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

13.6

The role of functional foods in promoting gut health

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

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

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

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

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

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Table 13.1 The definition of probiotics ± development of the current definition (adapted from Isolauri et al., 2002) Definition

Source

Specific bacteria in yoghurt fermentation balance intestinal microbiota Substances excreted by one protozoan to stimulate the growth of another Substances that have a beneficial effect on animals by contributing to the balance of the intestinal microbiota Live microbial feed supplements which beneficially affect the host animal by improving the intestinal microbial balance Mono- or mixed cultures of live micro-organisms which when applied to man affect beneficially the host by improving the properties of the indigenous microbiota Live microbial food ingredients that are beneficial to health (efficacy and safety scientifically documented) Specific live or inactivated microbial cultures that have documented targets in reducing the risk of human disease or in their adjunct treatment

Metchnikoff (1907) Lilly and Stillwell (1965) Parker (1974) Fuller (1989) Huis in't Veld and Havenaar (1991) Salminen et al. (1998) Isolauri et al. (2002b)

irritable bowel syndrome, colorectal cancer (Salminen et al., 1998) and food allergy (Laiho et al., 2002). Another alleviating food ingredient for inflammatory bowel diseases is n-3 fatty acids. These can modulate the dysfunctions found in the immune system (Kelley, 2001; Donnet-Hughes et al., 2001; Anderson and Fritsche, 2002; Ziegler et al., 2003). For gut-related surgery and trauma patients, a mixture of arginine, glutamine, n-3 fatty acids and nucleotides has been tested and applied successfully (Alexander, 2002). However, optimising the treatment is still needed. According to Prosky (2000) dietary fibre can be considered a functional food when it imparts a special function to that food aside from the normal expected function and similarly when the dietary fibre is used as an additive to foods.

13.7

Future trends

13.7.1 Gut renewal and absorption Malabsortion of nutrients, impairment of the ability of the intestine to adapt, and immunosenescence has been described in the elderly. Enhancing absorption by dietary manipulation to prevent malnutrition may prove useful (Gill et al., 2001; Woudstra and Thomson, 2002). As the body of knowledge surrounding the molecular processes involved in the regulation of intestinal adaptation and transport grows, it should be possible to develop novel agents to improve the quality of life of patients suffering from inadequate nutrient absorption (Ray et

Table 13.2 Current probiotic bacteria and their important reported effects in human studies Strain

Reported effects in clinical studies

Selected key references and reviews

L. johnsonii LA1

Adherence to human intestinal cells, balances intestinal microbiota, immune enhancement, adjuvant in H. pylori treatment.

L. acidophilus NCFB 1748 L. casei Shirota

Lowering of faecal enzyme activity, decreasing faecal mutagenicity, prevention of radiotherapy-related diarrhoea. Prevention of intestinal disturbances, balancing intestinal bacteria, lowering faecal enzyme activities, positive effects on reducing the recurrence of superficial bladder cancer.

Salminen et al. (1998); MacFarland (2000); Fonden et al. (2000); Ouwehand et al. (2003) Fonden et al. (2000)

Ouwehand et al. (2003); Salminen et al. (1998); Aso and Akaza (1992); Aso et al. (1995); Ohashi et al. (2002) S. thermophilus; L. No effect on rotavirus diarrhoea, no immune enhancing effect during rotavirus Fonden et al. (2000); Ouwehand et bulgaricus diarrhoea, no effect on faecal enzymes, strain dependent improvement of al. (2003) lactose intolerance symptoms. L. acidophilus La-5 Balancing intestinal microbiota, protection against traveller's diarrhoea, Fonden et al. (2000); Ouwehand et immune enhancement. al. (2003) B. Lactis Bb-12 Treatment of viral diarrhoea including rotavirus diarrhoea, alleviation of Saavedra et al. (1994); Salminen et symptoms of food allergy, balancing of intestinal microbiota. al. (1998); Isolauri et al. (2002b) L. reuteri Shortening of rotavirus diarrhoea, colonising the intestinal tract. De Roos and Katan (2000); Fonden et al. (2000); Ouwehand et al. (2003) S. boulardii Prevention of antibiotic associated diarrhoea, treatment of C. difficile colitis. MacFarland (2000) Probiotic mix VSL 3 Positive effect in inflammatory bowel disease and irritable bowel syndrome; Delia et al. (2002a,b); Kim et al. (L. bulgaricus, L. plantarum, treatment and prevention of pouchitis, prevention and alleviation of (2002) S. thermophilus , B. longum, radiotherapy associated diarrhoea B. infantis, and B. breve)

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al., 2002). As gut health, digestion and absorption have an impact on metabolic effect in other organs (Verschuren, 2002), functional foods for this area might be appealing for each age group. Colostrum-derived preparations have already been tested in the clinical management of gastrointestinal diseases (Playford et al., 2000). They seem to be useful at least in infancy and childhood, and the possibilities for also developing products for adults and the elderly are also interesting. Since nutrients affect hormone secretion and release, they also affect mucosa, nerves and muscles. Specific nutrients might also be useful as enhancers of gut renewal. Even gut microbes might be employed ± they may stimulate nerves (Hooper et al., 2001). Hardman (2002) assumes that after the appropriate cancer therapy, consumption of n-3 fatty acids might slow or stop the growth of metastatic cancer cells, increase longevity of patients and improve their quality of life. N-3 fatty acids seem to alter the eicosanoid metabolism of cancer cells, slow cancer cell mitosis, increase cancer cell death, induce differentiation, suppress angiogenesis and alter estrogen metabolism. Animal studies also suggest that n-3 fatty acids might increase the efficacy of chemotherapy and reduce its side effects. One of the mechanisms in the treatment of colon cancer might be the suppression of the increased production of COX-2 typical to the disease. 13.7.2 Gut microbiota and allergy Our studies have indicated significant differences in the Bifidobacterium and Clostridium microbiota composition of children who later develop atopic diseases (KalliomaÈki et al., 2001). The result has been confirmed in Estonian and Swedish infants (BjoÈrksten et al., 2001). Taken together the results suggest that the focus on intestinal microbiota and the search for new probiotics for the newborn and infants should be centred on the lactic acid bacteria and Bifidobacteria composition and modulating the ratio of bifidobacteria to clostridia in the intestinal contents of infants. We have obtained similar results in studies among very old Finnish and Japanese subjects (He et al., 2000). 13.7.3 Gut microbiota and intestinal mucus or age-specific intestinal microbiota We have also shown that the mucus microbiota is not necessarily reflected by faecal microbiota. This finding offers a basis for further assessing the influence of mucosal microbiota on the immune system and mucosal cell gene expression. The role and crosstalk between specific bifidobacteria and the gut epithelium has been further clarified by understanding the genome sequences of specific bifidobacteria. Schnell and co-workers (2002) reported the genome sequence of B. longum. They identified polypeptides with homology to most major proteins needed for production of glycoprotein-binding fimbriae. Such fimbriae were also tentatively for the first time identified for a Bifidobacterium by electron microscopy. B. longum was also shown to have mucin utilising properties and it

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has a eukaryotic-type serine protease inhibitor possibly involved in earlier reported immunomodulatory activity of bifidobacteria. Interestingly, B. longum also has numerous high-affinity oligosaccharide transporters, including the use of over 80 oligosaccharides found in breast milk, which may enhance its presence and competition for nutrients in the newborn infant colon during breast feeding. In the same manner, B. adolescentis, the dominant bifidobacterium species among the adults and the elderly, may have adapted to changing nutrient sources during old age. This is an area of future research as the host physiology may have an influence on the gut health of the elderly and the microbiota may have a significant role in preserving the gut health of the elderly. High antioxidative activity may be useful for survival and defensive properties of probiotics (Kullisaar et al., 2002). Starch encapsulation of probiotics may in future enable utilisation of new probiotic strains (PuupponenPimiaÈ et al., 2002). 13.7.4 `Novel prebiotics' Chemical modification of starch and other cereal components (Topping and Clifton, 2001; Charalampopoulos et al., 2002) and inulin (Stevens et al., 2001) offers possibilities for modifying their digestibility and thus efficiency as prebiotics. According to Das (2002) the combination of LCPUFA and probiotics could offer significant protection against atopy. 13.7.5 Nutrigenomics Genetic polymorphism may be a contributing factor in the inconsistensies found between diet and risk of colorectal cancer (Cotton et al., 2000; Houlston and Tomlinson, 2001). Van Ommen and Stierum (2002) stress the importance of multiple minor changes in genomic responses to food. Co-operation of nutritional sciences with systems biology is needed for proving health claims of functional food in future. Weststrate et al. (2002) predict that within ten years there will be some functional foods on the market that take into account gene polymorphism relevant to the development of chronic diseases. However, Van Ommen and Stierum (2002) state that prevention or intervention of a disease may be fundamentally different from maintaining or improving good health. 13.7.6 Biotechnology Genetic engineering has been applied to improve the nutritional quality of plants. Broun et al. (1999) have reviewed applications on plant lipids and Grusak (2002) on phytochemicals. Several tools and useful applications already exist. Food-grade microbes have been modified to produce low-energy sugars, vitamins, prebiotics and antioxidants. Constructed strains might be used in novel fermented foods with increased nutritional value (Hugenholtz and Smid, 2002).

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Also, therapeutic use of genetically engineered lactic acid bacteria (Lactococcus lactis) against IBD in animal models has already been described. The L. lactis strain was engineered to secrete biologically active interleukin IL-10, and daily treatment with the microbe reduced significantly intestinal inflammation (Steidler et al., 2000). Van der Werf and Venema (2001) describe tools for developing improved probiotic Bifidobacterium strains or increasing their stability during processing and viability upon storage. Chronic stress initiates mucosal inflammation in rat intestine by impairing mucosal defences (SoÈderholm et al. 2002). Is it possible in future to alleviate stress disorders by special probiotics?

13.8

Sources of further information

Ageing and nutrient needs are discussed with reference to nutrition recommendations in the following articles: Russel R, Rasmussen H and Lichtenstein A (1999), `Modified food guide pyramid for people over seventy years of age', J Nutr 129: 751±753 and Lichtenstein A (1999), `Optimal nutrition for mature adult in health and disease', in Rippe J, Lifestyle Medicine, Blackwell Science, Malden MA, USA, 160±167. Nutritional effects of pre- and probiotics are reviewed in Teitelbaum J E and Walker A (2002), `Nutritional Impact of Preand Probiotics as Protective Gastrointestinal Organisms', Annu Rev Nutr 22: 107±138. Functional foods and gastrointestinal physiology and function are discussed in detail in the following article: Salminen S, Bouley M C, Boutron-Rualt M C, Cummings J, Franck A, Gibson G, Isolauri E, Moreau M-C, Roberfroid M, Rowland (1998), `Functional food science and gastrointestinal physiology and function', Br J Nutr 80 Suppl 1: 147±171. Functional foods and different aspects of physiology are extensively described in two ILSI Europe working group proceedings: British Journal of Nutrition, (1998) vol 80 (Suppl. 1) `Functional Food Science in Europe' and British Journal of Nutrition, (2002) vol 88 (Suppl. 2) `Functional Foods: Scientific and Global Perspectives'. Several new reviews discuss gut defence systems: Berkes J, Viswanathan V K, Savkovic S D, Hecht G (2003), `Intestinal epithelial responses to enteric pathogens: effects on the tight junction barrier, ion transport, and inflammation', Gut 52: 439±451; Mowat A M (2003), `Anatomical basis of tolerance and immunity to intestinal antigens', Nature Rev Immunol 3: 331±341; Fagarasan S and Honjo T (2003), `Intestinal IgA synthesis: regulation of front-line body defences', Nature Rev Immunol 3: 63±72; Baumgart D C and Dignass A U (2002), `Intestinal barrier function' Curr Opin Clin Nutr Metab Care 5: 685± 694. Effects of glutathione on gut defence has been studied in: Hoensch H,

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Morgenstern I, Petereit G, Siepmann M, Peters W H M, Roelofs H M J, Kirch W (2002) `Influence of clinical factors, diet, and drugs on the human upper gastrointestinal glutathione system', Gut 50: 235±240.

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(2002), `The genome sequence of Bifidobacterium longum reflects its adaptation to the human gastrointestinal tract', Proc. Natl. Acad. Sci. 99: 14422±14427. SCHLEY P D, FIELD C J (2002), `The immune-enhancing effects of dietary fibres and prebiotic', Brit J Nutr 87 (Suppl 2): S221±S230. SCHNEEMAN B (2002), `Gastrointestinal physiology and functions', Brit J Nutr 88 (Suppl2): S159±S163. SHANAHAN F (2000), `Nutrient Tasting and Signaling Mechanisms in the Gut V. Mechanisms of immunologic sensation of intestinal contents', Am J Physiol Gastrointestinal Liver Physiol 278: G191±G196. SHANAHAN F (2002), `Probiotics and inflammatory bowel disease: from fads and fantasy to facts and future', Brit J Nutr 88(Suppl. 1): S5±S9. SHAO L, SERRANO D, MAYER L (2001), `The role of epithelial cells in immune regulation in the gut', Semin Immunol 13: 163±175. SHIMOTOYODOME A, MEGURO S, HASE T, TOKIMITSU I, SAKATA T (2000), `Short chain fatty acids but not lactate or succinate stimulate mucus release in the rat colon', Comparat Biochem Physiol Part A 125: 525±531. DESIERE F, BORK P, DELLEY M, PRIDMORE R D, ARIGONI F

È DERHOLM J D, YANG P C, CEPONIS P, VOHRA A, RIDDELL R, SHERMAN P M, PERDUE M H SO

(2002), `Chronic stress induces mast cell-dependent bacterial adherence and initiates mucosal inflammation in rat intestine', Gastroenterology 123(4): 1099± 1108. STEIDLER L, SCHOTTE H W, NEIRYNCK S, OBERMEIER F, FALK W, FIERS W, REMAUT E (2000), `Treatment of murine colitis by Lactococcus lactis secreting interleukin-10', Science 289: 1352±1355. STEINMAN R M, HAWIGER D, NUSSENZWEIG M C (2003), `Tolerogenic dendritic cells', Annu Rev Immunol 21: 685±711. STEVENS C V, MERIGGI A, BOOTEN K (2001), `Chemical Modification of Inulin, a Valuable Renewable Resource, and Its Industrial Applications', Biomacromolecules 2: 1±16. TAPPENDEN K A, MCBURNEY M I (1998), `Systemic short-chain fatty acids rapidly alter gastrointestinal structure, function, and expression of early response genes', Endocrinology 140: 1687±1694. TAPPENDEN K A, DROZDOWSKI L A, THOMSON A B, MCBURNEY M I (1998), `Short-chain fatty acid-supplemented total parenteral nutrition alters intestinal structure, glucose transporter 2 (GLUT2) mRNA and protein, and proglucagon mRNA abundance in normal rats', Am J Clin Nutr 68: 118±25. TEITELBAUM J E, WALKER A (2002), `Nutritional Impact of Pre- and Probiotics as Protective Gastrointestinal Organisms', Annu Rev Nutr 22: 107±138.

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THOMSEN C, RASMUSSEN O, LOUSEN T, HOLST J J, FENSELAU S, SCHREZENMEIER J, HERMANSEN K (1999), `Differential effects of saturated and monounsaturated fatty acids on postprandial lipemia and incretin responses in helathy subjects', Am J Clin Nutr 69: 1135±1143. THE DIETARY FIBER DEFINITION COMMITTEE (2001), `The definition of dietary fiber', CFW 46(3): 113±126. THULESEN J, HARTMANN B, NIELSEN C, HOLST J, POULSEN S S (1999), `Diabetic intestinal growth adaption and glucagons-like peptide 2 in the rat: effects of dietary fibre' Gut 45: 672±678. TOPPING D L, CLIFTON P M (2001), `Short-Chain Fatty Acids and Human Colonic Function: Roles of Resistant Starch and Nonstarch Polysaccharides', Physiol Rew 81(3): 1031±1064. VAN DER MEER R, BOVEE-OUDENHOVEN I (1998), `Dietary modulation of intestinal bacterial infections', Int Dairy J 8: 481±486. VAN DER WERF M J, VENEMA K (2001), `Bifidobacteria: Genetic modification and the study of their role in the colon', J Agric Food Chem 49: 378±383. VAN OMMEN B, STIERUM R (2002), `Nutrigenomics: exploiting systems biology in the nutrition and health arena', Curr Opin Biotechnol 13: 517±521. VAN POPPEL G (2002), `Nutrition and cancer ± a role for functional foods?' in Angus F and Miller C `Functional Foods', Surrey, Leatherhead Publishing, 216±225. VAUGHAN E, DE VRIES M, ZOETENDAL E, BEN-AMOR K, AKKERMANS A, DE VOS W (2002), `The intestinal LABs', Anth van Leeuwenh 82: 341±352. VERSCHUREN P M (2002), `Summary Report. Functional foods: Scientific and global perspectives', Brit J Nutr 88 (Suppl. 2): S125±S130. WESTSTRATE J A, VAN POPPEL G, VERSCHUREN P M (2002), `Functional foods, trends and future', Brit J Nutr 88 (Suppl.2): S233±S235. WILLIE J T, CHEMELLI R M, SINTON C M, YANAGISAWA M (2001), `To eat or to sleep? Orexin in the regulation of feeding and wakefulness', Annu Rev Neurosci 24: 429±458. WITTIG B M, ZEITZ M (2003), `The gut as an organ of immunology', Int J Colorectal Dis 18(3): 181±187. WOUDSTRA T, THOMSON A B R (2002), `Nutrient absorption and intestinal adaptation with ageing', Bailliere's Best Practice and Research Clinical Gastroenterology 16(1): 1±15. XIAO Q, BOUSHEY R P, DRUCKER DJ, BRUBAKER P L (1999), `Secretion of the intestinotrophic hormone glucagons-like peptide 2 is differentially regulated by nutrients in humans', Gastroenterology 117: 99±105. Â NDEZ-ESTIVARIZ C, JONES D P (2003), `Trophic and cytoZIEGLER T R, EVANS M E, FERNA protective nutrition for intestinal adaptation, mucosal repair, and barrier function', Annu Rev Nutr 23: 229±261.

14 Analysing gut microbiota M. Blaut, German Institute of Human Nutrition PotsdamRehbruecke

14.1

Introduction

The human gastro-intestinal (GI) tract is populated by a large and diverse community of up to 1014 micro-organisms which belong to an estimated 400± 500 different species (Finegold et al., 1983). Interest in gut micro-organisms has mainly been driven by the discovery that they have an impact on structure and function of the GI-tract and thereby influence host health. However, the exact role of the various bacterial population groups present in the GI-tract has been difficult to define. This requires a better knowledge of the makeup of the gut microbiota. Early investigations on the composition of the human gut microbiota missed the majority of bacteria present in the GI-tract because the techniques to grow strict anaerobes in the laboratory had not yet been fully developed. The introduction of the roll tube technique (Hungate, 1969) and of anaerobic cabinets (Aranki et al., 1969) was a major breakthrough in the cultivation of anaerobes which triggered the isolation and characterisation of previously uncultured strict anaerobes. Pioneers who laid the foundation for our present knowledge of the human gut microbiota included Moore and Holdeman (Moore and Holdeman, 1974) and Finegold and co-workers (Finegold et al., 1974). These researchers isolated a large number of bacterial species from human faeces and determined their distribution in humans. The in-depth analysis of the human gut microbiota as performed by these authors was laborious, time consuming and tedious. In particular, the identification of colonies to the species level required differentiation of large numbers of colonies on selective and differential media. This may explain why the number of studies involving the analysis of large numbers of subjects is limited. All studies indicated a high inter-individual variation in the microbiota composition, while a clear

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correlation between dietary habits and the presence of certain micro-organisms could not be found in most of these studies (Finegold, 1986). An important statement, which has been made previously by many other researchers, needs to be repeated here: The microbial composition of faeces does not necessarily reflect the microbial composition of lumenal contents from other sections of the gastro-intestinal tract. However, although quantitative differences exist there is a relatively high similarity of the bacterial composition in the colon and in the faeces (Bentley et al., 1972). Following the introduction of culture independent methods for bacterial identification it soon became clear that there is a large discrepancy between the number of micro-organisms that can be detected by virtue of their ability to form colonies on agar plates and the number of micro-organisms really present in a given habitat. Depending on the habitat, the proportion of culturable bacteria may be as low as 0.001%, as reported for seawater, and therefore the term `great plate count anomaly' was coined by Amann and co-workers (Amann et al., 1995). The application of culture independent methods to gut microbiology revealed that only 15±58% of the bacterial species resident in the human intestinal tract have been cultured (Langendijk et al., 1995; Suau et al., 1999) and that many microbiological data based on culture dependent techniques are biased because certain bacterial population groups have been underestimated while others have been overestimated. This is due to the fact that the specific growth requirements for many bacteria are not known and most media are not really selective for certain bacteria.

14.2 Molecular based methods for identifying gut micro-organisms Owing to the limited number of simple phenotypic traits, the identification of micro-organisms has always been a challenge. Moreover, most of the phenotypic characters used for bacterial identification not only lack the resolving power but also do not reflect the phylogenetic position of an organism. In recent years, ribosomal RNA (rRNA) has become the preferred marker for the identification and affiliation of new isolates because it is an excellent tool for measuring evolutionary relationships among organisms (Woese et al., 1990). Ribosomal RNAs are constituents of the ribosomes which in prokaryotes have an overall size of 70S (S=Svedberg units). They contain 2 subunits, of which the large one (50S) consists of 34 proteins, the 5S rRNA (120 bases), and the 23S rRNA (approximately 2,900 bases), while the 30S subunit contains 21 proteins and the 16S rRNA (approximately 1,500 bases). Even though all three ribosomal RNAs (5S, 16S, and 23S) have been employed in comparative sequence analysis, the 16S rRNA has become the most preferred molecule for bacterial identification (Amann et al., 1995).

Analysing gut microbiota 327 14.2.1 Generation of a database of small subunit ribosomal RNA sequences: a culture independent approach Woese was the first one to propose the use of the 16S rRNA sequence for the elucidation of bacterial phylogeny (Woese, 1987). Sequencing the 16S rRNA of a newly isolated micro-organism and subsequently comparing the retrieved sequence with the 16S rRNAs in the database is an excellent way of identifying this organism and determining its phylogenetic position. However, the 16S rRNA sequences can also be retrieved from uncultured bacteria, provided the bacterial DNA can be extracted. Owing to the fact that the 16S rRNA contains regions whose sequence is highly conserved in all bacteria and other regions whose sequence shows a high degree of variability among various organisms, it is possible to amplify the variable regions by polymerase chain reaction (PCR) by using primers that hybridise with the well-known conserved regions of the 16S rRNA gene (Olsen et al., 1986). Subsequent sequencing of the PCR product reveals the sequence of the adjacent variable region(s), the so-called signature sequence motifs, which are of diagnostic value and can be used for identification of bacteria (Amann et al., 1995). In practice, the retrieval of 16S rRNA sequences requires the isolation of nucleic acids from pure cultures of intestinal bacteria or from faecal samples. Ideally, the collection of retrieved 16S rRNA sequences should be a reflection of the microbial community composition. In other words, the frequency of a given 16S rRNA species should give an indication for the proportion of the corresponding target organism(s) in the microbial community. It is therefore important to make sure that the extraction of microbial DNA from a faecal sample is as complete as possible. Mechanic, enzymatic and/or chemical methods have been used for the disruption of the bacterial cell wall and the extraction of nucleic acids from faecal samples (Suau et al., 1999; Wilson and Blitchington, 1996). In particular humic acids, bile salts and complex polysaccharides present in faeces can inhibit PCR (Kreader, 1996). Incomplete DNA extraction or inhibition of PCR by inhibitory substances not removed during purification, may lead to a 16S rRNA gene library that is not representative of the microbial ecosystem studied. Bias in the resulting gene library may also be introduced by differences in the efficiency of the PCR amplification which may differ from template DNA to template DNA. It has been shown that the rRNA fragments of less abundant organisms tend to be underrepresented (Lee et al., 1996; Muyzer et al., 1993). Another important variable that influences the coverage of the microbial diversity is the number of amplification cycles applied in PCR: the higher the number of amplification cycles the smaller the degree of biodiversity coverage (Bonnet et al., 2002; Wilson and Blitchington, 1996). Bonnet and co-workers therefore proposed to keep the number of PCR cycles used for the amplification of ribosomal genes as small as possible (Bonnet et al., 2002). The amplified 16S rRNA genes are cloned into Escherichia coli to create a 16S rRNA gene library (Fig. 14.1). An ever-growing database of 16S rRNA sequences has substantially increased knowledge of bacterial phylogeny. The culture independent retrieval

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Fig. 14.1 Steps involved in generating a database of small subunit ribosomal RNA sequences of faecal bacteria.

of 16S rRNA sequences has been used by a number of researchers to investigate the microbial diversity in the human gut ecosystem (Suau et al., 1999; Wilson and Blitchington, 1996). Only 24% of the 16S rRNA sequences recovered in the study of Suau et al. (1999) could be assigned to described species indicating that a large proportion of the bacterial species present in the human gut microbiota have not yet been isolated and described. Analysis of the 16S rRNA-gene sequence of new faecal isolates is also the quickest and most efficient way to find out whether the isolate is a new or a previously described species. In general, bacteria exhibiting 98% sequence similarity are considered to be the same species. Based on such analyses, a number of recent isolates from human faeces have been shown to represent new diversity. These include Dorea longicatena (Taras et al., 2002), Anaerostipes caccae (Schwiertz et al., 2002a), Ruminococcus luti (Simmering et al., 2002), Roseburia intestinalis (Duncan et al., 2002) and Clostridium bolteae (Song et al., 2003). 14.2.2 Design and validation of ribosomal RNA-targeted oligonucleotide probes In recent years, the number of 16S rRNA sequences in general and of faecal micro-organisms in particular has increased considerably. A database of 16S rRNA sequences as complete as possible facilitates the design of oligonucleotide probes. They can be designed in such a way that they target bacteria at different levels of the phylogenetic hierarchy by hybridising to the target region of the

Analysing gut microbiota 329

Fig. 14.2 Alignment of the probe sequence (S-S-Acac-0194-a-A-18) and its 16S rRNA target sites in the gut anaerobe Anaerostipes caccae and closely related organisms.

RNA, where they form an RNA/DNA hybrid. While highly conserved stretches within the 16S rRNA sequence can be used to detect all bacteria, others are common to larger or smaller phylogenetic groups or even specific to a species and can therefore be used to detect the corresponding target organisms. Once it has been decided which organism or which group of organisms is to be targeted, suitable regions in the 16S rRNA sequence have to be identified that are present in the target organism but absent from the non-target organisms. A number of useful and easy-to-use computer programs for probe design have become available. These tools are often integrated into programs used for the storage, alignment and phylogenetic analysis of 16S rRNA sequences. The length of most 16S rRNA-targeted probes is in the range of 15 to 23 nucleotides. An example showing an in silico analysis for a probe targeting the gut anaerobe Anaerostipes caccae is given in Fig. 14.2. The optimal hybridisation conditions have to be determined for each probe experimentally because the dissociation temperature of an oligonucleotide probe depends on a number of parameters, including length and base composition. Whether the probe can form a hybrid with the target sequence depends primarily on the stringency of the hybridisation conditions. The stringency is determined by the hybridisation temperature and the composition of the buffer, in particular the salt concentration. Formamide is often used to increase stringency of the buffer. The specificity of a newly designed probe should be tested with rRNAs from as many faecal bacteria as possible. Optimally the organisms chosen should be representative of the ecosystem studied and include close relatives which should not be recognised by the probe. Between 45 and over 100 reference organisms have been used for testing the specificity of probes targeting human gut bacteria (Harmsen et al., 2000, 2002; Hold et al., 2003; Schwiertz et al., 2002b; Simmering et al., 1999). It is sometimes not easy to find experimental conditions under which the designed probe hybridises with the 16S rRNA of the target organism but not with the 16 rRNA of closely related bacteria with only one mismatch in their target sequence. The oligonucleotides have to be radioactively or fluorescently labelled to detect their hybridisation to the targeted 16S rRNA.

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14.2.3 Culture independent identification of gut bacteria There are several experimental approaches to identifying bacteria in a cultureindependent manner: dot blot hybridisation and whole cell in situ hybridisation. Both methods afford the determination of the relative abundance of the targeted bacterial group. Dot blot hybridisation Dot blot hybridisation and all methods derived from it do not detect bacterial cells but determine the proportion of rRNA originating from the target organism(s) relative to the amount of rRNA originating from all organisms present in the ecosystem. In practice, total RNA is isolated from an environmental sample such as human faeces and aliquots are blotted in parallel onto two nylon membranes or other suitable surfaces. One of the blots is hybridised with an oligonucleotide probe specific for the target group while the other blot is hybridised with a probe that detects all bacteria. The probes are usually radioactively labelled to quantify their binding to the blotted RNA spots. The signal intensity of a given sample on the blot hybridised with the specific probe is correlated with the signal intensity of the same sample blotted onto the other membrane and hybridised with the probe that detects all bacteria. The relative intensity of the signal may be taken as an indirect measure of the size of the targeted bacterial population group and has been referred to as RNA index (Sghir et al., 2000). Using this method, predominant bacterial groups were analysed in human faecal samples from 27 adults. The mean values determined for the following target groups were as follows: Bacteroides group, 36.8 Ô 16%; Clostridium leptum group, 16.3 Ô 7%; Clostridium coccoides group, 14.5 Ô 6%, Bifidobacterium spp., 0.7 Ô 1%; Lactobacillus spp. group, 0.6 Ô 1%; Enterobacteriaceae, 0.7 Ô 1% (Sghir et al., 2000). These signals added up to 70% of the total 16S rRNA detected by the probe targeting all bacteria. The percentages for the various groups are not comparable to the relative proportions of bacterial groups based on bacterial counts (see below). This may be explained by differences in the efficiency of the RNA recovery from the various target organisms, or by differences in the cellular RNA content. Fluorescence in situ hybridisation The most popular and widely used culture independent method is whole cell fluorescence in situ hybridisation which affords the specific detection of bacterial cells with fluorescently labelled oligonucleotide probes, often also referred to as FISH (Amann et al., 1995). Properly designed probes bind to their potential target sites in the 16S rRNA. Since one cell may contain 103 to 105 ribosomes the number of potential target sites for the labelled probe is sufficiently high to render the whole bacterial cell fluorescent so that it can be easily visualised in the epifluorescence microscope. In practice, this method requires the cells to be prepared in such a way that the target site becomes accessible to the oligonucleotide probes while preserving cell morphology. An aliquot of a homogenised faecal sample is tenfold diluted with buffer and

Analysing gut microbiota 331 thoroughly mixed. Since faecal samples contain not only bacterial cells but also particles originating from ingested food, which could interfere with the visualisation of the fluorescing bacteria at a later stage, a brief centrifugation step at reduced centrifugation force (1 min. at 300 g) is usually employed. The cells are subsequently fixed which also affords the permeabilisation of the bacterial cell envelope. Gram-negative bacteria are preferentially fixed with a freshly prepared paraformaldehyde solution whereas ethanol is used for grampositive bacteria (Amann et al., 1995). Some bacteria are more difficult to permeabilise because of their cell wall. In particular a number of grampositive bacteria additionally require lysozyme treatment to weaken the cell wall. Once the cells are fixed, they can be easily stored at ÿ20 or ÿ80ëC until analysis. For the identification and enumeration of target organisms by epifluorescence microscopy, the fixed bacterial cells have to be hybridised with the oligonucleotide probe of choice depending on which bacteria are to be targeted (see section 14.2.3). To be detectable, the oligonucleotides are labelled with a fluorochrome. Commonly used fluorochromes include tetramethylrhodamin (TMR), fluoresceinisothiocyanate (FITC), and the indocarbocyanine dyes Cy3 and Cy5. For that purpose, an aliquot of a diluted and fixed faecal sample is transferred into a well on a special type of microscopic slide (Fig. 14.3). It is important to ensure that the cells are distributed as evenly as possible to avoid a high variability between microscopic fields which are inspected for enumeration

Fig. 14.3 Enumeration of fluorescently labelled cells by epifluorescence microscopy taking advantage of custom-made slides.

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Fig. 14.4 Steps involved in culture independent detection of uncultured bacteria in faecal samples by whole cell fluorescent in situ hybridisation.

at a later stage. The cells in the well are air-dried and subsequently dehydrated by immersing the slide in 60%, 80% and 96% ethanol. After drying, the oligonucleotides are added and hybridisation is performed overnight at the optimal hybridisation temperature as determined experimentally with probe validation experiments (see section 14.2.3). After washing with buffer at 2ëC above the hybridisation temperature, mounting medium is added, which usually contains some anti-fading solution to reduce fluorescence bleaching. The cells can then be visualised by epifluorescence microscopy. It is even possible to identify several bacterial target groups simultaneously in the same sample provided each oligonucleotide employed is labelled with a different fluorochrome and the appropriate fluorescence filter is selected. A major advantage of FISH lies in the possibility of detecting bacteria that have not yet been cultured but whose 16S rRNA sequence has been retrieved in a culture independent way (14.2.1). Based on the retrieved sequence it becomes feasible to design an oligonucleotide probe which enables the detection of this particular organism by applying FISH to the original sample (Fig. 14.4). However, in this case it is not possible to test the specificity of the probe experimentally with the target organism. In particular obligate syntrophic bacteria may become detectable by this approach as shown previously for ectosymbiotic bacteria of the gastro-intestinal tract of surgeonfish (Angert et al., 1993) or of the bovine rumen (Stahl et al., 1988).

Analysing gut microbiota 333 Limitations of FISH Although FISH is a reliable technique, there are a number of limitations that have to be taken into consideration. The first limitation concerns the limit of detection. The total number of bacterial cells in 1 g human faeces (wet weight) is in the range of 1011 cells. If it is assumed that the average number of total bacterial cells per microscopic field is 1,000 it becomes clear that a species present at a concentration of 108 cells/g wet faeces will result on the average in only 1 cell per microscopic field. If a tenfold lower faecal dilution is used, the limit of detection is in the range of 107 cells/g wet faeces. If the number of cells per microscopic field is so low, a large number of microscopic fields have to be inspected to keep the statistical error small. In comparison, the detection limit for a given target organism in faeces may be considerably lower with the classical plating technique, provided less diluted samples are used and the growth of the background microbiota is suppressed by the use of selective media. Poor fluorescence may be another limitation of FISH. This may be due to an insufficient permeability of the cell envelope (Bidnenko et al., 1998) or due to a low content of ribosomes, which indicates that the cells are in a metabolically inactive state (Fegatella et al., 1998). Another explanation can be found in the fact that some probes do not bind effectively to the rRNA of the target organism although the in silico analysis indicates a perfect match with the target region. It has been demonstrated that the accessibility of the oligonucleotide probes depends on the target site within the 16S rRNA (Fuchs et al., 1998). It has been proposed that the dependence of probe binding on the target site is due to the intra- and intermolecular interactions of the ribosomal RNAs and proteins. The use of unlabelled so-called helper nucleotides, which are complementary to the neighbouring regions of the target site, may improve the accessibility of the labelled oligonucleotide probes to their target site considerably (Fuchs et al., 2000). Efforts have also been made to enhance the signal intensity in FISH. The signal-to-noise ratio not only depends on the quantum yield and molar absorbance of the fluorochrome used but also on the auto-fluorescence of the microbial cells and of the material present in the sample. Unspecific binding of the label may add to the background noise. Several alternatives for enhancing the probe signals have been tested. Indirect assays take advantage of reporter molecules such as biotin or digoxigenin bound to the oligonucleotide probe (Amann et al., 1992; Zarda et al., 1991). Following hybridisation, these reporter molecules may be detected by using avidin in the case of biotin, or specific antibodies in the case of digoxigenin. The antibodies are labelled with several molecules of a fluorescent dye or coupled to an enzyme which catalyses the formation of a product which can be visualised. A problem sometimes encountered with this labelling method stems from limitations in the accessibility of the bulky labelling complexes to their binding site in the fixed cell. It should be noted that these methods of signal enhancement have not found wide application. Even if a probe has been tested experimentally with representative faecal bacteria it is impossible to exclude the possibility that organisms other than the

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targeted one are detected. This may be the case if an unknown organism whose 16S rRNA sequence is not yet in the database happens to have the same target site and becomes fluorescently labelled although it is phylogenetically unrelated. This possibility always exists although it is probably a relatively rare event. If the targeted organisms are rod-shaped, the presence of fluorescently labelled cocci indicates that the latter are non-target organisms which have been labelled un-specifically.

14.3

Methods of characterising human gut microbiota

The human intestinal microbiota may have beneficial or adverse effects on human health. However, manipulating this complex system by dietary intervention requires better knowledge of this microbial community. To identify links between nutrition and microbiota mediated effects on health and disease, detailed microbial studies in human populations are urgently needed. On the one hand, the culture independent methods described above are ideally suited to perform such analyses, on the other hand they are time consuming as long as the enumeration is performed manually. The manual counting of bacteria is tedious and requires skilled personnel. There is therefore a need for more rapid, automated analyses of faecal samples on the basis of 16S rRNA targeted oligonucleotide probes. Two methods have been used to enumerate fluorescently labelled cells; image analysis and flow cytometry. 14.3.1 Automated microscopic detection of single cells by image analysis Image analysis aims to automate the counting of fluorescently labelled cells in faecal dilutions. In principle, the preparation of the faecal sample and the hybridisation are performed in the same way as described above. However, the fluorescent bacteria are not visualised and counted manually, but rather identified by image analysis. In principle this analysis consists of image acquisition, image storage, object recognition and object enumeration. Automated image acquisition requires a high precision motorised microscope stage whose movements in three dimensions can be programmed. Images are captured at predetermined positions using a charge coupled device (CCD) camera and stored as electronic files. The automatic image capture and the analysis routines require an optimal distribution of the bacterial cells in a faecal suspension on the slide. The slide co-ordinates for image capture may be chosen randomly or follow a programmed movement pattern within a well. In order to obtain representative cell counts, the concentration of cells on the surface should not vary excessively. One of the most difficult tasks during image capture is auto-focusing. Unfortunately, it is not sufficient to focus manually at one position of each well of a slide and to store the z-axis position, which determines the vertical position, i.e., the distance between the objective and the microscope stage, and to subsequently start the automated analysis routine. Owing to the fact that the z-axis position delivering a focused

Analysing gut microbiota 335 image may vary considerably from one x/y-axis position (movement in the horizontal plane) to another, it is necessary to recover a given z-axis position with an accuracy of only a few micrometres. Jansen and colleagues were the first ones who introduced an automated microscopy based image analysis system in gut microbiology for the analysis of intestinal bacteria in faecal samples (Jansen et al., 1999). Auto-focusing by this system involves the acquisition of a stack of images taken at various z-positions differing by 1 m. With this approach the proportion of out-of-focus images was approximately 1%. At the time when the protocol was published, the system afforded the analysis of 1,200 images within 20 h, which corresponds to 24 samples, each analysed twice, i.e., two wells per sample. In view of the improvements in computer technology since that time it is certain that the speed and accuracy of this analysis has greatly improved. Other microscopy-based operator-independent image analysis systems for the counting of multiple samples have been optimised for the analysis of marine (Pernthaler et al., 2003) or oral microbial ecosystems (Gmur et al., 2000; Singleton et al., 2001). Once the captured images have been stored in digital format, the fluorescing cells in the image have to be identified based on their fluorescence, size and shape. Commercial and non-commercial systems afford the adjustment of the recognition parameters to the type of samples analysed and to the actual oligonucleotide probe used. Potential problems may arise from the presence of weakly fluorescent non-target cells. To exclude these cells from being recognised as target cells a threshold of fluorescence intensity may be defined. The fidelity of the enumeration procedure may also be hampered by a high proportion of target cells whose fluorescence intensity is below the threshold. This problem not only impairs the automated cell recognition but also the manual identification of target cells. Poor specificity of a probe or unfavourable hybridisation conditions may account for this problem (see 14.3.3). However, poor fluorescence may also be due to reduced metabolic activity of the target cells which is associated with a lower content of ribosomal RNA. 14.3.2 Detection of single cells by flow cytometry (FCM) Flow cytometry (FCM) has found widespread application in medical diagnostics. It is used primarily for the enumeration of blood cells but has also been applied to other cells. One of the main advantages of using FCM is its unsurpassed speed of operation enabling the enumeration of up to 1,000 cells per second. However, the use of FCM in microbiology has been limited. This is primarily due to the small size of bacterial cells and the low signal intensity they produce (Steen, 1992). In spite of these disadvantages of bacterial cells in comparison to blood cells, a combination of FCM and FISH was successfully optimised for use in conjunction with rRNA targeted oligonucleotide probes (Wallner et al., 1993). While this study was done on pure cultures, FCM has also been applied to the analysis of environmental samples including faeces (Amann et al., 1990; Simon et al., 1995; Zoetendal et al., 2002).

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Fig. 14.5 Detection of fluorescently labelled bacteria with flow cytometry.

In FCM, cells in solution are passed through a small orifice so that they flow in single file through an illuminated volume where they can be registered electronically based on the light they scatter and the fluorescence they emit (Fig. 14.5). The features used for the discrimination of the cells include cell size, which is indicated by the forward angle light scatter (FSC), internal structure (granularity), which is indicated by the 90ë-angle light scatter (SSC) and intensity of fluorescence at one or several wavelengths depending on the available lasers and fluorochromes (Wallner et al., 1997). Using FISH combined with FCM (FISH-FCM), Rigottier-Gois and coworkers quantified five Bacteroides species in faecal samples from 20 healthy adults using species specific fluorescently labelled oligonucleotide probes (Rigottier-Gois et al., 2003) relative to all bacteria detected with the EUB338 probe, which essentially detects all members of the domain Bacteria. Bacteroides fragilis and Bacteroides vulgatus were detected in 90% and 85% of the individuals tested and represented on average proportions of 3.9 and 4.2% of total cells, respectively, with large variations between individuals. The other three species (B. ovatus, B. distasonis and B. putredinis) were considerably less frequent. The authors of this work used the NON338 probe, which is complementary to the EUB338 probe (Wallner et al., 1993) as a negative control because it does not bind to the 16S rRNA. Both EUB338 and NON338 were labelled with fluorescein isothiocyanate (FITC), while the oligonucleotide probes targeting the five Bacteroides species were labelled with Cy5. An argon ion laser (488 nm) was used for determining the FSC and the SSC and for exciting FITC labelled probes, while a red diode laser (635 nm) was employed to excite the Cy5 labelled probes. In addition to enumerating fluorescently labelled cells, FISH-FCM also makes it possible to determine the labelling

Analysing gut microbiota 337 efficiency for a given probe (Fuchs et al., 1998; Rigottier-Gois et al., 2003). The labelling efficiency may be deduced from the fluorescence intensity conferred by the species specific probes relative to the fluorescence intensity conferred by the EUB338 probe using the same fluorochrome for both probes. Rigottier-Gois and co-workers reported that the relative fluorescence intensity for the five Bacteroides species specific probes varied from 46 to 102% (Rigottier-Gois et al., 2003). In another study, which took advantage of FISH-FCM for investigating the faecal microbiota, an oligonucleotide probe targeting an uncultured Ruminococcus obeum-like bacterium was used to enumerate this organism in faecal samples collected from three healthy adults on three occasions during a period of four weeks (Zoetendal et al., 2002). In contrast to Rigottier-Gois et al. (2003), who determined the proportion of their target organisms relative to all bacteria as detected with the EUB338 probe, these researchers determined the cell counts. This was possible because they added fluorescent beads at known concentrations to the bacterial suspensions to be analysed. They also compared the bacterial counts obtained by FISH-FCM to the counts obtained by FISH combined with microscopic image analysis. The ratio between the two numbers varied between 0.64 and 1.67 and the mean proportions of the Clostridium coccoides-Eubacterium rectale group, which were also determined, were 16.9% as determined by microscopy and 13.5% as determined by FISH-FCM (Zoetendal et al., 2002). These results indicate that the two techniques give similar results. Taken together, FISH-FCM is a very powerful method for the enumeration of faecal bacteria, the main advantage being the speed of analysis. This feature makes FISH-FCM really suitable for high throughput analysis of faecal samples. Another advantage lies in the possibility to sort cells and thereby enrich targeted bacteria, including uncultured organisms, although this may be limited by cell aggregation. It is even possible to amplify ribosomal DNA fragments from sorted cells (Wallner et al., 1997). 14.3.3 Microbiota characterisation by DNA arrays The FISH methods described so far have the disadvantage that the number of organisms that can be targeted simultaneously is limited by the number of fluorochromes. As a consequence, the analysis of a complex microbial community like the one in the human intestinal tract is usually restricted to a panel of selected target organisms. Community analysis at a high level of resolution with FISH is very time consuming. DNA arrays or DNA chips may circumvent these limitations as they allow the parallel application of multiple probe sets. In principle, oligo- or polynucleotide probes are immobilised on a matrix such as glass, silicon or nylon membranes (Ramsay, 1998) and serve to capture labelled RNA or DNA molecules containing complementary sequences which allow the formation of DNA-DNA or DNA-RNA hybrids (Fig. 14.6).

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Fig. 14.6 Detection of 16S rRNAs with DNA arrays. Each spot on the array may target 16S rRNAs from different organisms. Labelled RNAs that hybridise with the immobilised capture probes will result in a signal.

So far, DNA chip technology has been used primarily on eukaryotic cells for monitoring the expression of target genes, large-scale gene discovery and the detection of mutations, but DNA chips have also been tested for their applicability to the analysis of microbial ecosystems (Guschin et al., 1997; Rudi et al., 2000; Small et al., 2001). However, the advantage of the array technology to detect a large number of target molecules simultaneously is seriously hampered by a number of problems that have to be solved. These include the necessity in microbial ecology to identify nucleic acids against a largely unknown genetic background (Peplies et al., 2003). Moreover, the recognition of single mismatches is crucial for the analysis of microbial ecosystems but difficult to realise as the optimal hybridisation conditions may differ between probes within a large set of probes. The adjustment of stringency for optimal discrimination as usually applied to conventional hybridisation assays is difficult to apply to DNA micro-array assays because one set of conditions cannot provide optimal target discrimination for all probes (El Fantroussi et al., 2003). Efficient labelling of the target DNA is a prerequisite for the identification of captured DNA or RNA. The use of labelled primers for the amplification by PCR of 16S rRNA genes is a possible way to label the target DNA (Peplies et al., 2003). However, it is well known that biases may be introduced by PCR (Speksnijder et al., 2001) and, therefore, the direct labelling of RNA isolated from a microbial community is preferable although the detection sensitivity may be considerably reduced. Multiple biotin labels introduced along the target RNA molecule were found to be ineffective while the use of labelled detector probes, which specifically bind to the target near the region that hybridises to immobilised capture probe was reported to be more efficient (Fig. 14.7); with

Analysing gut microbiota 339

Fig. 14.7 Detection of unlabelled 16S rRNAs by the combined use of capture probes and labelled indicator probes, which can bind to the captured target RNAs.

this strategy it was possible specifically to detect 16S rRNA from Geobacter chapellii in RNA extracts from soil (Small et al., 2001). Systematic studies have been performed to define the parameters that are crucial for specific hybridisation. The monitoring of the association/dissociation conditions would be a solution to this problem as the thermal dissociation analysis of all probe hybrids performed in parallel on a single DNA array would provide information to achieve a better discrimination between matched and mismatched duplexes (Liu et al., 2001). It was demonstrated that the analysis of the melting profile for each duplex provides information on the specificity of hybridisation, thus enabling the discrimination between target and non-target sequences (El Fantroussi et al., 2003). Applying neural network analyses, Urakawa and co-workers systematically investigated the effects of the position of a single base pair mismatch and the type of mismatch on the dissociation temperature and signal intensity of short DNA duplexes using oligonucleotide micro-arrays (Urakawa et al., 2002). The trained neural networks predicted the dissociation temperature with relatively high precision, while the prediction of the signal intensity, whose variability could partly be explained by the formamide concentration used, was poor. The analyses also indicated that the position of the mismatch at or near the 50 end is more important for determining the dissociation temperature than the type of mismatch. A systematic investigation defined a number of determinants for specific and highly discriminative DNA micro-array hybridisations and devised means for optimising the specific detection of target DNA or RNA (Peplies et al., 2003). It was shown that the standard FISH protocol can be transferred to the DNA micro-array analysis. Best hybridisation results were obtained by applying moderately stringent conditions, while discrimination of a mismatch near the terminus of a duplex was difficult to achieve. Steric hindrance was identified as an important factor that influences the binding efficiency of target molecules to

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the capture probes immobilised on a solid matrix. The size of the target DNA or RNA and the distance between the solid surface and its distance to the capture probe were demonstrated to be important determinants for the signal intensity. The application of poly(A) spacers of various lengths (6 to 24 nucleotides) and the addition of helper nucleotides that bind near the target sites of the capture probes improved the signal intensity in most cases considerably (Peplies et al., 2003). The results obtained by these researchers also suggested that fragmentation of the target DNA or RNA may lead to false positive signals. So far there is only one example for the application of the DNA array technology to the analysis of faecal microbiota (Wang et al., 2002). Three redundant 16S rRNA targeted 40-mer oligonucleotides were designed for each of 20 predominant human intestinal species and immobilised on glass slides. DNA was extracted from faecal samples of three healthy individuals and the complete 16S rRNA gene was amplified using Cy5 labelled dCTP in PCR. The labelled PCR products were applied to the oligonucleotide micro-arrays for hybridisation analysis. The results indicated some variability in the composition of the target organisms between the three individuals. In most cases, the redundant probes for a given target organism were well in agreement. Moreover, the bacteriocidal effect of an antibiotic treatment of an individual was clearly reflected in the results of the micro-array analysis performed on faecal samples from this individual. After antibiotic treatment of the targeted bacteria only Escherichia coli and Bacteroides fragilis could be detected. It may be concluded that micro-array based methods for the detection of bacteria with 16S rRNA target probe has been shown to work in principle. However, it is also clear that there are many parameters that have to be taken into consideration and optimised to avoid false positive or false negative signals. There is no doubt that an essentially error-free micro-array based detection method would greatly facilitate the microbial analysis of large numbers of samples. However, this method has to be further optimised for use on human faecal samples.

14.4 Using denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) for characterising microbiota DGGE and TGGE have become very popular tools among microbial ecologists since they allow the molecular characterisation of the diversity of microbial ecosystems and the monitoring of population dynamics (Muyzer, 1999). The technique is based on the amplification by PCR of 16S rRNA genes present in DNA extracted from environmental samples such as soil or faeces. However, in contrast to a normal PCR, one of the primers employed in the reaction differs from a normal PCR primer in that it has a long stretch of 40±50 G and C nucleotides attached to it. Following amplification, this so-called GC-clamp prevents the two DNA strands from dissociating completely, even if highly

Analysing gut microbiota 341 denaturing conditions are applied. The polyacrylamide gels employed for separating the amplification products are characterised by an increasing gradient of temperature (TGGE) or denaturing agents such as urea or formamide (DGGE). In principle, these gels afford the separation of DGGE-PCR products that differ in as little as one nucleotide (Sheffield et al., 1989). DGGE and TGGE have been employed to follow changes in microbiota composition with time and space. Each band in a D/TGGE-gel represents the population groups whose target sequence was amplified. To identify the species corresponding to a band in the gel, the DNA fragment of this band can be recovered from the gel and subjected to sequence analysis. Alternatively, bacteria isolated from the same sample are also subjected to D/TGGE-PCR and applied to the same gel. There are a number of excellent examples of the usefulness of this technique, including an investigation on the succession of microbial populations during colonisation of the intestinal tract of newborn babies (Favier et al., 2002). However, it is noteworthy that the use of a GCclamp primer may cause artefacts during annealing and that the heteroduplexes formed in PCR may lead to unspecific bands because the mismatched base pairs are less stable (Ruano and Kidd, 1992).

14.5

Future trends

14.5.1 The need for the isolation and phenotypic characterisation of new gut micro-organisms The knowledge of the identity and the numerical importance of microbial population groups in the human GI-tract are certainly important aspects for characterising this microbial ecosystem but insufficient to appreciate their possible role in host health and disease. To define the potential role of a given organism in a complex bacterial community it is necessary to culture the key organisms of this microbial community. Only then is it possible to investigate their physiology and biochemistry which are important determinants for their respective contribution to the metabolic processes catalysed by the gut microbiota. The same applies to the study of bacteria-bacteria and bacteriahost interactions. In view of the fact that the culture independent methods indicate that a large proportion of gut bacteria have eluded cultivation, it is obvious that the isolation and characterisation of new gut bacteria is still an important task. There is an urgent need for fast, reliable and easy-to-perform methods for analysing the composition of the human gut microbiota with high resolution and precision. The principal experimental approaches described above have the potential to be applied as high-throughput methods. There is no doubt that DNA arrays enable the quickest microbial analysis of faecal samples because the number of probes that can be encompassed on a microarray and hybridised with target nucleic acids is so high that a high resolution analysis is feasible. However, as mentioned above, there are still a number of serious problems to be

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solved to make this an easy-to-use method that will find widespread application in gut microbiology. It can be expected that the existing problems will be solved in the near future and that highly discriminative oligonucleotide probes targeting all relevant bacterial populations of the human gut will become available for large-scale human studies. These probes will have to be optimised for use on DNA arrays. The number of probes available for analysis of the human gut microbiota is still limited and needs to be increased. On the one hand, it will remain difficult to transform the signals of an oligonucleotide array into cell numbers of the corresponding target populations. On the other hand, the knowledge of the presence or absence of a bacterial population group is valuable information per se and, moreover, this method has the potential to be automated, which is an invaluable advantage. At present, FISH-FCM is the quickest available method for analysing human gut microbiota. The number of available and validated probes is sufficient to cover the microbial community of the gut at the group level to approximately 90%. In future, the proportion of probes targeting faecal bacteria at species level will increase and hence the resolution and the potential relevance of the results will be improved. While the FISH-FCM analysis itself is essentially automated, the sample preparation is not, but could be facilitated and accelerated by automation. It can also be expected that the possibility of sorting bacteria with FCM may lead to the isolation of previously undescribed bacteria. Future efforts will also be directed at the in situ detection of bacterial activities. The ultimate objective is to study the in vivo effects of dietary and host factors on interactions of microbial populations in the human gastrointestinal tract. Principal approaches include the in situ detection of mRNA which has been shown to work in model systems (Hodson et al., 1995; Holmstrom et al., 1999; Tolker-Nielsen et al., 1997). Since enzyme expression usually correlates with enzyme activity, the level of specific mRNAs may reflect the activity of the corresponding enzyme. However, a number of obstacles have to be overcome to make mRNA detection a widely applied method. In contrast to 16S rRNA, mRNA has a shorter lifetime and is usually present in smaller concentrations. Hence, the detection of mRNAs with fluorescently labelled oligonucleotide probes, which works well with rRNA, is difficult to achieve because the number of target mRNA molecules in a cell is too low to render the cell fluorescent (Hodson et al., 1995). The in situ amplification of mRNAs is a more promising approach, but it depends on the effective permeabilisation of the cell wall so that nucleotides, primers and enzymes can enter the cell where they catalyse the reverse transcription and subsequent linear amplification of the resulting c-DNA. To render the cells detectable, the primers or the nucleotides used in PCR have to be labelled. For example, digoxigenin (DIG)-labelled dUTP has often been used because it can be recognised by antibodies conjugated to enzymes (e.g. alkaline phosphatase) capable of converting suitable precursors into detectable molecules. Alternatively DIG may be recognised with anti-DIG antibodies labelled with a fluorescing dye such as fluorescein. The loss of label from the

Analysing gut microbiota 343

Fig. 14.8 In situ detection of metabolic activity using fluorogenic substrates.

permeabilised cells by diffusion may be a major problem of detection (Hodson, 1995). Methodological improvements in the future may help to make the in situ detection of mRNA applicable to the analysis of gene expression in gut microorganisms. The detection of enzyme activity at the cellular level may also be accomplished by the use of fluorogenic substrates whose uptake into the cell and the ensuing cleavage results in the accumulation of the cleaved-off fluorescence moiety rendering the cell detectable by FISH or FISH-FCM (Lewis et al., 1994). The formation of a fluorescent dye in the cytoplasm requires that the transport system and the enzyme catalysing the cleavage recognise the fluorogenic substrate in the same way as the natural substrate (Fig. 14.8). The loss of the fluorescent product by leakage out of the cell may be a problem which can be largely prevented by the use of mildly thiol-reactive fluorochromes which upon their cleavage from the fluorogenic substrate may react with cysteine-containing cellular proteins. Presently, the application of these functional probes is restricted to a small number of enzymes such as galactosidase, -glucosidase and -glucuronidase because the spectrum of the available fluorogenic substrates is limited. Nevertheless, some of these enzymes are of some relevance for the human gut ecosystem. The use of fluorogenic substrates has a lot of potential because their application can be combined with the 16S rRNA approach which reveals at the same time the metabolic activity and the identity of bacteria at the cellular level. It should be noted, however, that cells already fixed for the application of 16S rRNA targeted oligonucleotides, usually lose enzymatic activity. Consequently, the metabolic labelling has to be

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done with live cells first and the phylogenetic labelling has to be applied in a second step. It can be assumed that the combination of phylogenetic and metabolic labelling will find a more widespread application in the future and that the spectrum of the available fluorogenic substrates will be increased.

14.6

Sources of further information and advice

The phylogenetic analysis of ribosomal RNA sequences and the design of rRNA targeted oligonucleotide probes require the use of up-to-date sequence data sets and analysis tools. The internet sites shown below provide access to databases of 16S rRNA sequences and enable the user to perform sequence alignments, integrate new sequences into the database, obtain phylogenetic trees and design diagnostic oligonucleotide probes. These internet sites contain additional links related to phylogeny and microbial ecology. GenBank EMBL The European Ribosomal Database The Ribosomal Database Project The Arb project probeBase.net

14.7

http://www.ncbi.nlm.nih.gov http://www.ebi.ac.uk http://oberon.fvms.ugent.be:8080/ rRNA/index.html http://rdp.cme.msu.edu/html/ http://www.arb-home.de/ http://www.microbial-ecology.net/ probebase/index2.html

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Eubacterium formicigenerans Holdeman and Moore 1974 as Dorea formicigenerans gen. nov., comb. nov., and description of Dorea longicatena sp. nov., isolated from human faeces'. Int J Syst Evol Microbiol, 52, 423±428. TOLKER-NIELSEN T, HOLMSTROM K, MOLIN S (1997) `Visualization of specific gene expression in individual Salmonella typhimurium cells by in situ PCR'. Appl Environ Microbiol, 63, 4196±4203. URAKAWA H, NOBLE P A, EL FANTROUSSI S, KELLY J J, STAHL D A (2002) `Single-base-pair discrimination of terminal mismatches by using oligonucleotide microarrays and neural network analyses'. Appl Environ Microbiol, 68, 235±244. WALLNER G, AMANN R, BEISKER W (1993) `Optimizing fluorescent in situ hybridization with rRNA-targeted oligonucleotide probes for flow cytometric identification of microorganisms'. Cytometry, 14, 136±143. WALLNER G, FUCHS B, SPRING S, BEISKER W, AMANN R (1997) `Flow sorting of microorganisms for molecular analysis'. Appl Environ Microbiol, 63, 4223±4231. WANG R F, BEGGS M L, ROBERTSON L H, CERNIGLIA C E (2002) `Design and evaluation of oligonucleotide-microarray method for the detection of human intestinal bacteria in fecal samples'. FEMS Microbiol Lett, 213, 175±182. WILSON K H, BLITCHINGTON R B (1996) `Human colonic biota studied by ribosomal DNA sequence analysis'. Appl Environ Microbiol, 62, 2273±2278. WOESE C R (1987) `Bacterial evolution'. Microbiol Rev, 51, 221±271. WOESE C R, KANDLER O, WHEELIS M L (1990) `Towards a natural system of organisms: proposal for the domains Archaea, Bacteria and Eucarya'. Proc Natl Acad Sci USA, 87, 4576±4579. ZARDA B, AMANN R, WALLNER G, SCHLEIFER K-H (1991) `Identification of single bacterial cells using digoxigenin-labelled, rRNA-targeted oligonucleotides'. J General Microbiol, 137, 2823±2830. ZOETENDAL E G, BEN-AMOR K, HARMSEN H J, SCHUT F, AKKERMANS A D, DE VOS W M (2002) `Quantification of uncultured Ruminococcus obeum-like bacteria in human fecal samples by fluorescent in situ hybridization and flow cytometry using 16S rRNAtargeted probes'. Appl Environ Microbiol, 68, 4225±4232.

15 Dietary lipids and immune function P. C. Calder, University of Southampton, UK

15.1 Introduction: the immune system in health, disease and ageing 15.1.1 The immune system in health The immune system acts to protect the host from infectious agents that exist in the environment (bacteria, viruses, fungi, parasites). It serves to distinguish `non-self ' from `self '. In addition the immune system plays an important role in the identification and elimination of tumour cells. Thus, an effective and efficient immune system is central to host defence against infectious diseases and cancer. A detailed description of the components of the immune system, how they work and how they interact is beyond the scope of this chapter. However, details may be found in standard immunology textbooks (e.g. Abbas et al., 1994). Suffice it to say that the immune system responds to challenge (e.g. a bacterial infection) with an increase in the activity of certain components that act to eliminate the source of the challenge. The human immune system has the capacity to respond to millions of antigens (these are the precise peptide components that are recognised as `non-self ' by the immune system and which trigger an immune response). This response will include both non-specific and specific actions that will involve a variety of cell types, mediators and chemical agents. The exact nature of the response will depend upon the origin and nature of the antigen (and so the origin and nature of the challenge). The immune response is, by definition, highly destructive and therefore it must be regulated in order that it does not proceed for a longer duration than is required and that it does not cause excessive damage to host tissues. Clearly, because of the many differing components involved in the immune response its regulation will be highly complex. However, one paradigm that has developed

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Fig. 15.1 The Th1, Th2 paradigm. Solid arrows indicate producers; broken arrows indicate regulators. IFN, interferon; IL, interleukin; Th, T helper cell.

to explain the promotion and regulation of an appropriate response is the socalled Th1, Th2 paradigm (Mossman and Sad, 1996). This describes the differentiation of the helper T lymphocyte (Th cell; defined by the appearance of the protein CD4 on its surface) along one of two phenotypic pathways (Fig. 15.1). The pathway followed depends upon the nature of the antigen being presented to the undifferentiated T helper cell and the cytokines present; cytokines are peptide mediators that have a hormone-like regulatory role within the immune response. An intracellular antigen, which will result from the phagocytic uptake of a bacterium, and the presence of interleukin (IL)-12, produced by the antigen-presenting cell, will promote differentiation along the Th1 pathway (Fig. 15.1). The Th1 cells thus produced will generate IL-2 and interferon (IFN)- . IL-2 will promote the proliferation of antigen-specific T lymphocytes while IFN- will activate the cells involved in elimination of bacteria, viruses, fungi and tumour cells (e.g. monocytes, macrophages, cytotoxic T lymphocytes, natural killer cells) and will promote the generation of antigen-specific immunoglobulin (Ig) G by B lymphocytes. Such antibodies can coat pathogens to facilitate their recognition and uptake by phagocytes. Extracellular antigens, for example due to infection with helminthic worms, and the presence of IL-4 will promote differentiation along the Th2 pathway (Fig. 15.1). The Th2 cells thus produced will generate IL-4, IL-5 and IL-13 (amongst other cytokines). IL-4 will promote the generation of antigen-specific IgE by B lymphocytes. The IgE will bind to and cross-link IgE receptors on the

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surface of mast cells and induce the extracellular release of the contents of mast cell granules. IL-5 promotes the activation of eosinophils. Mast cells and eosinophils are the mediators of host defence against extracellular parasites. Thus, Th1 and Th2 cells are involved in promoting an appropriate immune response to different types of infectious organisms. The final element of the Th1, Th2 paradigm is cross-regulation; IFN- inhibits the differentiation and activity of Th2 cells while IL-4 inhibits the differentiation and activity of Th2 cells (Fig. 15.1). Clearly this serves to cause a transient `skewing' of the immune response while an infectious agent is present. However, once the source of the infection is eliminated the system will return to the basal state in preparation for any subsequent infections. 15.1.2 The immune system in disease Whilst the capacity to mount vigorous Th1 or Th2 type responses is central to effective host defence against the range of possible infectious organisms, a number of human diseases are associated with inappropriate activation or activity of the immune response. These diseases appear to be associated with an inherent, inappropriate `skewing' towards either a Th1 or Th2 type response. There is a genetic predisposition towards such skewing, with susceptibility genes frequently being associated with antigen presentation (Howell et al., 2002). It is likely that the combination of genetic predisposition and certain environmental and/or lifestyle factors allows the disease to manifest itself. In some individuals the immune system responds to presentation of a host antigen (or a normally benign foreign one) rather than a foreign antigen; this triggers a Th1±type response with the destructive actions of the immune system targeted at those body sites where the antigen (or similar ones) is expressed. An example of such a disease is rheumatoid arthritis. In rheumatoid arthritis susceptibility genes map to antigen presentation proteins and this somehow results in host antigen being presented to T cells, which differentiate along the Th1 path. The Th1-type cytokines promote the activity of macrophages and the generation of auto-antibodies by B lymphocytes. The Th1type cytokines also suppress the Th2-type response and so skew the system towards the Th1 phenotype (Panayi, 1999). The host's synovial joints become infiltrated by activated T cells, macrophages and B cells. The macrophages produce a range of cytokines including tumour necrosis factor (TNF)-, IL-1 and IL-6 (Feldmann and Maini, 1999). Some effects of these so-called inflammatory cytokines are listed in Table 15.1. Clearly they have a role in innate immunity, in activating cellular immune responses and in co-ordinating the whole body response to infection and injury. However, in excess, they are involved in causing a range of local and systemic responses that are detrimental to the host. Macrophages will also produce the matrix proteases that degrade the cartilage and bone of the host. Other diseases in which an inappropriate Th1-type response appears to be involved include Crohn's disease, multiple sclerosis and atherosclerosis. Together these diseases are termed chronic inflammatory diseases.

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Table 15.1 List of some effects of the classic pro-inflammatory cytokines TNF, IL-1 and IL-6. Note that not all cytokines affect all responses listed equipotently Site of action

Biological effect

Hyothalamus Liver Vascular endothelium

Induction of fever Synthesis of acute phase proteins Activation ÿ! upregulation of adhesion molecules, chemokines, inducible nitric oxide synthase Activation ÿ! bone resorption Activation ÿ! matrix metalloproteinase synthesis ÿ! extracellular matrix degradation Activation of proteolysis ÿ! muscle wasting Activation of lipolysis; induction of insulin resistance Synthesis of TNF, IL-1, IL-6 and IL-8

Osteoclasts Fibroblasts Skeletal muscle Adipose tissue Monocytes and macrophages T cells B cells Bone marrow Monocytes, macrophages Neutrophils Kidney mesangial cells

Activation ÿ! proliferation and synthesis of IFN- Activation ÿ! antibody production Induction of haematopoiesis Induction of phospholipase A2 and COX-2 ÿ! synthesis of prostaglandins Chemotaxis Activation ÿ! degranulation, respiratory burst Chemotaxis Reactive oxygen species production

An inappropriate predisposition to skewing towards a Th2 response also occurs. Here the antigens involved are frequently normally benign foreign antigens, such as those present in cow's milk, egg, house dust mite, tree or grass pollen and are referred to as allergens since they can trigger allergy in sensitised individuals (Romagnani, 2000). The Th2 response results in production of allergen-specific IgE, which, when and where allergen is present, will induce mast cell degranulation, and in activation of eosinophils. This process can be termed allergic inflammation. 15.1.3 Ageing and the immune system Changes in the immune system appear to be associated with ageing. A full description of these is beyond the scope of this chapter and may be found elsewhere (Lesourd and Mazari, 1999; Lesourd et al., 2002). It appears that these age-related changes are not a general decline but rather a dysregulation. There is a decrease in the number of T cells, Th cells and cytotoxic T cells in the bloodstream with increasing age, and there appears to be a decrease in some T cell functions such as proliferation and production of IL-2. Whether IFN- production declines with age is not clear at present, although there are some reports that it does. Primary antibody responses to vaccination decline with age and the antibodies produced have lower antigen affinity. These changes are in part due to changes in B cell phenotypes; ageing is associated with a decline in

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the B cells that make antibodies against foreign antigens and with an increase in those that make auto-antibodies. There are a number of reports of increased production of Th2-type cytokines with age. Together these observations suggest that ageing is characterised by a progressive switch towards Th2-type responses. This may mean that elderly subjects are more susceptible to bacterial, viral and fungal infections, since they may not be able to mount a sufficiently strong Th1type response, and that they become progressively more responsive to allergens. Despite the apparent switch towards a Th2-type phenotype with ageing, monocyte/macrophage responses, particularly production of inflammatory cytokines, appear enhanced with ageing. For example, TNF- concentrations were higher in the plasma of 81-year-old subjects than in plasma from 18 to 30 year olds (Bruunsgaard et al., 2000). Likewise, there was a significant positive linear correlation between age and plasma TNF- concentration among subjects aged 20 to 100 years (Paolisso et al., 1998). Endotoxin-stimulated blood monocytes from older subjects produce greater amounts of IL-1 and IL-6 than those from young subjects (Meydani et al., 1991). Thus, ageing appears to be associated with an increased tendency towards inflammatory cytokine production. Given the effects of these cytokines (Table 15.1), this proinflammatory tendency might contribute to age-related chronic diseases and might also promote adverse responses to injury and surgery. 15.1.4 Adhesion molecules in health, disease and ageing Adhesion molecules are cell surface proteins involved in the cell-to-cell communication which contributes to the movement of leukocytes between body compartments. Such interactions involve adhesion molecules on the vascular endothelial cell surface and their ligand adhesion molecules on the leukocyte surface. These molecules serve to bind circulating leukocytes and to promote their movement into the sub-endothelial space. While this movement of leukocytes is important in mounting appropriate inflammatory and immune responses and in the homing of leukocytes to lymphoid organs, it also appears to play an important role in the development of atherosclerosis and chronic inflammatory diseases (Munro, 1993; Faull, 1995). Among the key adhesion molecules are vascular cell adhesion molecule 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1) and E-selectin. The expression of these molecules is up-regulated by inflammatory stimuli such as TNF- and reactive oxygen species. Soluble forms of VCAM-1, ICAM-1 and E-selectin (termed sVCAM-1, sICAM-1 and sE-selectin) are found in plasma, most likely as the result of shedding from the surface of activated endothelial cells. Thus, soluble adhesion molecule concentrations are believed to reflect endothelial inflammation. Indeed, the plasma concentrations of sVCAM-1, sICAM-1 and sE-selectin are elevated in inflammatory conditions (e.g. rheumatoid arthritis) in which there is increased expression of the cellular forms on endothelial and other cells (Gearing and Newman, 1993). Plasma concentrations of sICAM-1, sVCAM-1

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and sE-selectin are higher in individuals with cardiovascular disease than in controls and there is a positive correlation between the extent of atherosclerosis and the plasma concentrations of sICAM-1 (Rohde et al., 1998) and sVCAM-1 (Peter et al., 1997; de Caterina et al., 1997; Rohde et al., 1998). It was reported that sICAM-1 concentration predicts future myocardial infarction (Ridker et al., 1998) and that sICAM-1 concentration is a molecular marker of atherosclerosis and of other forms of coronary heart disease that is independent of other risk factors (Hwang et al., 1997). Ageing appears to be associated with increased endothelial inflammation as indicated by circulating soluble adhesion molecule concentrations. Positive correlations between age and the concentrations of sVCAM-1, sICAM-1 and sEselectin have been reported in Japanese (Morisaki et al., 1997), American (Hwang et al., 1997; Rohde et al., 1999) and British (Miles et al., 2001) subjects. For example, plasma concentrations of sICAM-1, sVCAM-1 and sEselectin were on average 60%, 38% and 14% higher in the blood of apparently healthy British males aged 55 to 75 years than in the blood of those aged 18 to 40 years (Miles et al., 2001).

15.2

Dietary fatty acids: nomenclature, sources and intakes

In Western countries an adult eats on average 75 to 150 g of fat each day and fat contributes 30 to 45% of dietary energy. By far the most important component of dietary fat in quantitative terms is triacylglycerol, which in most diets constitutes > 95% of dietary fat. Each triacylglycerol molecule is composed of three fatty acids esterified to a glycerol backbone. Other components of dietary fat, such as phospholipids, also contain fatty acids in their structure. Thus, fatty acids are major constituents of dietary fat. Because of the wide range of foods consumed, the human diet contains a great variety of fatty acids. The most abundant fatty acids have straight chains of an even number of carbon atoms. The chain lengths vary from 4 (e.g. in milk) to 30 (e.g. in some fish oils) and may contain double bonds (unsaturated fatty acids; Fig. 15.2). It is the nature of the constituent fatty acids (their chain length and degree of unsaturation) that gives a fat its physical properties. Fatty acids are often referred to by their common names, but are more correctly identified by a systematic nomenclature (Table 15.2). This nomenclature indicates the number of carbon atoms and the number and position of double (unsaturated) bonds in the chain (see Fig. 15.2). It is the position of the first double bond in the hydrocarbon chain that is indicated by the n-7, n-9, n-6 or n-3 part of the shorthand notation for a fatty acid. Note that n-6 and n-3 are sometimes referred to as omega-6 and omega-3. Mammalian cells are able to synthesise (from non-fat precursors) saturated fatty acids and unsaturated fatty acids of the n-9 and n-7 series but lack the delta12 and delta-15 desaturase enzymes (found in most plants) for insertion of a double bond at the n-6 or n-3 position (Figs 15.2 and 15.3). Thus, mammalian

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Fig. 15.2 The structure of some fatty acids.

cells cannot synthesise n-6 or n-3 polyunsaturated fatty acids (PUFAs) de novo. The n-6 and n-3 fatty acids are essential substrates for many of the major regulatory lipids in the body and as they cannot be synthesised in the body, they must be obtained from the diet. The commonly consumed PUFAs are linoleic acid (18:2n-6) and -linolenic acid (18:3n-3; ALA). Plant tissues and plant oils tend to be rich sources of linoleic and -linolenic acids. For example, linoleic acid contributes over 50% and often up to 80% of the fatty acids fond in corn, sunflower, safflower and soybean oils. Rapeseed and soybean oils are also good sources of ALA since this fatty acid contributes between 5 and 15% of the fatty acids present. However, the richest source of ALA is linseed oil (also known as flaxseed oil) in which ALA can contribute as much as 60% of the fatty acids present. Once consumed in the diet, linoleic acid and ALA can be converted to the longer chain, more unsaturated derivatives (Fig. 15.3). This process occurs mainly in the liver. Thus linoleic acid is converted via -linolenic (GLA; 18:3n-6) and dihomo- -linolenic (DGLA; 20:3n-6) acids to arachidonic acid (20:4n-6) (Fig. 15.2). Likewise, ALA is converted to eicosapentaenoic acid (EPA; 20:5n-3) (Fig. 15.2). There is some controversy about the extent to which docosahexaenoic acid (DHA; 22:6n-3) can be synthesised from EPA in humans. According to the United Kingdom Adult Survey conducted in 1986, the daily diet of the average adult male in the United Kingdom contains 42 g saturated fatty acids, 31 g monounsaturated fatty acids (mainly oleic acid) and 15.8 g

Table 15.2 Fatty acid nomenclature and sources Systematic name

Trivial name

Shorthand notation

Sources

Decanoic Dodecanoic Tetradecanoic Hexadecanoic

Capric Lauric Myrsitic Palmitic

10:0 12:0 14:0 16:0

Octadecanoic 9-Hexadecenoic 9-Octadecenoic

Stearic Palmitoleic Oleic

18:0 16:1n-7 18:1n-9

9,12-Octadecadienoic

Linoleic

18:2n-6

9,12,15-Octadecatrienoic

-Linolenic

18:3n-3

6,9,12-Octadecatrienoic 11,14,17-Eicosatrienoic

-Linolenic Mead

18:3n-6 20:3n-9

8,11,14-Eicosatrienoic 5,8,11,14-Eicosatetraenoic

Dihomo- -linolenic Arachidonic

20:3n-6 20:4n-6

5,8,11,14,17-Eicosapentaenoic 7,10,13,16,19-Docosapentaenoic 4,7,10,13,16,19-Docosahexaenoic

Eicosapentaenoic Docosapentaenoic Docosahexaenoic

20:5n-3 22:5n-3 22:6n-3

De novo synthesis; coconut oil De novo synthesis; coconut oil De novo synthesis; milk De novo synthesis; milk; eggs; Animal fats; meat; cocoa butter; Palm oil (other vegetable oils contain lesser amounts); fish oils De novo synthesis; milk; eggs; animal fats; meat; cocoa butter Desaturation of palmitic acid; fatty fish; fish oils Desaturation of stearic acid; milk; eggs; animal fats; meat; cocoa butter; Most vegetable oils especially olive oil Cannot be synthesised in mammals; some milks; eggs; animal fats; meat; most vegetable oils especially corn, sunflower, safflower and soybean oils; green leaves Cannot be synthesised in mammals; green leaves; some vegetable oils especially rapeseed, soybean and linseed oils Synthesised from linoleic acid; borage and evening primrose oils Synthesised from oleic acid; indicator of essential fatty acid deficiency Synthesised from -linolenic acid Synthesised from linoleic acid via -linolenic and dihomo- -linolenic acids; meat Synthesised from -linolenic acid; fatty fish; fish oils Synthesised from -linolenic acid via eicosapentaenoic acid Synthesised from -linolenic acid via eicosapentaenoic acid; fatty fish; fish oils

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Fig. 15.3

357

The biosynthesis of unsaturated fatty acids; CLA, conjugated linoleic acid.

PUFAs (Department of Health, 1994). Table 15.3 shows intakes of various PUFAs in adult men and women in the United Kingdom. The main PUFA in the diet is linoleic acid followed by ALA. Longer chain PUFAs are consumed in lower amounts than linoleic and -linolenic acids (Table 15.3; British Nutrition Foundation, 1999). Estimates of the intake of arachidonic acid intakes in Western populations vary between 50 and 300 mg/day for adults (Table 15.3). Table 15.3 Habitual intakes of PUFAs in adults in the United Kingdom (g/day). Data are from Kew et al. (2003a) Men (n = 88) Median Linoleic acid Arachidonic acid Total n-6 PUFAs ALA EPA Docosapentaenoic acid DHA Total n-3 PUFAs

Women (n = 62)

10th 90th percentile percentile

Median

10th 90th percentile percentile

13.2 0.21 13.7 1.4 0.15 0.09

7.2 0.10 7.5 0.1 0.04 0.04

24.1 0.35 24.7 2.5 0.42 0.17

11.1 0.17 11.5 1.3 0.11 0.08

6.8 0.05 7.1 0.1 0.06 0.02

18.1 0.31 18.5 1.9 0.39 0.15

0.23 2.0

0.07 1.2

0.45 3.5

0.17 1.7

0.09 1.1

0.42 2.9

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EPA and DHA are found in high quantities in `fatty' fish (e.g. herring, mackerel, fresh tuna, sardines, salmon) and in the commercially available products known as fish oils. The latter have two origins: the body oils of fatty fish or the oils extracted from the livers of fish which live in warmer waters (e.g. cod). EPA and DHA comprise 20% to 30% of the fatty acids in a typical preparation of fish oil, which means that a one gram fish oil capsule can provide 200 to 300 mg of EPA plus DHA. In the absence of fatty fish or fish oil consumption, ALA is the main dietary n-3 PUFA (Table 15.3). Average intake of the long-chain n-3 PUFAs in the United Kingdom is estimated at < 250 mg per day (British Nutrition Foundation, 1999). Conjugated linoleic acid (CLA) is a term used to describe the mixture of isomers of linoleic acid with conjugated double bonds (i.e. the two double bonds are separated by only one single bond; see Fig. 15.2). The double bonds can be in either the cis or trans configuration (all double bonds in the fatty acids described above and in Table 15.1 are in the cis configuration) and can be in any position in the carbon chain. Thus, there are a large number of isomers of CLA. The most frequently encountered isomers have the double bonds in positions 8 and 10, 9 and 11, 10 and 12 or 11 and 13. CLA is formed as a result of the metabolism of linoleic acid and ALA in the rumen, and so CLA is found in the milk and meat of ruminants. The predominant (> 90%) CLA in cow's milk is the cis-9, trans-11 isomer. Estimates of intakes of CLA from the Western diet range from 15 to several hundred mg/day. However, average intakes of CLA appear to be 100 to 200 mg/day, with milk and dairy products contributing > 60% of this (Ens et al., 2001; Ritzenthaler et al., 2001; Voorrips et al., 2002). 15.2.1 The fatty acid composition of cells of the immune system can be modulated by dietary fatty acids The principal role of fatty acids in cells of the immune system is as components of cell membrane phospholipids, and human inflammatory and immune cells tend to be rather rich in arachidonic acid. However, there is some variation in the fatty acid composition of human immune cells, which may reflect, in part, variation in the dietary intake of various fatty acids. Table 15.4 presents the fatty acid composition of blood mononuclear cells (an 85:15 mixture of lymphocytes and monocytes) taken from 67 healthy males aged 20 to 40 years living in the southern United Kingdom. As expected, arachidonic acid comprises about 20% of total fatty acids but varies from 12.6 to 27.6%. In contrast to the high content of arachidonic acid, EPA is a fairly minor constituent of human mononuclear cells comprising an average of < 1% of total fatty acids. The data in Table 15.4 are consistent with those recently reported for 150 men and women living in the southern United Kingdom (Kew et al., 2003a). If some of the variation in the fatty acid composition of human immune cells is related to dietary variation, then the fatty acid composition of human immune cells should be amenable to dietary manipulation, as seen with experimental and farm animal studies. A number of studies have investigated this possibility.

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Table 15.4 Fatty acid composition of human peripheral blood mononuclear cells. Data are for 67 males aged 20 to 40 years and are previously unpublished g/100 g total fatty acids Fatty acid

Mean

Standard deviation

10th percentile

90th percentile

Myristic acid Palmitic acid Stearic acid Oleic acid Linoleic acid GLA ALA DGLA Arachidonic acid EPA Doco sapentaenoic acid DHA

0.55 19.9 21.3 17.2 8.6 0.08 0.17 2.7 19.5 0.8 2.9 2.3

0.33 2.5 3.1 3.1 0.9 0.22 0.38 1.1 3.0 0.9 2.1 1.1

0.18 16.5 18.9 14.1 7.3 0 0 1.7 15.4 0.1 1.2 1.2

1.02 22.4 23.8 20.7 9.9 0.35 0.66 4.6 23.7 1.5 4.7 3.9

A dietary intervention in which healthy human volunteers consumed a diet providing extra oleic acid at the expense of saturated fatty acids for eight weeks resulted in a significant increase in the content of oleic acid in peripheral blood mononuclear cells: 20.3 Ô 1.0% of total fatty acids vs. 24.5 Ô 0.8% of fatty acids (Yaqoob et al., 1998). However, in another study, increased intake of oleic acid (6.5 g/day for 12 weeks) from olive oil capsules did not significantly alter the oleic acid content of human blood mononuclear cells (Yaqoob et al., 2000). The different findings of these two studies may relate to the amount of oleic acid provided. In the dietary intervention study oleic acid intake was increased from 11 to 18% of total energy intake, representing an increased intake of 15 to 20 g/day. An increased intake of linoleic acid (6.5 g/day for 12 weeks) from sunflower oil capsules did not significantly alter the fatty acid composition of human blood mononuclear cells (Yaqoob et al., 2000). Increased intake of GLA (1 g/day for 12 weeks) from evening primrose oil capsules increased the DGLA content of human blood mononuclear cells by about 50% but this was not statistically significant (Yaqoob et al., 2000). Arachidonic acid content did not increase. A more modest intake of GLA (0.7 g/day for 12 weeks) by elderly humans increased the DGLA content of mononuclear cells by about 50%, again without an increase in arachidonic acid content (Thies et al., 2001a). DGLA increased in human neutrophils following consumption of 3 or 6, but not 1.5, g GLA/day for three weeks (Johnson et al., 1997). Increased GLA consumption and appearance of DGLA was not associated with a change in the arachidonic acid content of human neutrophils (Johnson et al., 1997). Increased intake of arachidonic acid (0.7 g/day for 12 weeks) by elderly humans increased the arachidonic acid content of mononuclear cells from about 20% to about 23% of fatty acids (Thies et al., 2001a).

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Kelley et al. (2001) reported that intake of 3.9 g CLA/day in the form of mixed isomers in capsules resulted in significant incorporation of CLA isomers into blood mononuclear cells (total CLA content increased from about 0.12 to about 1% of total fatty acids) over nine weeks. In comparison, Albers et al. (2003) reported CLA contents of 0.9% of total fatty acids and about 1.3% of fatty acids in mononuclear cells before and after 85 days of supplementation of the diet with 1.6 g CLA/day. Supplementing the diet of human volunteers with fish oil in capsules results in incorporation of both EPA and DHA into blood neutrophils (Lee et al., 1985; Sperling et al., 1993; Gibney and Hunter, 1993; Luostarinen and Saldeen, 1996; Healy et al., 2000), monocytes (Lee et al., 1985; Fisher et al., 1990; Gibney and Hunter, 1993), T lymphocytes (Gibney and Hunter, 1993), B lymphocytes (Gibney and Hunter, 1993) and mononuclear cells (Endres et al., 1989; Molvig et al., 1991; Caughey et al., 1996; Yaqoob et al., 2000; Thies et al., 2001a). This incorporation is again largely at the expense of arachidonic acid. Both EPA and DHA are readily taken up, with near-maximal incorporation occurring within four weeks (Yaqoob et al., 2000; Thies et al., 2001a). Incorporation of both fatty acids occurs in a dose response manner (Healy et al., 2000). Once dietary supplementation ceases, EPA is lost from the cells within eight weeks, but DHA is better retained (Yaqoob et al., 2000). An ALA intake of about 18 g/day for eight weeks resulted in an increased content of ALA in human mononuclear cells (from 0.2 to 0.6% of fatty acids) as well as a small increase in the content of EPA and docosapentaenoic acid (Kelley et al., 1993). There was no change in content of arachidonic acid or DHA. In another study, an ALA intake of about 14 g/day for four weeks resulted in an increased content of ALA in human mononuclear cells (from 0.1 to 0.3% of fatty acids; Caughey et al., 1996) and neutrophils (from 0.4 to 0.6% of fatty acids; Mantzioris et al., 1994). This was associated with a small decrease in the content of arachidonic acid in mononuclear cells, but not in neutrophils, and with an increase in the content of EPA but not DHA (Mantzioris et al., 1994; Caughey et al., 1996). In contrast to these studies using large amounts of ALA, increased intake of smaller amounts of ALA from capsules (2 g/day for 12 weeks) by elderly human volunteers did not alter the fatty acid composition of blood mononuclear cells (Thies et al., 2001a). Likewise, an increase in ALA intake from capsules of 4 g/day for 12 weeks had little impact on the fatty acid composition of human neutrophils (Healy et al., 2000). A recent study showed that consumption of a spread providing a daily ALA intake of 4.5 g for six months did not influence the fatty acid consumption of blood mononuclear cells (Kew et al., 2003b). However, consuming a spread providing a daily intake of 9.5 g ALA/day significantly increased the EPA, but not the DHA, content of mononuclear cells by 30% (Kew et al., 2003b). Taken together these studies suggest that quite significant changes in fatty acid consumption might be required to alter the fatty acid composition of cells of the human immune system. Consistent patterns that have emerged from human studies are:

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· the oleic and linoleic acid contents of human immune cells are difficult to manipulate · increased intake of GLA increases DGLA content · markedly increased intake of arachidonic acid increases arachidonic acid content · markedly increased intake of CLA increases CLA content · fish oil intake increases EPA and DHA contents (and decreases arachidonic acid content) · an increase in intake of ALA of at least 4 g/day is necessary to alter fatty acid composition · a substantial increase in ALA intake increases EPA, but not DHA, content.

15.3 Fatty acid composition of immune cells and immune function: eicosanoids 15.3.1 Arachidonic acid as an eicosanoid precursor The principal functional role for arachidonic acid in inflammatory and immune cells is as a substrate for synthesis of the eicosanoid family of bioactive mediators. These include the prostaglandins (PG), thromboxanes, leukotrienes (LT), hydroxy-eicosatetraenoic acids and lipoxins. Arachidonic acid in cell membranes can be mobilised by various phospholipase enzymes, especially phospholipase A2, and the free arachidonic acid can subsequently act as a substrate for the enzymes which synthesise eicosanoids (Fig. 15.4). Metabolism by cyclooxygenase enzymes (COX) gives rise to the 2-series prostaglandins and thromboxanes (Fig. 15.4). There are two isoforms of COX: COX-1 is a constitutive enzyme and COX-2 is induced in inflammatory cells as a result of stimulation and is responsible for the markedly elevated production of prostaglandins that occurs upon cellular activation. Prostaglandins are formed in a cell-specific manner. For example, upon activation monocytes and macrophages produce large amounts of PGE2 and PGF2, neutrophils produce moderate amounts of PGE2, and mast cells produce PGD2. Lymphocytes are a poor source of prostaglandins, but may release arachidonic acid extracellularly for use by other cell types (Goldyne and Stobo, 1982). Metabolism of arachidonic acid by the 5-lipoxygenase (5-LOX) pathway gives rise to hydroxy and hydroperoxy derivatives, and the 4-series leukotrienes, LTA4, B4, C4, D4 and E4 (Fig. 15.4). 5±LOX is found in mast cells, monocytes, macrophages and granulocytes. Arachidonic acid-derived eicosanoids are involved in modulating the intensity and duration of inflammatory responses and in regulating immune responses (see Lewis et al., 1990; Tilley et al., 2001 for reviews). The effects of PGE2 and LTB4 are summarised in Table 15.5. LTB4 has a range of proinflammatory effects. PGE2 also has a number of pro-inflammatory effects. Note however, that PGE2 also acts to down-regulate the production of the classic inflammatory cytokines (TNF, IL-1 and IL-6) and inhibits the production of

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Fig. 15.4 Metabolism of 20 carbon polyunsaturated fatty acids to yield eicosanoids. Cox, cyclooxygenase; LOX, lipoxygenase; LT, leukotrienes; PG, prostaglandins.

4-series leukotrienes. PGE2 also induces the production of lipoxins (Levy et al., 2001), which are now recognised to be inflammation `stop' signals (Gewirtz et al., 2002; Vachier et al., 2002). Thus, PGE2 is both a mediator and a regulator of inflammation, and exerts both pro- and anti-inflammatory actions. PGE2 also promotes IgE production by B lymphocytes and so in this respect PGE2 is proallergic. Finally, PGE2 inhibits T cell proliferation and the production of the Th1-type cytokine IFN- . In these resects PGE2 is immunosuppressive. Table 15.5 Effects of PGE2 and LTB4 on inflammation and immunity PGE2

LTB4

Pro-inflammation Induces fever Increases vascular permeability Increases vasodilatation Causes pain Enhances pain caused by other agents

Pro-inflammation Increases vascular permeability Enhances local blood flow Chemotactic agent for leukocytes Induces release of lysosomal enzymes Induces release of reactive oxygen species by granulocytes Increases production of TNF, IL-1 and IL-6

Anti-inflammation Inhibits production of TNF, IL-1 and IL-6 Inhibits 5-LOX Induces lipoxin production Immunosuppression Inhibits production of IL-2 and IFN- Inhibits lymphocyte proliferation Pro-allergy Promotes IgE production

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The capacity to generate eicosanoids from arachidonic acid appears to be influenced by age. Production of PGE2 and TXA2 was higher by macrophages, splenocytes and lung tissue from 24-month old compared with three-month old mice (Meydani et al., 1988, 1992; Hayek et al., 1994; Wu et al., 1998). Furthermore, PGE2 production by blood mononuclear cells from older women was greater than by cells from younger women (Meydani et al., 1991). The agerelated increase in production of arachidonic acid-derived eicosanoids is related to increased COX activity (Hayek et al., 1997; Wu et al., 1998), which in turn is due to increased expression of the COX-2 gene (Hayek et al., 1997). The latter study showed that peak COX-2 mRNA levels in endotoxin-stimulated macrophages from old mice were twice those seen in young mice. This correlated with twice the peak COX-2 activity and twice the peak PGE2 production in these mice (Hayek et al., 1997; Wu et al., 1998). This age-related increase in the production of PGE2 and related eicosanoids may play a role in the immune and inflammatory changes seen in the elderly. 15.3.2 Di-homo- -linolenic acid and eicosanoid production DGLA is a substrate for COX-2 and for 5-LOX giving rise to derivatives which have a different structure from those produced from arachidonic acid (i.e. 1series PG and 3-series LT) (Fig. 15.4). PGE1 has a number of anti-inflammatory effects including inhibition of superoxide, elastase and myeloperoxidase production by neutrophils, and inhibition of TNF, IL-1 and IL-6 production by monocytes and macrophages. DGLA is also a substrate for 15-LOX giving rise to 15-hydroxy-DGLA, which is a 5-LOX inhibitor. DGLA levels in inflammatory cells are increased by GLA supplementation of the diet (see section 15.2) and GLA supplementation has been shown to result in increased production of PGE1 and decreased production of PGE2, LTB4, and LTC4 (Johnson et al., 1997; Wu et al., 1999). 15.3.3 Long-chain n-3 PUFAs and eicosanoid production Since significantly increased consumption of long-chain n-3 PUFAs results in a decrease in the amount of arachidonic acid in the membranes of inflammatory cells, there will be less substrate available for synthesis of eicosanoids from arachidonic acid. In accordance with this, fish oil feeding results in a decreased capacity of inflammatory cells to synthesise COX- and 5-LOX-derived eicosanoids from arachidonic acid (e.g. Lee et al., 1985; Endres et al., 1989; Meydani et al., 1991; Sperling et al., 1993; Caughey et al., 1996). However, the effects of n-3 PUFAs on eicosanoid production extend beyond simply decreasing the amount of substrate available. For example, EPA competitively inhibits the oxygenation of arachidonic acid by COX (Obata et al., 1999). Recent cell culture studies have demonstrated that n-3 PUFAs suppress cytokine-induction of COX-2 and 5-LOX gene expression (Curtis et al., 2000, 2002). It is the net result of these various actions, that accounts for the decreased

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generation of arahidonic-acid derived eicosanoids by n-3 PUFAs. The reduction in generation of arachidonic acid-derived mediators that accompanies fish oil consumption has led to the idea that fish oil is anti-inflammatory. In addition to inhibiting metabolism of arachidonic acid, EPA is able to act as a substrate for both COX and 5-LOX (Fig. 15.4), giving rise to derivatives which have a different structure from those produced from arachidonic acid (i.e. 3series PG and TX and 5-series LT). Thus, the EPA-induced suppression in the production of arachidonic acid-derived eicosanoids may be accompanied by an elevation in the production of EPA-derived eicosanoids. This is most evident for the 5-LOX products of EPA metabolism (Lee et al., 1985; Sperling et al., 1993). The eicosanoids produced from EPA are considered to be less biologically potent than the analogues synthesised from arachidonic acid, although the full range of biological activities of these compounds has not been investigated. Therefore, it is possible that EPA gives rise to eicosanoids that are less inflammatory, less pro-allergic and less immunosuppressive than those produced from arachidonic acid (see Miles et al., 2002, 2003 for further discussion). Interestingly recent studies have revealed that n-3 PUFAs give rise to novel anti-inflammatory eicosanoids generated via COX-2 (Levy et al., 2001).

15.4 Dietary fatty acids and immune function: mechanisms of action There is a large literature based upon cell culture and animal feeding studies investigating the effects of various fatty acids on inflammation and immune function. These studies have established a basis for effects that might be observed following manipulation of the fatty acid composition of the human diet and have been very useful in identifying mechanisms of action of different fatty acids. A description of these studies is beyond the scope of this chapter, which will focus upon results from human studies. However, a number of review articles serve to summarise, evaluate and discuss these studies (Calder 1996, 1997, 1998a,b, 2001b, 2002; Calder et al., 2002; Yaqoob, 1998, 2003; Kelley and Erickson, 2003; Harbige, 2003). 15.4.1 Oleic acid A dietary intervention study in which healthy middle-aged men consumed diets providing 11.3 (control) or 18.4% energy from oleic acid for eight weeks was performed by Yaqoob et al. (1998). There was no significant effect on the proportion of T cells, B cells, T helper cells, cytotoxic T cells, monocytes or natural killer cells in the circulation or on natural killer cell activity or mitogenstimulated proliferation of lymphocytes. There was however a significant reduction in the proportion of mononuclear cells (most likely monocytes) expressing ICAM-1 (Yaqoob et al., 1998). In another study providing 9 g encapsulated olive oil per day for 12 weeks did not affect the proportion of T

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cells, B cells, T helper cells, cytotoxic T cells, monocytes, or natural killer cells in the circulation; on natural killer cell activity, mitogen-stimulated proliferation of lymphocytes, or stimulated production of a range of cytokines by lymphocytes and monocytes (Yaqoob et al., 2000). 15.4.2 N-6 PUFAs Surprisingly few human studies have been conducted investigating the immunological effects of linoleic acid. The most detailed of those reported were those of Kelley et al. (1989, 1992) that involved providing volunteers with low fat diets (25% energy as fat) that were rich (12.9% of energy) or poor (3.5% of energy) in linoleic acid. No differences were observed in the response of lymphocytes to various T cell mitogens, in circulating concentrations of IgM, IgG, IgE or IgA, or in delayed-type hypersensitivity, an in vivo measure of cellmediated immunity. Yaqoob et al. (2000) included a group consuming 9 g encapsulated sunflower oil/day for 12 weeks in their study. This had no effect on T lymphocyte proliferation, natural killer cell activity, or production of TNF-, IL-1, IL-1 , IL-6, IL-2 and IFN- by mononuclear cells. These studies suggest a limited effect of linoleic acid (at a level 3.5% of dietary energy) on human immune function. In another study, 11.2 g linoleic acid/day from safflower oil for 12 weeks had no effect on the serum concentrations of IL-6, C-reactive protein or amyloid A (Rallidis et al., 2003). The habitual intake of linoleic acid in the subjects studied was an average of 11 g/day; thus the linoleic acid intake was substantially increased (by an average of about 100%) in these subjects, without any adverse effects on inflammatory markers. Supplementation studies using GLA-rich oils such as borage oil and providing volunteers with > 2.4 g GLA/day report decreased T lymphocyte proliferation (Rossetti et al., 1997), decreased production of TNF-, IL-1 and IL-6 by monocytes (de Luca et al., 1999), and decreased production of platelet-activating factor by neutrophils (Johnson et al., 1997). Recent studies using 1 or 0.8 g GLA/ day for 12 weeks report no effect on circulating immune cell numbers and types, T lymphocyte proliferation, natural killer cell activity, neutrophil and monocyte phagocytosis, neutrophil and monocyte respiratory burst, or production of a range of cytokines by lymphocytes and monocytes (Yaqoob et al., 2000; Thies et al., 2001a,b,c). Furthermore there was no effect of 0.8 g GLA/day on plasma soluble adhesion molecule concentrations (Thies et al., 2001c). These studies suggest that the lowest amount of GLA required to influence inflammation and immune function is somewhere between 1 and 2.4 g per day. Two studies of the influence of dietary arachidonic acid on human immune function have been performed. In the first of these 1.5 g arachidonic acid/day was included as part of a low-fat diet (27% energy from fat) consumed for eight weeks by healthy males aged 20 to 38 years (Kelley et al., 1997, 1998a). This level of arachidonic acid did not alter the proliferation of T cells in response to mitogens, natural killer cell activity, the production of TNF-, IL-1 , IL-6 or IL-2 by mononuclear cells, the delayed-type hypersensitivity response, or

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antibody responses to immunisation with vaccines against three strains of influenza virus (Kelley et al., 1997, 1998a). However, there was an increase in the production of PGE2 and LTB4 by endotoxin-stimulated mononuclear cells (Kelley et al., 1998a). The second study involved supplementing the diet of healthy elderly (aged 55 to 75 years) subjects with 0.7 g arachidonic acid/day for 12 weeks (Thies et al., 2001a,b,c). There was no significant effect on circulating immune cell numbers or on any of the immune parameters measured. The latter included T lymphocyte proliferation, natural killer cell activity, neutrophil and monocyte phagocytosis, neutrophil and monocyte respiratory burst, and the production of TNF-, IL-1 , IL-6, IL-2 and IFN- (Thies et al., 2001a,b,c). There was also no effect on plasma sICAM-1, sVCAM-1 or sE-electin concentrations (Thies et al., 2001c). These studies suggest that increasing arachidonic acid intake in healthy humans may not have adverse immunological effects. However, these studies had durations of 8 and 12 weeks, respectively, and the longer-term effects of arachidonic acid on inflammation and immune function in humans are not known. 15.4.3 Conjugated linoleic acid Two studies of CLA and human immune function have been reported to date (Kelley et al., 2000, 2001; Albers et al., 2003). Kelley et al. (2000, 2001) provided an encapsulated mix of CLA isomers (total CLA intake 3.9 g/day) to young women consuming a 30% energy from fat diet for nine weeks. The mix contained a number of isomers with no single isomer contributing more than 23.6% of total CLA; cis-9, trans-11 CLA and trans-10, cis-12 CLA contributed 17.6% (690 mg per day) and 22.6% (880 mg per day), of CLA, respectively. CLA did not affect the numbers of T lymphocytes, T helper cells, cytotoxic T cells, B cells, monocytes or natural killer cells in the circulation (Kelley et al., 2000). There was also no effect of CLA on T or B lymphocyte proliferation, on the production of TNF-, IL-1 , IL-2 and IFN- , on the serum antibody response to immunisation with vaccines against three strains of influenza virus, or on the delayed-type hypersensitivity response (Kelley et al., 2000, 2001). Finally there was no effect of CLA on production of PGE2 or LTB4 by endotoxin-stimulated mononuclear cells (Kelley et al., 2001). Although this study could be taken to indicate that CLA (at an intake of 3.9 g/day) does not affect human immune function, it is important to note that the study used a rather crude mixture of CLA isomers. Thus, the amount of a CLA isomer that does affect immune function may have been insufficient. Furthermore, different isomers may have opposing actions so that overall a mixture of isomers is without effect. Albers et al. (2003) provided about 1.6 g CLA/day as an 80:20 or a 50:50 mix of the cis-9, trans-11 and trans-10, cis-12 isomers to healthy males aged 31 to 69 years for 12 weeks; sunflower oil was used as the control. There was no effect of either mix on T lymphocyte or monocyte proliferation, natural killer cell activity, production of IL-2, IL-4 and IFN- by lymphocytes, and production of TNF-, IL-1 , IL-6 and PGE2 by monocytes. Furthermore the delayed-type

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hypersensitivity response was not affected. Subjects were immunised with a vaccine for hepatitis B. Although the mean concentration of anti-hepatitis B vaccine antibodies was not different between the groups, more subjects mounted a response to the vaccine in the 50:50 group. The lack of effect in the 80:20 group suggests that the trans-10, cis-12 CLA isomer is responsible for the improved response in the 50:50 group. Clearly this requires further investigation with studies using pure CLA isomers. Thus, at this stage the precise effects of different CLA isomers on human immune function remain unknown. 15.4.4 N-3 PUFAs Fish oil, EPA and DHA Studies investigating the effect of fish oil on human inflammatory and immune cell functions and responses date back to the early 1980s and there is a great deal of literature on this area. Effects on a number of cellular functions and on a range of mediators have been investigated. Although most studies have used fish oil which contains both EPA and DHA, some studies have tried to distinguish between the effects of these two long-chain n-3 PUFAs. The literature on n-3 PUFAs and inflammation and immune function in humans has been collated and reviewed a number of times and the reader is referred to such reviews for a full description and discussion of these studies (e.g. Calder 2001a,b). Chemotaxis, respiratory burst, phagocytosis and adhesion Fish oil, providing between 2.3 and 14.5 g EPA plus DHA/day has been reported to decrease neutrophil chemotaxis (Lee et al., 1985; Schmidt et al., 1989, 1992; Luostarinen et al., 1992; Sperling et al., 1993), neutrophil respiratory burst (Thompson et al., 1991; Varming et al., 1995; Luostarinen and Saldeen, 1996) and neutrophil binding to endothelial cells (Lee et al., 1985). Fish oil, providing 4.5 to 5.3 g EPA plus DHA/day, decreased monocyte chemotaxis (Schmidt et al., 1989, 1992; Endres et al., 1989) and respiratory burst (Fisher et al., 1990). However, lower doses of long-chain n-3 PUFAs (0.55 to 2.3 g/day) did not affect neutrophil or monocyte phagocytosis or respiratory burst (Schmidt et al., 1996; Healy et al., 2000; Thies et al., 2001c; Kew et al., 2003b) or neutrophil chemotaxis (Healy et al., 2000). Although Hughes et al. (1996) reported that 1.6 g EPA plus DHA/day for 3 weeks decreased ICAM-1 on the surface of blood monocytes, this effect was not confirmed in a recent study using 0.77 and 1.7 g EPA plus DHA/day for six months (Kew et al., 2003b). Natural killer cell activity There are relatively few studies of long-chain n-3 PUFAs and human natural killer cell activity. Fish oil providing 1.1 g EPA plus DHA/day significantly decreased natural killer cell activity in subjects aged 55 to 75 years (Thies et al., 2001b). In contrast, 3.2 g EPA plus DHA/day had no effect on natural killer cell activity (Yaqoob et al., 2000). It is possible that the age of the subjects studied may be a factor accounting for these differences.

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Monocyte-derived cytokines Fish oil providing > 2.4 g EPA plus DHA/day decreased the production of TNF (Endres et al., 1989; Meydani et al., 1991; Gallai et al., 1993; Caughey et al., 1996), IL-1 (Endres et al., 1989; Meydani et al., 1991; Gallai et al., 1993; Caughey et al., 1996) and IL-6 (Meydani et al., 1991) by mononuclear cells. Another study, providing subjects consuming a low-fat diet that included fatty fish (daily intake of 1.2 g EPA plus DHA), reported decreased production of TNF, IL-1 and IL-6 (Meydani et al., 1993). Intake of 4.55 g EPA plus DHA as ethyl esters/day for four weeks significantly decreased plate-derived growth factor (PDGF)-A and -B and monocyte chemotactic peptide-1 mRNA in monocytes (Baumann et al., 1999). This is consistent with the significant decrease in plasma PDGF concentration observed in women consuming 4 g fish oil/day (1.2 g EPA plus DHA/day) for four weeks (Wallace et al., 1995). In contrast to the above studies are a number reporting no effect of 0.55 to 3.4 g EPA plus DHA/day on production of TNF (Molvig et al., 1991; Cooper et al., 1993; Schmidt et al., 1996; Blok et al., 1997; Yaqoob et al., 2000; Thies et al., 2001c; Kew et al., 2003b; Wallace et al., 2003), IL-1 (Molvig et al., 1991; Cooper et al., 1993; Cannon et al., 1995; Schmidt et al., 1996; Blok et al., 1997; Yaqoob et al., 2000; Thies et al., 2001c; Kew et al., 2003b; Wallace et al., 2003) and IL-6 (Cooper et al., 1993; Schmidt et al., 1996; Thies et al. 2001c; Kew et al., 2003b). Possible reasons for the discrepancies in the literature are discussed in detail elsewhere (Calder, 2001a), but may relate to the dose of n-3 PUFAs used, the duration of the study, the age of the subjects studied, sample size, and differences in background diet. Duration appears not to be a factor because some relatively short studies report effects (e.g. Caughey et al., 1996), whereas other short (Cooper et al., 1993) and longer term (Blok et al., 1997; Kew et al., 2003b) studies report no effect. However, the dose of long-chain n-3 PUFAs provided is likely to be important; a recent study reported for the first time a dose-response relationship between EPA plus DHA and IL-6 production (Wallace et al., 2003). This study reported that the threshold for an effect of long-chain n-3 PUFAs on IL-6 production is between 0.44 and 0.94 g/day. However, dose cannot be the sole explanation for differences in the literature, because some studies providing as much as 3.2 g EPA plus DHA per day report no effect on cytokine production (e.g. Yaqoob et al., 2000). Another recent study has highlighted a further possible explanation. This study reports that polymorphisms in the promoter regions of the TNF- and TNF- genes play a role in conferring sensitivity of TNF- production to fish oil intervention (Grimble et al., 2002). Antigen presentation Hughes et al. (1996) reported that 1.6 g EPA plus DHA/day for three weeks decreased the level of expression of MHC-II (HLA-DP, -DQ and -DR) on the surface of blood monocytes. Lymphocyte proliferation and production of lymphocyte derived cytokines Supplementation of the diet with fish oil providing 2.4 g EPA plus DHA/day decreased the proliferation of lymphocytes from older (aged 51 to 68 years) but

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not young (aged 21 to 33 years) women (Meydani et al., 1991). IL-2 production was also decreased in the older, but not younger, group (Meydani et al., 1991). Molvig et al. (1991) reported decreased lymphocyte proliferation after providing 1.7 or 3.4 g EPA plus DHA/day to men, while Gallai et al. (1993) reported that 5.2 g EPA plus DHA/day decreased IL-2 and IFN- production. Inclusion of fatty fish providing 1.2 g EPA plus DHA/day to elderly subjects consuming a low fat diet decreased lymphocyte proliferation and IL-2 production (Meydani et al., 1993). Fish oil providing 1.1 g EPA plus DHA/day significantly decreased proliferation of T lymphocytes from subjects aged 55 to 75 years, although there was no effect on IL-2 or IFN- production (Thies et al., 2001a). In contrast to these observations there are several reports of no effect of 0.77 to 3.2 g EPA plus DHA/day on T lymphocyte proliferation or production of various T cell derived cytokines including IL-2 and IFN- (Yaqoob et al., 2000; Wallace et al., 2003; Kew et al., 2003b). These latter studies investigated mainly younger subjects. Putting these studies together suggests that it is difficult to influence production of cytokines by T lymphocytes except by using very high doses of long-chain n3 PUFAs (e.g. Gallai et al., 1993). Furthermore, proliferation of lymphocytes from older subjects appears to be more sensitive to increased availability of long-chain n-3 PUFAs than that of those from younger subjects. Delayed-type hypersensitivity The delayed-type hypersensitivity response to seven recall antigens was decreased by inclusion of fatty fish (1.2 g EPA plus DHA/day) in a low fat diet (Meydani et al., 1993). In another study providing 0.77 or 1.7 g EPA plus DHA/day for six months did not affect this response (Kew et al., 2003b). Soluble adhesion molecules The concentrations of sICAM-1, sVCAM-1 and sE-selectin were significantly negatively correlated with the concentration of non-esterfied EPA in the bloodstream of elderly males at high risk of coronary heart disease (Yli-Jama et al., 2002). Furthermore the concentrations of sICAM-1 and sVCAM-1 were significantly negatively correlated with the concentration of non-esterfied DHA in the bloodstream (Yli-Jama et al., 2002). These observations suggest that an increase in long-chain n-3 PUFAs may decrease endothelial inflammation, as indicated by soluble adhesion molecule concentrations. In accordance with this, consumption of 1.1 g EPA plus DHA/day for 12 weeks by subjects aged 55 to 75 years significantly decreased sVCAM-1 concentration, with non-significant decreases in sICAM-1 and sE-selectin concentrations (Thies et al., 2001c). Average decreases were 26% (sVCAM-1), 14% (sICAM-1) and 23% (sEselectin). A reduction in soluble adhesion molecule concentrations was not observed in young male subjects consuming 1.2 g EPA plus DHA/day for 12 weeks (Miles et al., 2001), suggesting that older subjects may be more sensitive to the effects of long-chain n-3 PUFAs. In contrast to the observation of Thies et al. (2001c), three studies report increases in these soluble adhesion molecules following fish oil administration (Seljeflot et al., 1998; Abe et al., 1998;

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Johansen et al., 1999). However, these studies used high doses of fish oil providing 3.3 to 5.1 g EPA plus DHA/day, studied subjects with much higher cardiovascular risk and used different study designs from that used by Thies et al. (2001c) (see Miles et al., 2001 for a discussion). C-reactive protein C-reactive protein (CRP) is an acute phase protein synthesised by the liver in response to infections and to certain inflammatory stimuli, most notably IL-6. Thus, plasma CRP concentrations are elevated in chronic inflammatory conditions and in individuals who are obese or with advanced cardiovascular disease. Since n-3 fatty acids affect inflammatory processes they might be expected to affect CRP concentrations. Indeed, the concentration of CRP was significantly negatively correlated with the proportion of DHA in the membranes of granulocytes of patients undergoing coronary angiography (Madsen et al., 2001). Fish oil (3.6 g EPA plus DHA/day for 12 weeks) significantly lowered (by 20%) plasma CRP concentrations in patients with rheumatoid arthritis, who have elevated concentrations (Nielsen et al., 1992). However, 4.4 g EPA plus DHA/day for six weeks did not lower CRP concentrations in obese or non-obese subjects (Chan et al., 2002). Furthermore, a recent study reported no effect of either 2 or 6.6 g EPA plus DHA/day for 12 weeks on serum CRP in healthy subjects (Madsen et al., 2003). EPA vs. DHA and Fish oil vs. DHA Recent studies have compared effects of EPA and DHA or of fish oil and DHA and have attempted to identify whether the effects of fish oil are due to EPA or to DHA. There was no effect of 3.8 g of either EPA or DHA/day for seven weeks on phagocytosis of E. coli by human monocytes (Halvorsen et al., 1997). Kelley et al. (1998b, 1999) reported the effects in men aged 20 to 40 years of including 6 g DHA/day in a 30% energy from fat diet for 90 days. There was no effect of DHA on lymphocyte proliferation, serum immunoglobulin G concentrations, the delayed-type hypersensitivity response or the serum antibody response to immunisation against three strains of influenza virus (Kelley et al., 1998b, 1999). Natural killer cell was unaffected at day 55 but was significantly decreased at day 80 (Kelley et al., 1999). Similarly, the production of TNF- and IL-1 tended to decrease at day 55 but was significantly decreased at day 80 (Kelley et al., 1999). More recently, 0.75 g DHA/day for 12 weeks did not affect phagocytosis or respiratory burst by neutrophils and monocytes, natural killer cell activity, lymphocyte proliferation, the production of cytokines by lymphocytes and monocytes, or the concentrations of circulating adhesion molecules (Thies et al., 2001a,b,c). Thus a low dose of DHA ( 0.75 g/day) does not affect inflammation or immune function even in elderly subjects. However a very high dose of DHA (6 g/day) exerts some anti-inflammatory and immunosuppressive effects. The limited nature of these effects at such a high dose is suggestive that DHA does not mediate the effects of fish oil upon inflammatory and immune processes.

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-Linolenic acid A high dose of ALA (13.7 g/day for four weeks) decreased the production of TNF and IL-1 by human monocytes by an average of 27 and 30%, respectively (Caughey et al., 1996). The effect of this dose of ALA compares with reductions of 70 and 78%, respectively, after four weeks of 2.7 g EPA plus DHA/day (Caughey et al., 1996). Adding linseed oil providing about 15 g ALA/day as part of a low-fat diet (29% of energy from fat) resulted in a decrease in T lymphocyte proliferation in response to some, but not all, mitogens and a decrease in the delayed-type hypersensitivity response (Kelley et al., 1991). Circulating antibody concentrations were not affected (Kelley et al., 1991). Supplementing the diet of healthy young males with encapsulated linseed oil providing 3.5 g ALA/day for 12 weeks did not alter superoxide production by neutrophils or neutrophil chemotaxis (Healy et al., 2000). Furthermore, the number of T lymphocytes, helper T lymphocytes, cytotoxic T lymphocytes, B lymphocytes and natural killer cells in the bloodstream was not affected (Wallace et al., 2003). Likewise there was no effect on the proliferation of T lymphocytes in response to a mitogen, the production of IL-2, IL-4, IFN- and IL-10 by lymphocytes, or the production of TNF-, IL-1 and IL-6 by monocytes (Wallace et al., 2003). A similar lack of effect of ALA (2 g/day for 12 weeks) on circulating immune cell numbers, T cell proliferation, production of cytokines by lymphocytes and monocytes and natural killer cell activity was reported in elderly subjects (Thies et al., 2001a,b,c). There was however, a significant decrease in sVCAM-1 and sE-selectin, but not sICAM-1, concentrations in these subjects (Thies et al., 2001c). The average decreases were 15% and 27%, respectively. The decrease in sE-selectin concentration was similar to that seen in subjects consuming 1.1 g EPA plus DHA/day (23%; Thies et al., 2001c), but the decrease in sVCAM-1 concentration was less than that seen in the subjects consuming long-chain n-3 PUFAs (26%; Thies et al., 2001c). A recent intervention study used margarines to provide dietary intakes of 4.5 or 9.5 g ALA/day for six months, largely at the expense of linoleic acid (Kew et al., 2003b). ALA did not affect the proportions of T lymphocytes, helper T lymphocytes, cytotoxic T lymphocytes, B lymphocytes or monocytes in the circulation. There was no effect of ALA on the phagocytic activity of neutrophils and monocytes, on the ability of neutrophils and monocytes to undergo respiratory burst, on T lymphocyte proliferation, or on the production of cytokines (IL-2, IL-4, IFN- , TNF-, IL-1 , IL-6) by mononuclear cells (Kew et al., 2003b). Finally, ALA did not alter the delayed-type hypersensitivity response (Kew et al., 2003b). In another dietary intervention study using margarine and other strategies to provide an average of 6.3 g ALA/day for one year, plasma fibrinogen concentration was lower (by approximately 5%) than in the control group (Bemelmans et al., 2002). In another study, 8.1 g ALA/day from linseed oil for 12 weeks significantly decreased the serum concentrations of IL-6 (average decrease 23%), C-reactive protein (25%) and amyloid A (26%) (Rallidis et al., 2003). The habitual intake of ALA in the subjects studied was an average of 1 g/day. Thus, a substantial

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increase in ALA intake in these subjects appears to have induced a marked antiinflammatory effect.

15.5 Other mechanisms of action of dietary fatty acids not involving eicosanoids Altered synthesis of eicosanoids is a biologically plausible and readily accepted mechanism by which to explain the actions of certain fatty acids, especially n-3 PUFAs upon inflammation and immunity. However, many of the effects reported are different from those that would be predicted if EPA was acting as a PGE2 antagonist. For example, n-3 PUFAs decrease production of TNF- by monocytes and macrophages, but so does PGE2. One explanation for this is that focusing on PGE2 is too simplistic. It may be that it is the overall impact of n-3 PUFAs on the entire range of arachidonic acid-derived mediators coupled with the increased production of EPA-derived mediators (whose effects are not known and some of which are not yet even discovered) that accounts for the observed effects. Thus, the effects of n-3 PUFAs may still be related to eicosanoids. Another explanation is that n-3 PUFAs, and other active fatty acids, work through eicosanoid-independent mechanisms. There is some evidence for this from cell culture studies (Santoli and Zurier, 1989; Calder et al., 1992; Soyland et al., 1993). These other mechanisms also rely upon an altered fatty acid composition of membrane phospholipids (Fig. 15.5) and include effects on: · the physical nature of the membrane (often referred to as fluidity) · the ability of the membrane to undergo structural and functional changes in response to a cellular stimulus · the ability to generate intracellular signalling molecules. As will be seen below, these mechanisms are closely related to one another, since cell membrane composition, fluidity and function and the generation of signalling molecules following a cellular stimulus are interlinked. The fluidity of the plasma membrane, or of regions of the plasma membrane, is important in the functioning of cells (Stubbs and Smith, 1984). The fluidity of a membrane is determined by its lipid components and by their fatty acid composition (Stubbs and Smith, 1984). Membrane fluidity is an important regulator of phagocytosis (Calder et al., 1990). Cell culture experiments have demonstrated that changes in fatty acid composition of immune cells alter membrane fluidity (e.g. Calder et al., 1994), and that this is related to altered cell function (Calder et al., 1990, 1994). However, an alteration in membrane fluidity of cells of the immune system has been less easy to demonstrate after dietary manipulations (e.g. Yaqoob et al., 1995; Tappia et al., 1997). This may be because the fatty acid composition changes induced by dietary changes are less extreme than those seen in cell culture. In addition, in the intact animal, mechanisms to counter the fluidising effect of increasing the PUFA content of membranes (e.g. insertion of cholesterol) can be achieved more readily than in

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Fig. 15.5 Potential mechanisms by which fatty acids can affect inflammation and immune function.

culture. Therefore, alterations in the fluidity of the immune cell membranes may not be a mechanism by which fatty acids affect immune function. However, techniques used to determine membrane fluidity tend to measure `average' fluidity over a large surface and it may be that, while this is unaffected, fluidity of smaller regions of the membrane does change but is undetected. Lipid rafts are microenvironments in the outer leaflet of the phospholipid bilayer of plasma membranes. Lipid rafts are rich in unsaturated fatty acids, and they are considered to be more fluid than other regions of the membrane. Many proteins involved in cell signalling are located in lipid rafts (Brown and London, 1998; Simons and Toomre, 2000), and they appear to be particularly important in the signalling processes within immune cells (Katagiri et al., 2001). For example, the T cell receptor clusters within lipid rafts upon contact with an antigen presenting cell forming a contact zone within which intracellular signalling is initiated. Several signalling proteins including members of the src family of protein kinases such as lyk and fyn are concentrated on the cytoplasmic side of lipid rafts and become activated in response to signalling through the T cell receptor. Cell culture studies have demonstrated that provision of EPA to T cells results in marked enrichment of EPA in lipid rafts and in the displacement of certain proteins from those rafts (Stulnig et al., 2001). These displaced proteins included lck and the protein known as linker of activated T cells (LAT). LAT is involved in signalling subsequent to src kinases and among its substrates in T cells is phospholipase C 1. A recent study showed that EPA decreased the phosphorylation of LAT and of phospholipase C 1 in

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cultured T cells and the authors suggested that this was caused by selective displacement of LAT from lipid rafts (Zeyda et al., 2002). Interestingly, feeding fish oil to rats resulted in decreased PLC 1 phosphorylation in T lymphocytes subsequently stimulated in culture (Sanderson and Calder, 1998). Taken together these findings suggest that dietary n-3 PUFAs might modify the composition of lipid rafts in T cells and that this alters the subsequent cellular response to a stimulus. Many of the established cell signalling molecules are generated directly from membrane phospholipids. Examples of these signalling molecules include inositol-1,4,5±trisphosphate, diacylglycerol, phosphatidic acid, choline, and ceramide. These have important roles in regulating the activity of proteins involved in immune cell responses. The concentration and/or composition of lipid-derived signalling molecules have been shown to be sensitive to n-3 PUFA availability either in cell culture or through the diet (Miles and Calder, 1998 for references). This may be due to either altered activity of the enzymes that generate the signals or to altered composition of the substrate molecules. There is evidence to support each of these possibilities (Miles and Calder, 1998; Yaqoob, 2003). For example, lymphocyte phospholipase C 1 activity is reduced after feeding a diet rich in fish oil, which might account for the decreased generation of signalling molecules observed (Sanderson and Calder, 1998). A change in the generation of intracellular signalling molecules may have rapid effects on cellular responses. However, there may also be longer term effects such as alterations in the pattern of gene expression. The effects of fatty acids, especially PUFAs, on expression of genes coding for key regulatory proteins in various metabolic pathways has been most clearly described in hepatocytes and adipocytes (Jump et al., 1994). These effects of fatty acids are mediated by both indirect mechanisms (e.g. by eicosanoids, hormones) and direct effects on gene expression. There is now emerging evidence that PUFAs regulate the expression of genes involved in inflammation and immunity (Calder, 2002). Among the genes that are down-regulated by n-3 PUFAs are those encoding TNF-, IL-1, COX-2, 5-LOX, 5-LOX activating protein, certain matrix metalloproteinases, and VCAM-1. Since the expression of many of these genes is regulated by the transcription factor nuclear factor kappa B (NFB), these observations suggest that n-3 PUFAs might somehow affect the activity of this transcription factor. This might be through effects on cell signalling leading to NFB activation. There is recent evidence that dietary fish oil affects NFB activity (Lo et al., 1999; Xi et al., 2001), in a manner that is consistent with its ability to down-regulate the production of inflammatory mediators. A second group of transcription factors currently undergoing scrutiny for their role in inflammation are the peroxisome proliferator activated receptors (PPARs). The main members of this family are PPAR and PPAR . Although PPAR and play important roles in liver and adipose tissue, respectively (Schoonjans et al., 1996), they are also found in inflammatory cells (Chinetti et al., 1998; Ricote et al., 1998). PPARs can bind, and appear to be regulated by, PUFAs and eicosanoids (Kliewer et al., 1995; Devchand et al., 1996). PPAR

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deficient mice have a prolonged response to inflammatory stimuli (Devchand et al., 1996), suggesting that PPAR activation might be anti-inflammatory. More recently activators of both PPAR and have been shown to inhibit the activation of a number of inflammatory genes, including TNF-, IL-1 , IL-6, IL-8, COX-2 and VCAM-1 (see Calder, 2002 for references). Two mechanisms for the anti-inflammatory actions of PPARs have been proposed (Chinetti et al., 2000; Delerive et al., 2001). The first is that PPARs might stimulate the breakdown of inflammatory eicosanoids through induction of peroxisomal oxidation. The second is that PPARs might interfere with/antagonise the activation of other transcription factors, including NFB. Although the effect of fish oil on PPAR expression in inflammatory cells has not been reported, studies in other tissues (e.g. Berthou et al., 1995) suggest that n-3 PUFAs might act by increasing the level of these anti-inflammatory transcription factors in such cells.

15.6

Dietary fatty acids and inflammatory diseases

As indicated in section 15.1, inappropriate immune activity or immune dysregulation is a feature of a range of degenerative human diseases. Furthermore, ageing can be associated with diminished acquired immune function, with altered balances within the T cell phenotypes, and with increased inflammation. These changes might predispose the elderly to infections and/or to specific diseases including rheumatoid arthritis, allergic diseases, and inflammatory bowel disease. It is now recognised that atherosclerosis is an inflammatory disease (Ross et al., 1999). Furthermore, acute cardiovascular events are driven by inflammatory activities within the vessel wall (Plutzky, 1999). Given the effects of different fatty acids described in section 15.4, it is possible that the status of certain fatty acids may play a role in influencing the risk of inappropriate immune activity, immune dysregulation, immune decline and inflammation. It follows from this that decreased consumption of those fatty acids that increase risk and increased consumption of those that decrease risk should be associated with improved health. As far as diseases with an immunologic basis are concerned, there is evidence that oleic acid, GLA and long-chain n-3 PUFAs consumption is associated with improvements in outcome. An extrapolation from this is that they may lower the risk of developing the disease in the first place 15.6.1 Oleic acid Evidence for a protective effect of oleic acid comes from epidemiological and clinical studies involving rheumatoid arthritis. Linos et al. (1991, 1999) compared the relative risk of development of rheumatoid arthritis to lifelong consumption of olive oil in a Greek population. They found that individuals in the lowest category of consumption had 2.5±times higher risk of having arthritis

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than those in highest category of consumption. This study has several limitations including its retrospective nature and the difficulty of assessing lifelong consumption accurately. Nevertheless, Mediterranean populations have a lower prevalence of rheumatoid arthritis than those in Northern Europe (Cimmino et al., 1998) and where it occurs it appears to be less severe (Drosos et al., 1992). In a clinical trial investigating the potential benefit of fish oil in patients with rheumatoid arthritis, Kremer et al. (1990) used olive oil (9 g/day) as the placebo. They identified that olive oil improved five out of the 45 clinical measures made, and suggested that olive oil itself may be of some benefit in these patients. 15.6.2 GLA GLA has been used in a variety of diseases involving immune dysregulation or inflammation. It has been found to be efficacious in some of these. Indeed, in the United Kingdom certain preparations of GLA are licensed as medicines for use in atopic dermatitis, a disease in which it is particularly effective (see Burton, 1990 for a review). GLA also appears to be effective in rheumatoid arthritis. At least five double-blind, placebo-controlled studies of GLA in rheumatoid arthritis have been reported. These are reviewed elsewhere (Zurier, 1998; Belch and Muir, 2000) and so will only be summarised here. These studies provided between 0.36 and 2.8 g GLA/day for between 12 and 52 weeks. Each study reported some form of clinical improvement with GLA including a reduction in duration of morning stiffness and in the number and painfulness of tender and swollen joints. Three of the studies reported decreased use of non-steroidal antiinflammatory drugs (NSAIDs). 15.6.3 Long-chain n-3 PUFAs Aspirin and NSAIDs are widely used for symptom relief in inflammatory disease. These drugs act as COX inhibitors specifically targeting 2-series prostaglandins and thromboxanes. The discovery that long-chain n-3 PUFAs also act to reduce formation of these mediators promoted studies in animal models and clinical trials in a range of human diseases. Dietary fish oil has been shown to increase survival and decrease proteinuria and anti-DNA antibody formation in mice with autoimmune glomerulonephritis (a model of lupus), to decrease joint inflammation in rodent models of arthritis, and to decrease inflammation in rat models of colitis and of type-1 diabetes (see Calder 1997, 2001b for references). The efficacy of fish oil has been studied in several inflammatory diseases including rheumatoid arthritis, Crohn's disease, ulcerative colitis, psoriasis, lupus, multiple sclerosis, cystic fibrosis and asthma. Although there are clinical benefits reported from trials in each of these diseases (e.g. see Belluzzi and Miglio, 1998; Rodgers, 1998; Ziboh, 1998; Beckles Willson et al., 2003), the only one for which there is really strong evidence of benefit is rheumatoid arthritis. This may be a reflection of the large number of well designed and well

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conducted studies in arthritis; there have been at least 14 randomised, placebocontrolled, double-blind studies of fish oil in rheumatoid arthritis. These trials have been reviewed in some detail elsewhere (James and Cleland, 1997; Geusens, 1998; Calder, 2001c; Calder and Zurier, 2001), and so will be discussed only briefly here. They used between 1 and 7.1 g EPA plus DHA per day (average dose was 3.3 g/day) with a duration of 12 to 52 weeks. A variety of improvements in clinical outcome were reported. These include reduced duration of morning stiffness, reduced number of tender or swollen joints, reduced joint pain, reduced time to fatigue, increased grip strength and decreased use of NSAIDs. It has been concluded that the evidence for benefit from long-chain n-3 PUFAs in rheumatoid arthritis is robust (Cleland and James, 2000). There is currently considerable interest in the relative effects of n-3 and n-6 PUFAs in asthma (and other atopic diseases) (Hodge et al., 1994; Black and Sharp, 1997; Calder and Miles, 2000). The discussion centres on the roles of various eicosanoids produced from arachidonic acid in mediating allergic inflammation and in programming T lymphocytes to a phenotype that predisposes to such inflammation. Arachidonic acid-derived eicosanoids such as PGD2, LTC4, D4 and E4 are produced by the cells that mediate pulmonary inflammation in asthma (e.g. mast cells) and are believed to be the major mediators of asthmatic bronchoconstriction. Thus, it is considered that provision of n-3 PUFAs to asthmatics might be beneficial because of the resulting decrease in production of 4-series leukotrienes and other arachidonic acidderived mediators. However, the situation is complicated by the fact that different eicosanoids have different effects, some antagonising others. For example, the observations that PGE2 inhibits 5-LOX and promotes generation of lipoxins that act as inflammation `stop signals', indicate that PGE2 could, in fact, be protective in active asthma. Thus, interventions that aim to suppress PGE2 production could be counterproductive, at least in some asthmatics. Nevertheless, a number of trials of fish oil in asthma and related atopic diseases have been performed (Calder and Miles, 2000). Most of these studies reveal limited clinical impact, despite significant biochemical changes, although some have shown clinical improvements at least in some patient groups (Hodge et al., 1996; Broughton et al., 1997; Nagakura et al., 2000). A recent meta-analysis of fish oil in asthma concluded that there was no evidence of benefit (Woods et al., 2003). However, trying to intervene in the disease once it has developed may be the wrong approach, and n-3 PUFAs may still have a role in prevention. Since, PGE2 regulates T lymphocyte differentiation promoting the development of the Th2-type phenotype that underlies sensitisation to environmental allergens, it is possible that early exposure to long-chain n-3 PUFAs may be protective towards allergy, asthma and related diseases. There is some epidemiological evidence in support of this (Calder, 2003). However, despite a biologically plausible mechanism and supportive biochemical and epidemiological data, the key to demonstrating a protective effect of increased long-chain n-3 PUFA consumption towards allergic-type

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diseases must come from well-designed placebo-controlled intervention studies. It is now recognised that sensitisation to allergens occurs early in life, and so the characteristics of the maternal diet may be very important in determining predisposition to these diseases. Therefore, studies addressing this question need to be performed in pregnant women. Several such studies are currently under way and their findings are eagerly anticipated. Consumption of long-chain n-3 PUFAs lowers the risk of mortality from cardiovascular disease (see Calder and Yaqoob, 2003 for references). Since inflammation within the vessel wall contributes to the development of atherosclerosis (Ross, 1999), anti-inflammatory effects might play a role in the observed protective effect of n-3 PUFAs. N-3 PUFAs lower risk of myocardial infarction (Calder and Yaqoob, 2003) which is induced by inflammatory activity within the vessel wall (Plutzky, 1999). A recent study has provided evidence that long-chain-3 PUFAs from fish oil act to decrease inflammation within advanced atherosclerotic plaques (Thies et al., 2003). Therefore, the anti-inflammatory effects of n-3 PUFAs may make an important contribution to their cardioprotective effects. The improved appetite, increased dietary intake and weight gain in advanced pancreatic patients supplementing their diet with 2 g EPA/day (Barber et al., 1999) appears to relate to decreased production of mediators that give rise to cachexia such as IL-6 (Barber et al., 1999, 2000, 2001). Thus, the applications of the anti-inflammatory effects of n-3 PUFAs may extend beyond those disorders tradionally considered as `inflammatory'.

15.7 Targeting the immune function and inflammation: fatty acid-enriched functional foods Most experimental studies investigating dietary fatty acids, inflammation and immunity have used encapsulated oils, although a small number have used dietary change and/or specially modified foodstuffs. One of the great challenges in using functional foods to modulate the immune system will be to deliver sufficient amounts of the active fatty acids to have the desired effects. From the foregoing discussion it is clear that oleic acid, GLA, CLA and n-3 PUFAs are each of interest and it may be that different approaches will need to be used for each of these. 15.7.1 Oleic acid Western populations consume a significant amount of oleic acid (see section 15.1) with meat, cereals, milk and milk products, spreads and vegetables each supplying significant amounts. Olive oil is especially rich in oleic acid and other oleic acid rich oils such as high-oleic sunflower oil have been developed. Spreads that include a substantial proportion of oleic acid have become available in some countries. The use of oleic acid-rich oils and spreads in cooking and

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baking can lead to a substantial increase in oleic acid intake at the expense of either saturated or n-6 polyunsaturated fatty acids, or both, depending upon the exact nature of the replacement. However, the increase in oleic acid intake that appears to be required in order to influence immune function and inflammatory processes is quite large (Yaqoob et al., 1998), although this approach may have a significant impact on cardiovascular risk factors (Williams et al., 1999). 15.7.2 GLA GLA is found in oils that are commonly sold as dietary supplements, such as evening primrose oil and borage (also called starflower) oil. GLA is also found in blackcurrant seed oil. The dose of GLA required to elicit changes in cell fatty acid composition appears to be of the order of 1 to 1.5 g/day, and this may not be associated with changes in cell function. The incorporation of GLA into foods to deliver a biologically effective dose may be difficult. 15.7.3 CLA Preparations of CLA that are available in capsules are almost exclusively mixtures of several isomers, some of which will be biologically inactive and some of which may even be detrimental to human health (see Gaullier et al., 2002). The cis-9, trans-11 isomer of CLA is naturally found in ruminant milks, and is by far the most commonly consumed of the CLA isomers. Current intake of CLA is < 250 mg with most of this coming from milk and dairy products and much of the rest from meat. The cis-9, trans-11 CLA content of cows' milk and of beef can be increased by altering dairy feeding strategies (Lawson et al., 2001). Therefore, CLA-rich milk, dairy products and meat can be produced. Through consumption of a range of such modified products it will be possible for intake of cis-9, trans-11 CLA to be significantly increased. However, it is not yet clear whether this isomer exerts immunologic effects (see section 15.4.3). There are suggestions that it is the trans-10, cis-12, rather than the cis-9, trans11, isomer that is immunologically active. Currently it is not possible to produce milk containing trans-10, cis-12 CLA (Lawson et al., 2001), so that dairy products enriched with this isomer do not seem to be a probability. However, it is possible to produce trans-10, cis-12 CLA in pure form chemically, so this may be a viable alternative for use in foodstuffs, such as spreads. However, the whole range of health effects of this isomer need be examined in humans before it can be considered for widespread use. To date there are no studies of CLA-enriched foods and immune function in humans. 15.7.4 N-3 PUFAs From the foregoing discussion it is evident that long-chain n-3 PUFAs are more biologically potent than the precursor ALA. The only food sources that are naturally rich in long-chain n-3 PUFAs are the meat and blubber of marine

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mammals such as whale and seal; the flesh of fatty fish such as herring, mackerel, tuna, salmon, trout and sardines; and the DHA-rich brains and neural tissue of mammals. Of these sources, only fatty fish present a suitable option for most consumers. An intake of long-chain n-3 PUFAs of at least 1 g/day, and probably more (perhaps as much as 2 g/day), is required to exert immunologic effects. Fatty fish provide 1.5 to 3.5 g long-chain n-3 PUFAs per portion (British Nutrition Foundation, 1999). Thus, regular consumption of fatty fish would deliver amounts of these fatty acids to exert immunologic effects. Whitefish and shellfish are also sources of long-chain n-3 PUFAs, but one portion of these delivers between 0.1 and 0.5 g long-chain n-3 PUFAs (British Nutrition Foundation, 1999). Some consumers are unable to access or prepare fatty fish or dislike it. Therefore, although fatty fish is an excellent mode of delivery of long-chain n-3 PUFAs, it is not suitable for all consumers. Therefore, there is a need for alternative strategies for delivery of these fatty acids. One strategy is to enrich foods that do not normally contain long-chain n-3 PUFAs. There are two approaches to this. The first is to enrich existing foods with fish oil through food-processing technologies. One difficulty with this is that fish oil has a characteristic smell and taste that may not be appealing to consumers. More importantly, long-chain n-3 PUFAs oxidise very readily on exposure to air and so the enriched foods may be unstable and, at best, they will have a fairly short shelf life. One way to circumvent these problems is to use `microencapsulated' fish oil, in which the n-3 PUFAs are protected from air. There is still a limitation to how much n-3 PUFA can be incorporated through this route. However, this approach was recently used to prepare a spread that provided 0.8 g EPA plus DHA/day when consumed as part of the habitual diet (Kew et al., 2003b). The spread contained 0.56 g EPA plus DHA per 25 g. The increased intake of longchain n-3 PUFAs did not affect either immune cell fatty acid composition or function (Kew et al., 2003b). This is most likely because the dose of n-3 PUFAs provided was insufficient. The second approach to providing long-chain n-3 PUFAs is to enrich traditional foods with n-3 PUFAs through farming practices. For example, feeding pigs on a diet containing fish oil results in a marked increase in the amounts of EPA, DPA and DHA in the meat and fat (Leskanich et al., 1997; Irie and Sakimoto, 1992). Furthermore, feeding pigs on linseed, a source of ALA, resulted in increased amounts of EPA and DPA, but not DHA, in muscle, liver and kidney (Matthews et al., 2000). However, again one problem that arises when the level of enrichment of these tissues with n-3 PUFAs gets too high is lipid peroxidation, spoiling, and poor consumer acceptance. The immunologic effect of meats enriched in long-chain n-3 PUFAs, in the absence of other fatty acid interventions, has not been examined. Since markedly increasing the long-chain n-3 PUFA content of single foods presents a technical difficulty, an alternative approach may be to enrich many foods but to a lesser extent, such that consumption of the combination of foods will increase long-chain n-3 PUFA intake. Candidate foods would be those

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mentioned above (spreads, animal meats), eggs, bread, yoghurt, and milk-based drinks. An alternative to this could be to increase ALA consumption, since ALA can be converted to the longer chain fatty acids (Fig. 15.3). The study of Kelley et al. (1991, 1993) described earlier provided 18 g ALA/d from linseed oil mixed into salads, yoghurts, spreads and vegetables. Mantzioris et al. (1994) and Caughey et al. (1996) provided 14 g ALA/day through spreads and cooking oil. Both of these studies reported increases in the EPA content of immune cells and alterations in immune cell function (see sections 15.2.4 and 15.4.6). Therefore, foods enabling high ALA consumption may be useful to modifying immune function in humans. Kew et al. (2003b) used spreads to increase ALA intake by human volunteers to 4.5 or 9 g/day for a six-month period. The lower level of intake did not alter mononuclear cell fatty acid composition. However, the higher level of intake increased the EPA content of mononuclear cells by 30% (Kew et al., 2003b). This did not induce any alteration in any of the wide range of immune parameters studied. This observation, coupled with similar negative findings from studies with encapsulated linseed oil (Healy et al., 2000; Thies et al., 2001a,b,c; Wallace et al., 2003) suggests that quite marked increases in ALA intake are required to affect immune function, and that the only way to achieve these will be to produce a range of foods with increased ALA content. One factor restricting the effectiveness of ALA may be its limited conversion to longer chain-3 PUFAs (Burdge et al., 2002; Burdge and Wootton, 2002). This is believed to be due to the low activity of the -6 destaurase enzyme (Fig. 15.3). If this really is a limitation then a novel approach to increasing EPA status of human immune cells may to consume an increased amount of the product of 6 destaurase, stearidonic acid (Fig. 15.3). Very recently, James et al. (2003) reported that consumption of 0.75 or 1.5 g stearidonic acid/day (from capsules) increased the EPA and DPA content of red blood cells and plasma phospholipids, whereas consumption of the same amounts of ALA did not. They also stated that stearidonic acid increased the amount of EPA and DPA in mononuclear cells, although that data was not presented. However, stearidonic acid did not influence the production of TNF-, IL-1 or PGE2 by LPS-stimulated whole blood (James et al., 2003). This study highlights a novel approach to enriching human immune cells with long-chain n3 PUFAs, but it reinforces the conclusion that increases in intake of fatty acids that will induce functional changes are difficult to achieve. One final approach to increasing intake of n-3 PUFAs has been the combined use of an ALA-rich spread, salad dressing and mayonnaise made with linseed oil, linseed oil for cooking, canned fatty and fresh lean fish, and sausages and French onion dip containing microencapsulated fish oil (Mantzioris et al., 2000). This combination was used to increase the average intakes of ALA, EPA and DHA to 9.2, 0.8 and 1 g/day, respectively. The average proportion of ALA in blood mononuclear cells increased from 0.02 to 0.1% of fatty acids after two weeks, while the average proportion of EPA increased from 0.4 to 1.1% of fatty acids after four weeks. The proportions of DPA and DHA also increased

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significantly (Mantzioris et al., 2000). The production of PGE2, TXB2, TNF- and IL-1 by LPS-stimulated whole blood decreased by 26%, 34%, 40% and 20%, respectively. Thus, this study indicates that food vehicles can be used to deliver n-3 PUFAs to human immune cells in sufficient quantities to induce functional changes. 15.7.5 Combinations of nutrients A recent study used a novel semi-skimmed milk formulated to provide 5.1 g oleic acid, 0.13 g EPA, 0.2 g DHA, as well as vitamin E, vitamin B12 and folic acid, in a daily portion of 500 ml (Baro et al., 2003). The milk contained more linoleic acid than normal milk and contained only 30% of the normal amount of saturated fatty acids. Young adult volunteers consumed the milk (500 ml/day) for eight weeks. After this time the plasma concentrations of sICAM-1 and sVCAM-1 were significantly decreased by 10% and 15%, respectively, perhaps an indication of decreased inflammation.

15.8

Conclusions

Cells of the human immune system are amenable to altered fatty acid composition via the diet. This can lead to altered function. The effects of the long-chain n-3 PUFAs are the most studied and the best described. These fatty acids have been demonstrated to be efficacious in the treatment of some diseases related to immune dysfunction and to induce effects that might promote healthier ageing. Other fatty acids of interest in this context are oleic acid, GLA, CLA and n-3 PUFAs that are precursors to EPA (e.g. ALA, stearidonic acid). The levels of dietary intake at which these fatty acids modify immune cell function are quite high and this presents a challenge for the development of functional foods.

15.9

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(2001a), Dietary supplementation with gamma linolenic acid or fish oil decreases T lymphocyte proliferation in healthy older humans, J Nutr, 131, 1918±1927. THIES F, NEBE-VON-CARON G, POWELL J R, YAQOOB P, NEWSHOLME E A, CALDER P C (2001b), Dietary supplementation with eicosapentaenoic acid, but not with other long chain n-3 or n-6 polyunsaturated fatty acids, decreases natural killer cell activity in healthy subjects aged > 55 years, Am J Clin Nutr, 73, 539±548. THIES F, NEBE-VON-CARON G, POWELL J R, YAQOOB P, NEWSHOLME E A, CALDER P C

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VOORRIPS L E, BRANTS H A M, KARDINAAL A F M, HIDDINK G J, VAN DEN BRANDT P A, GOLDBOHM R A (2002), Intake of conjugated linoleic acid, fat, and other fatty acids in relation to postmenopausal breast cancer: the Netherlands Cohort Study on Diet and Cancer, Am J Clin Nutr, 76, 873±882. WALLACE F A, MILES E A, CALDER P C (2003) Comparison of the effects of linseed oil and different doses of fish oil on mononuclear cell function in healthy subjects, Brit J Nutr, 89, 679±689. WALLACE J M W, TURLEY E, GILMORE W S, STRAIN J J (1995), Dietary fish oil supplementation alters leukocyte function and cytokine production in healthy women, Arterioscler Thromb Vasc Biol, 15, 185±189.

WILLIAMS C M, FRANCIS-KNAPPER J A, WEBB D, BROOKES C A, ZAMPELAS A, TREDGER J A, WRIGHT J, MEIJER G, CALDER P C, YAQOOB P, ROCHE H, GIBNEY M J (1999), Cholesterol reduction using manufactured foods high in monounsaturated fatty acids: a randomized crossover study, Brit J Nutr, 81, 439±446. WOODS R K, THIEN F C K, ABRAMSON M J (2003) Dietary marine fatty acids (fish oil) for asthma in adults and children, in The Cochrane Library, Issue 2, Oxford, Update Software. WU D, MURA C, BEHARKA A A, HAN S N, PAULSEN K E, HWANG D, MEYDANI S N (1998), Ageassociated increase in PGE(2) synthesis and COX activity in murine macrophages is reversed by vitamin E, Am J Physiol, 275, C661±C668. WU D, MEYDANI M, LEKA L S, NIGHTINGALE Z, HANDELMAN G J, BLUMBERG J B, MEYDANI S N

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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.

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Successional development of gastrointestinal microflora

Normally, the gastrointestinal tract is sterile at birth and we derive our gut microflora during conventional delivery from the faecal and vaginal microflora of our mother and from the post-natal environment. Early colonisers, taking advantage of readily available nutrients within the first few days of life, (e.g. facultative streptococci/enterococci, lactobacilli and the Enterobacteriaceae) lower lumenal redox potential, allowing subsequent colonisation by more strictly anaerobic species of bacteria such as Bacteroides spp., Clostridium spp. and the bifidobacteria (Finegold et al., 1983). Upon breast feeding, a microflora dominated by bifidobacteria establishes. Indeed, human breast milk contains an array of bifidogenic oligosaccharides (e.g. N-acetylated aminosugars), and peptides (e.g. glycomacropeptide) which have been shown to selectively encourage the growth of bifidobacteria within the gastrointestinal tract. Breast milk also contains a significant immunological component, which also impacts on the composition and activity of the gut microflora (Mountzouris et al., 2002). A much more diverse collection of bacterial species has been shown to colonise the gastrointestinal tract of formula-fed infants, with the dominance of bifidobacteria often supplanted by Bacteroides spp. and the clostridia (Mackie et al., 1999). Fortification of infant milk formula feeds with bifidogenic agents such as prebiotics (see below) is an active area of research and development within many formula feed and health care companies. The gut microflora becomes more diverse during weaning onto solid foods, and it has been proposed that a microflora resembling that of the adult becomes established by the age of two, although it is likely that successional development continues until much later into childhood. Indeed, a lower microbial diversity in children aged 16 months to seven years compared to adults was observed using proportionality of 16S rRNA species compared to total faecal 16S rRNA using dot-blot oligonucleotide probing (Hopkins et al., 2001, 2002). It is likely that as bacterial numbers and competition for nutrients increase, ecological niches become occupied by more specialised groups of bacteria, eventually resulting in climax microbial populations where potentially all ecological niches are occupied (Falk et al., 1998). Such species and ecological diversity affords the adult gut microflora a high degree of homeostasis and greatly limits the ability of invading bacteria to colonise the gut. In health, the adult gut microflora has been shown to be remarkably stable in species composition within individuals over time (Zoetendal et al., 1998). It has been estimated that this microflora is made up of some 500 different species of bacteria with maybe 50 different species representing the dominant populations within the gut microflora (Moore and Holdeman, 1974). Other bacterial species occupy specific and often unique ecological niches within this complex microbial ecosystem, often cross-feeding on the by-products of more dominant members of the microflora.

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16.2.1 The microflora of the gastrointestinal tract and interactions with host physiology As well as changing temporally from birth to adulthood, the gut microflora changes spatially within the gastrointestinal tract. The human stomach is relatively sparsely colonised by bacteria with numbers of facultative anaerobes such as lactobacilli, streptococci and yeast occurring in numbers as low as 102 colony-forming units (CFU) per ml contents. Gastric acid is the chief factor limiting microbial colonisation of the stomach, and acts as a crucial first line of defence in limiting the growth of invading microbial pathogens ingested from the external environment. Indeed, it is likely that many of the bacteria isolated from the stomach are allochthonous, originating in the oral cavity or being ingested with food (Gorbach, 1993). One important exception is Helicobacter pylori, evolved for gastric colonisation and occurring in approximately 40% of the adult population in developed countries. H. pylori possesses a number of unique ecological adaptations which allow it to colonise the stomach. This spiral shaped, flagellated, Gram negative organism, burrows into the gastric mucosa and adheres onto the gastric epithelium. Here, it may escape to some degree from the acidic environment of the stomach lumen. Specific physiological characteristics which enable H. pylori to cope with high acid environments include the ability to reduce cytoplastic H+ concentrations through hydrolysis of urea to ammonia, secretion of carbonate and production of outer membrane proteins with higher isoelectric points (Sachs et al., 2002). H. pylori has been shown to be the causative agent of gastric ulcers and is considered to be a major risk factor for the development of gastric cancer. Indeed, the organism was recognised by the International Agency for Cancer Research as a biological carcinogen in 1994, for its role in the inducing atrophic gastritis leading to the gastritis-metaplasia-carcinoma sequence (IARC Working Group, 1994; Baldini et al., 1999). Carriage of H. pylori can reach up to 80% of the population in developing countries and this has been attributed mainly to poor sanitation and spread via the faecal-oral route. In developed countries, where sanitation has been much improved over the last 50±60 years, the incidence of H. pylori infection is much higher in the elderly often reaching up to 80% of the over sixties. This cohort effect is probably due to infection of this population subset early in life, when sanitation was rudimentary and on a par with present-day developing countries, e.g., inner-city slums and under-developed rural areas. The small intestine is also relatively sparsely populated, with the rapid transit of digesta driven by peristalsis limiting microbial colonisation. The digestive capabilities of the small intestine and biliary secretions also inhibit microbial colonisation in this region of the gut. Indeed it is not until the distal ileum that microbial populations start to increase in number significantly. Here numbers of facultative lactobacilli, streptococci and enterobacteria, as well as some anaerobic bifidobacteria, Bacteroides spp. and clostridia reach population levels of about 104±108 CFU/ml. The ileocecal valve, in health, effectively limits the

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spread of a more diverse and dense microflora moving proximally from the colon (Gorbach, 1993; Conway, 1995). The colon is the major site of microbial colonisation within mono-gastric mammals with microbial numbers reaching up to 1011±1012 CFU/ml contents. This climax microbial population is also the most diverse within the human host, and a large proportion of species present are new to science (Suau et al., 1999; Blaut et al., 2002). Indeed many of the bacteria present defy cultivation by classical microbiological techniques and have only recently been recognised with the application of modern molecular microbiological tools to study this rich microbial habitat. The colonic microflora is dominated by the strict anaerobes such as Bacteroides spp., the clostridia and other families within the Clostridium mega-genus (including Ruminococcus spp., Butyrovibrio spp., Fusobacterium spp., Eubacterium spp., Peptostreptococcus), Bifidobacterium spp., Atopobium spp. and the peptococci. Facultative bacteria occur in much lower numbers, typically 1,000-fold lower, and include species of lactobacilli, enterococci, streptococci, the Enterobacteriaceae. In health, yeasts are also found in low numbers (about 102±104 CFU/ml), due to competitive exclusion by bacteria. This complex microflora, by its very nature, serves as an important barrier to the establishment of allochthonous microorganisms which may be pathogenic to the host. They effectively limit their colonisation potential through competition for nutrients, ecological niches, attachment sites on the gut wall and production of inhibitory metabolites such as short chain fatty acids (which also lower colonic pH) and bacteriocidal compounds such as bacteriocins. 16.2.2 The gut microflora in old age The composition of the gut microflora, both in relative numbers of different populations and in species present has been reported to be altered in old age. Indeed, it appears that microbial succession within the gastrointestinal tract does not stop with the relatively stable adult gut microflora, but may be thought of as entering senescence in the elderly. Gorbach et al. (1967) observed that elderly people had lower numbers of bifidobacteria and elevated population levels of yeasts and enterobacteria compared to adults. Mitsuoka and Hayakawa (Mitsuoka and Hayakawa, 1973; Mitsuoka, 1992) reported that numbers of bifidobacteria decreased in the aged, while numbers of clostridia, lactobacilli, streptococci and enterobacteria were present in high population levels. More recent studies have confirmed these observations. Hopkins et al. (2001) using a combination of traditional microbiological culture based techniques, a molecular technique which estimates relative abundance of 16S rRNA populations compared to total faecal 16S rRNA (dot-blot hybridisation), and community cellular fatty acid profiles, investigated the composition of the gut microflora in four different groups of individuals. The cohorts included children (16 months to seven years, n 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

References

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POOL-ZOBEL B L, NEUDECKER C, DOMIZLAFF I, JI S, SCHILLINGER U, RUMNEY C, MORETTI M, VILARINI I, SCASELLATI-SFORZOLINI R and ROWLAND I R (1996), `Lactobacillus and Bifidobacterium mediated antigenotoxicity in the colon of rats', Nutr Cancer, 26, 365±380. POOL-ZOBEL B, VAN LOO J, ROWLAND I R and ROBERFROID M B (2002) Experimental evidence on the potential of prebiotic fructans to reduce the risk of colon cancer. Brit J Nutr, 87 (Suppl. 2), S273±S281. POTHOULAKIS C and LAMONT T (2001), `Microbes and microbial toxins: paradigms for microbial mucosal interactions. II. The integrated response of the intestine to Clostridium difficile toxins', Am J Physiol (Gastroinest Liver physiol), 280, G178± G183. RAFTER J (2002), `Lactic acid bacteria and cancer: mechanistic perspective', Brit J Nutr 88 (Suppl. 1), S89±S94. RASTALL R A and MAITIN V, `Prebiotics and synbiotics: towards the next generation' Curr Opin Biotech, 13, 490±496. RATNAIKE R N and JONES T E (1998), `Mechanisms of drug-induced diarrhoea in the elderly', Drugs Aging, 13, 245±253. ROBERFROID M B, VAN LOO J A and GIBSON G R (1998), `The bifidogenic nature of chicory inulin and its hydrolysis products', J Nutr, 128, 11±19. ROWLAND I R, RUMNEY C J, COUTTS J T and LIEVENSE L C (1998), `Effect of Bifidobacterium longum and inulin on gut bacterial metabolism and carcinogen-induced aberrant crypt foci in rats', Carcinogenesis, 19, 281±285. RUSSELL R M, KRASINSKI S D, SAMLOFF I M, JACOB R A, HARTZ S C and BROVENDER S R (1986), `Folic acid malabsorption in atrophic gastritis', Gastroenterol, 91, 1476±1482. È HTEENMAÈKI L, CRITTENDEN R, SALMINEN S and MATILLA-SANDHOLM T SAARELA M, LA (2002), `Gut bacteria and health foods the European perspective', Int J Food Microbiol, 78, 99±117. SACHS G, MOO SHIN J, VAGIN O, MUNSON K, WEEKS D, SCOTT D R and VOLAND P (2002), `Current trends in the treatment of upper gastrointestinal disease', Baillieres Best Pract Res Clin Gastroenterol, 16 (6), 835±849. SAMUEL S C, HANCOCK P and LEIGH D A (1991), `An investigation into Clostridium perfringens enterotoxin-associated diarrhoea', J Hospit Infect, 18, 219±230. SAUNIER K and DOREÂ J (2002), `Gastrointestinal tract and the elderly: functional foods, gut microflora and healthy ageing', Dig Liver Dis, 34, S19±S24. SCHIFFRIN E J, ROCHAT F, LINK-AMSTER H, AESCHLIMANN J M and DONNET-HUGHES A (1995), `Immunomodulation of human blood cells following the ingestion of lactic acid bacteria', J Dairy Sci, 78, 491±497. SCHLEY P D and FIELD C J (2002), `The immune-enhancing effects of dietary fibres and prebiotics', Brit J Nutr, 87, (Suppl. 2), S221±S230. SCHMUCKER D L, TOREUX K and OWEN R L (2001), `Aging impairs intestinal immunity', Mech Ageing Dev, 122, 1397±1411. SILVI S, VERDENELLI M C, ORPIANESI C and CRESCI A (2003), `EU project CROWNALIFE: functional foods, gut microflora and healthy ageing isolation and identification of Lactobacillus and Bifidobacterium strains from faecal samples of elderly subjects for a possible probiotic use in functional foods', J Food Eng, 56, 195±200.

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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 `organisms 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 `a 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 <55 mol% in the DNA (Holzapfel et al., 2001). However, traditionally, physiologically, and also for practical purposes, bifidobacteria are still considered to form part of the LAB. Relevant Bifidobacterium spp. that act as probiotics (Table 17.1) are in general strict anaerobes, and are difficult to cultivate in milk or other food substrates (Gomes and Malcata, 1999). Bifidobacteria have been shown to be the predominant species in the GIT of infants, and represent the third most numerous species encountered in the colon of adult humans, considerably outnumbering other LAB groups such as Lactobacillus species. They play a vital role therefore in carbohydrate

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fermentation in the colon. They are considered to have a significant role in maintaining the good health of the human host. Furthermore, there is mounting evidence pointing to the activity of these bacteria in mediating other positive health attributes such as the alleviation of lactose intolerance, stimulation/ potentiation of the immune system, and production of vitamins and antimicrobials (Ballongue, 1998; Gomes and Malcata, 1999). Bifidobacteria were first isolated and described over 100 years ago from human faeces and were quickly associated with a healthy GIT due to their numerical dominance in breast-fed infants compared to bottle-fed infants (Tissier, 1906). They are among the first colonisers of the sterile GIT of newborns and predominate until weaning, when other bacterial groups surpass them (Tannock, 1999). While they were first grouped in the genus Bacillus, the genus Bifidobacterium was proposed in the 1920s (Orla-Jensen, 1924). However, there was not a taxonomic consensus for this new genus, and for much of the twentieth century they were classified in the genus Lactobacillus (e.g. as Lactobacillus bifidus) due to their rod-like shapes and obligate fermentative characteristics. Later, the accumulation of studies detailing DNA hybridisations, G + C content, and unique metabolic capabilities resulted in the resurrection of the Bifidobacterium genus (Rogosa, 1974). The most important species with regard to their application as probiotic cultures are B. longum and Bifidobacterium lactis. However, using genomic methods some authors could show that most strains isolated from probiotic dairy products belong to B. animalis. Indeed, the taxonomic position of B. lactis must be evaluated in more depth. Moreover, it should be ruled out if B. lactis strains are in fact B. animalis strains that are adapted to milk (Klein et al., 1998). The most common species found in the infant gut are Bifidobacterium infantis (now B. longum), Bifidobacterium breve, and B. longum, whilst Bifidobacterium adolescentis and B. longum are thought to predominate within the adult colon (Modler et al., 1990). In general, B. longum is often the dominant species detected in humans (Klaenhammer et al., 2002). Interestingly, preliminary studies have shown that B. longum is able to exert stronger antimicrobial activities than B. bifidum (Gibson and Wang, 1994). 17.2.2 Prebiotics Any dietary ingredient that reaches the colon unchanged, e.g., non-digestible carbohydrates (from small sugar alcohols and disaccharides to oligosaccharides and large polysaccharides), some peptides and proteins, as well as certain lipids, is a candidate prebiotic. The prebiotics developed so far are the non-digestible oligosaccharides (NDOs) (Roberfroid, 1997; Gibson and Fuller, 2000): fructooligosaccharides (FOS), galacto-oligosaccharides (GOS), transgalacto-oligosaccharides (TOS), isomalto-oligosaccharides (IMO), xylo-oligosaccharides (XOS), gentio-oligosaccharides (GEO), and soybean oligosaccharides (SOS). In particular, non-digestible inulin-type fructans, i.e., FOS and inulin, are known to lead a specific fermentation towards a community predominated by bifido-

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bacteria (Wang and Gibson, 1993; Buddington et al., 1996; Kleesen et al., 1997; 2001; McBain and Macfarlane, 1997), and reproducibly demonstrate prebiotic effects in humans (Gibson et al., 1995; Roberfroid et al., 1998; Bouhnik et al., 1999; Kruse et al., 1999; Harmsen et al., 2002). Indeed, there is a general consensus that inulin and FOS modify the bowel habit, causing faecal bulking, normalisation of stool frequency, and a prebiotic effect, i.e., a food-induced increase in the numbers and/or activity of bifidobacteria and other LAB in the GIT (van Loo et al., 1999). Other claims are increased calcium absorption, modulation of lipid metabolism, and preventive effect against colon cancer (Roberfroid, 1997; Cummings et al., 2001; Van Loo and Jonkers, 2001; Buddington et al., 2002). Inulin and FOS are the market leaders in Europe and the USA. GOS are also on the market in the EU and USA, but have yet to become as widely used as the fructans. Almost all NDOs are on the Japanese market. To date, there is only one comparative study of the fermentation properties of these commercial prebiotics (Rycroft et al., 2001). It indicates that oligosaccharides differ in their fermentation characteristics. IMO and GOS are most effective at increasing bifidobacterial numbers and lactate while generating the least gas. Importantly, also resistant starch displays a prebiotic nature. Resistant starch is starch that resists small intestinal digestion and enters the large bowel in normal humans. The amount of resistant starch is dependent of the source. It is thought that approximately 10% of the total dietary starch may escape digestion in the human small intestine. This resistant starch will contribute to the shortchain fatty acid production, increase stool weight, and decrease faecal pH. Several studies have shown that resistant starch favours butyrate production (Cummings and Macfarlane, 1991). Butyrate is the preferred substrate for the colonocytes, even when competing substrates such as glucose are present, and it is probably protective against colorectal cancer (Silvi et al., 1999; Topping and Clifton, 2001).

17.3 Investigating the effectiveness of probiotics and prebiotics: the case of antimicrobial function 17.3.1 Mechanisms of action To have an effect that leads to disease, a possible pathogen will have to overcome a series of hurdles. The first hurdle consists of adhesion to the host intestinal epithelium. This first step is very essential, otherwise the pathogen is flushed out by the fluid flow in the gut lumen and by the gut peristalsis (Lu and Walker, 2001; Reid et al., 2001). Once bound, the pathogen can start to replicate and to colonise the epithelial surface. Therefore, it must resist the environmental conditions of the gut, created both by the host that will counteract via the immune system and by the other microbes present in the intestine, an interaction also referred to as colonisation resistance (barrier effect). This is the phenomenon by which microbes already present in the ecosystem display properties (competitive and antagonistic) that prevent or minimise the

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replication of newly introduced strains (Tannock, 1999). Thirdly, the pathogen must induce its pathogenic effect in the intestine, such as disruption of the tight junctions of epithelial cells by production of enterotoxins, disruption of the fluid and electrolyte absorption with diarrhoea as a result of chloride secretion, and by inciting inflammation of the gastrointestinal mucosa (Berkes et al., 2003). Probiotics can interfere in each of these steps and thus prevent infection and pathogenesis. First of all, pathogens and probiotics must compete for nutrients available in the gut lumen (Fuller and Gibson, 1997). However, given the complex, nutrient-rich environment of this ecosystem it may be assumed that this is not really the case. Further, epithelial and mucosal adherence of pathogens may be inhibited as well as their epithelial invasion (Servin and Coconnier, 2003). Probiotics can block the adhesion of the pathogen to the epithelial cells by steric hindrance (Chan et al., 1985; Lee and Puong, 2002), competition for host-cell-binding sites (Neeser et al., 2000), or changing the intestinal mucus production (Bourlioux, 1997; Mattar et al., 2002, Mullie et al., 2002; Mack et al., 2003), and by secretion of anti-adhesive molecules (Velraeds et al., 1996; Fujiwara et al., 1997; 1999; 2001b). Also, they can directly interfere with growth and even kill the invading pathogen by the production of antimicrobial compounds (section 17.3.2). All these functionalities will contribute to the colonisation resistance or barrier effect. Inhibitory compounds that are produced by lactobacilli are lactic acid and volatile acids (acetic acid and formic acid) that will decrease the luminal pH. The production of organic acids, in particular acetic acid and lactic acid, by bifidobacteria appears to be the underlying mechanism of the antagonistic activity against pathogenic bacteria (Ibrahim and Bezkorovainy, 1993; Asahara et al., 2001a,b; Bruno and Shah, 2002; Rokiah et al., 2000; Fooks and Gibson, 2000; Setoyama et al., 2003). The inhibitory effect of the organic acids is primarily due to the undissociated molecules that lower the pH of the cytoplasm. This effect leads to the destruction of the transmembrane potential of the proton motive force and the loss of active transport of nutrients through the membrane, resulting in cell death. Other primary metabolites that can interfere with the growth of other bacteria and pathogens are hydrogen peroxide, carbon dioxide, and diacetyl, and specific antimicrobial compounds like reuterin, reutericyclin, and bacteriocins (Vandenbergh, 1993; De Vuyst and Vandamme, 1994). The ability to synthesise bacteriocins is widely distributed among lactobacilli of the human GIT (Toba et al., 1991; Itoh et al., 1995; Kawai et al., 2001). In contrast, the only bacteriocin produced by a Bifidobacterium strain that has been purified and characterised till now is bifidocin B, which is produced by a B. bifidum strain and is active against pathogenic Listeria spp., but not against any of the Gram-negative indicators tested (Yildirim and Johnson, 1998; Yildirim et al., 1999). However, bacteriocins of LAB are active only against Gram-positive bacteria, and the frequency of gastrointestinal infectious disorders caused by Gram-positive bacteria is relatively low compared with that of gastrointestinal infections caused by Gram-negative bacteria, which are insensitive to bacteriocins. Further,

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given their peptide nature, it is still unclear if bacteriocins would have a significant activity in the gut where host and microbially produced proteases are active. However, it is reasonable to assume that the host may control the production of such substances and that the functionality of bacteriocins may be restricted to local niches. Only one study reports the presence of a bacteriocin in the faeces of gnotobiotic rats mono-associated with a human Ruminococcus gnavus strain, inhibiting several Clostridium species, inluding Clostridium perfringens (Ramare et al., 1993). The bacteriocin probably needs trypsin processing or induction for developing its antimicrobial activity (Ramare et al., 1993; Dabard et al., 2001; Gomez et al., 2002). Also, other antimicrobial compounds, often referred to as bacteriocin-like compounds, can be produced by LAB (De Vuyst and Vandamme, 1994). They may exert direct inhibitory effects against Gramnegative pathogenic bacteria like Campylobacter, Escherichia coli, and Salmonella (Silva et al., 1987; Gibson and Wang, 1994; Coconnier et al., 1997; LieÂvin et al., 2000). Probiotics may also prevent disease by interfering at the onset of the disease, for instance through substrate detoxification and stimulation of mucosal immunity. For instance, toxin binding can be inhibited by S. boulardii (Pothoulakis et al., 1993; BrandaÄo et al., 1998). Probiotics can enhance the specific immune response to a pathogen by modulation of the immune system, thus improving the resistance of the host against the pathogen (de Waard et al., 2001; 2002; Shu et al., 2000; 2001; Shu and Gill, 2001). Otherwise, lactobacilli can down-regulate inflammation by altering the tissue cytokine secretion from a proinflammatory (e.g. secretion of interleukin 8 or IL-8) to an anti-inflammatory profile (e.g. secretion of interleukin 10 or IL-10) (Madsen et al., 2001; Mercenier et al., 2003). 17.3.2 Antibacterial activity of probiotics against gastrointestinal pathogens The main pathogen in the upper GIT is Helicobacter pylori (Mobley et al., 2001; Achtman and Suerbaum, 2001). Helicobacter pylori is a spiral bacterium infecting the stomach of 50% of the population. The infection leads to gastritis and in about 10% of those infected to the development of gastric ulcers. Furthermore, decades of stomach colonisation by this bacterium can lead to gastric cancer in about 1% of infected people. The bacterium can be eradicated from the human stomach by administration of a cocktail of antibiotics and drugs, affecting the stomach acidity, which leads to healing of peptic ulcers and consequent reduction in cancer risk development. Problems encountered with the current eradication of H. pylori are the increasing rates of eradication failure, mainly due to increased rates of resistance of H. pylori to the administered antibiotics. The use of probiotics can be an interesting alternative for the standard multiple antibiotic therapy. Several LAB have been shown to inhibit H. pylori (see below). However, the fundamental basis of the inhibition of H. pylori has not been elucidated.

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The lower GIT can be infected by many pathogens, such as E. coli, Salmonella, Bacteroides fragilis, Campylobacter jejuni, C. difficile, C. perfringens, Klebsiella pneumoniae, Listeria monocytogenes, Pseudomonas aeruginosa, Shigella dysenteriae, Shigella flexneri, Vibrio cholerae, Vibrio parahaemolyticus, and Yersinia enterocolitica (Berkes et al., 2003). Gastroenteritis can be caused by a variety of pathogens including rotavirus, E. coli, Campylobacter, and Salmonella, due to overgrowth of the intestines (McDaniel et al., 1995; Sansonetti and Phalipon, 1999; Lu and Walker, 2001). Enteropathogenic (EPEC) and enterohemorrhagic (EHEC) E. coli mediate their effect on the host cell by attachment to, and effacement lesions of, intestinal cells. This is characterised by localised destruction of brush border microvilli, intimate bacterial adhesion, and major cytoskeletal rearrangement. Probiotics can interfere in the pathogenesis of these bacteria by several mechanisms, thus preventing or counteracting infection. Salmonella enterica serovar Typhimurium is an enteroinvasive pathogen that causes infectious diarrhoea. In a first step, the bacterium will attach onto M-cells of the Peyer's patches. Subsequently, it will cross the epithelial barrier by entering via M-cells. Later, the M-cells will be destroyed and bacteria will invade subepithelial macrophages. They will induce an enhanced macrophage apoptosis and an increased immune and inflammatory response. It is a worldwide health problem among children and infants. Certain probiotics have been found to be helpful in preventing and treating some types of bacterialinduced diarrhoea because of their ability to alter the activity of the intestinal microflora and compete with potential pathogens (see below). The underlying mechanism is not known. In this chapter, we will focus on the interactions between probiotic lactobacilli or bifidobacteria and E. coli, Salmonella, and H. pylori, in particular with respect to their expression of antimicrobial activities. Antibacterial activity of lactobacilli Helicobacter pylori: in vitro and animal model investigations Since 1989, several investigators have studied the interactions between probiotics and H. pylori in vitro and in animal models (Table 17.2). Lactic acid is the major end metabolite of the carbohydrate metabolism of lactobacilli and has a strong inhibitory activity against H. pylori (Bhatia et al., 1989; Midolo et al., 1995; Aiba et al., 1998; Lorca et al., 2001). For instance, Lorca et al. (2001) showed that H. pylori was sensitive to L-lactic acid concentrations from 60 to 100 mM. They could also show that the antibacterial activity as observed for 17 different Lactobacillus species was lost upon pH neutralisation. Aiba et al. (1998) found that among three tested strains, L. salivarius was the most inhibiting and also produced the highest amount of lactic acid. Furthermore, the same strain was capable of completely inhibiting colonisation of the stomach by H. pylori in germ-free mice (Kabir et al., 1997). Similar observations were made with the probiotic L. gasseri OLL 2716. This strain was capable of inhibiting both H. pylori in vitro and in mice. Furthermore, it inhibited the production of IL-8, a pro-inflammatory cytokine released by

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human gastric cells upon infection with H. pylori. In addition, both clarithromycin-sensitive and -resistant H. pylori were inhibited both in vitro and in mice. In the mice trials, the clarithromycin-resistant H. pylori were even more susceptible towards L. gasseri OLL 2716 than the clarithromycin-sensitive ones. However, one has to bear in mind that the stomach of germ-free mice is completely different from the human situation. In humans, colonisation of the stomach by lactobacilli is very low because of the low pH in human stomachs whereas the pH in mice stomachs is higher and hence densely colonised by lactobacilli (Tannock, 1999). Some differences in antagonistic activity towards H. pylori between Lactobacillus strains can occur that cannot be attributed to differences in lactic acid production (Coconnier et al., 1998; Wendakoon et al., 1998; Michetti et al., 1999; Lorca et al., 2001). As an example, Michetti et al. (1999) compared the antibacterial activity of two strains, L. johnsonii La1 and L. acidophilus La10. Although both strains produced similar amounts of lactic acid, the first showed clear antibacterial activity and the second did not. Therefore, antimicrobials other than lactic acid have to be produced as well. This production of anti-H. pylori substances was strain-dependent given the big differences in antibacterial activity between the strains (Michetti et al., 1999). Lorca et al. (2001) discovered a proteinaceous substance from L. acidophilus CRL 639 that was released into the culture medium after cell lysis and that displayed a strong anti-H. pylori activity. The inhibitory activity produced by L. johnsonii La1 was heat-resistant and dialysable, but was lost when the pH of the cell-free culture supernatant (CFCS) was raised to pH 6.0 (Michetti et al., 1999). In addition, a very strong inhibitory activity was observed with CFCS of a culture of L. acidophilus LB (Coconnier et al., 1998). One hour of contact between CFCS of a culture of L. acidophilus LB and H. pylori cells caused a decrease of 5.5 log of H. pylori. The antibacterial activity withstood heating at 100ëC for 1 h. In the same study, CFCS of a culture of L. rhamnosus GG gave a decrease of 2.5 log in H. pylori viability. After heat treatment, the antibacterial activity of L. rhamnosus GG was lost. When mice infected with Helicobacter felis were treated with CFCS of a culture of L. acidophilus LB, a significant decrease of inflammation was observed without eradication of the pathogen (Coconnier et al., 1998). Sgouras et al. (2001) made similar findings with L. casei ACA-DC6002 during a treatment of mice infected with H. pylori. Both the colonisation of the stomach and the associated gastritis were significantly reduced in the presence of the probiotic strain. The question whether bacteriocins might be involved in the observed antibacterial effects of lactobacilli against H. pylori remains unanswered. Although the production of bacteriocins, which are generally active only against other closely related Gram-positive bacteria by lactobacilli is well-known, their in vivo activity has never been shown. One group investigated the effect of nisin, a well-known lantibiotic produced by Lactococcus lactis, towards H. pylori (Projan and Blackburn, 1993). They found that this bacteriocin displayed no activity against H. pylori when tested solely. However, when nisin was

Table 17.2 In vitro and animal model studies with probiotic lactobacilli towards Helicobacter pylori. The papers are listed chronologically Microorganism

End point

L. paracasei subsp. paracasei M3 L. casei Shirota L. acidophilus DDS-1J

H. pylori colony suppression

L. gasseri OLL2716

9 L. reuteri strains 17 lactobacilli

Results

Isolation of a bacteriocin-like proteinaceous compound. H. pylori colony suppression Inhibition of H. pylori by living L. casei cells. H. pylori colony suppression Inhibition of H. pylori when grown in a 1:1 ratio or higher with L. acidophilus. Suppression of growth; Both clarithromycin-sensitive and production of IL-8 by human clarithromycin-resistant H. pylori were gastric carcinoma cells; in vivo inhibited in coculture and in mice with activity L. gasseri. IL-8 response was decreased. Inhibition of binding to 2 out of 9 L. reuteri strains bound, and gangliotetraosylceramide and inhibited binding of H. pylori as well. sulfatide H. pylori colony suppression; Lactic acid-correlated inhibition of H. pylori. decrease in cell numbers after Bactericidal effect of a proteinaceous contact compound released after cell lysis by L. acidophilus CRL 639.

Reference Atanassova et al. (2003) Cats et al. (2003) Chatterjee et al. (2003) Ushiyama et al. (2003)

Mukai et al. (2002) Lorca et al. (2001)

L. casei ACA-DC6002 L. johnsonii La1, L. acidophilus La10 L. salivarius WB1004 L. acidophilus LB, L. rhamnosus GG 63 dairy starter cultures L. salivarius WB1004 12 LAB L. acidophilus

In vivo activity Urease activity; H. pylori colony suppression In vivo activity

Reduction of colonisation and gastritis. Inhibition of H. pylori by L. johnsonii culture and supernatant. Suppression of H. pylori in mice via lactic acid production. Decrease in cell numbers after Suppression of H. pylori in vitro. Protection contact; urease activity; in vivo against infection in vivo with L. acidophilus activity LB. Decrease of urease activity in vitro and in vivo. H. pylori colony suppression 28 out of 63 strains were inhibitory. Organic acids were at least partially the responsible agents. In vivo activity Inhibition of adhesion and colonisation of H. pylori in mice. H. pylori colony suppression Inhibition of H. pylori for 7 out of 12 strains and correlated with lactic acid production. Decrease in cell numbers after Inhibition of H. pylori probably due to lactic contact acid.

Sgouras et al. (2001) Michetti et al. (1999) Aiba et al. (1998) Coconnier et al. (1998)

Wendakoon et al. (1998) Kabir et al. (1997) Midolo et al. (1995) Bhatia et al. (1989)

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combined with chelating agents that interact with the cell envelop of Gramnegative bacteria, nisin showed a bactericidal effect towards H. pylori within seconds or minutes. Therefore, bacteriocin production might play a role in the inhibition of H. pylori when combined with other harsh conditions such as those occurring in the stomach, i.e., low pH and in the presence of defensins (Hamanaka et al., 2001). These in vivo conditions might make H. pylori susceptible for bacteriocins from lactobacilli (GaÈnzle et al., 1999). Two very recent studies underline the potential of bacteriocins as anti-H. pylori agents (Atanassova et al., 2003; Kim et al., 2003). The first study describes a bacteriocin-producing L. paracasei strain that inhibited not only H. pylori, but that was also capable of inhibiting Bacillus, Lactobacillus delbrueckii, several Candida species, and Saccharomyces cerevisiae (Atanassova et al., 2003). Another paper describes the inhibition of H. pylori by partially purified bacteriocins. Lacticins, especially those produced by Lactococcus species, displayed an activity that was stronger than that of nisin. Pediocins and leucocins, produced by Pediococcus and Leuconostoc, respectively, showed a relatively low inhibitory activity (Kim et al., 2003). Finally, the inhibition of the binding of H. pylori to glycolypids, including gangliotetraosylceramide and sulfatide, has been investigated as well (Osawa et al., 2001; Mukai et al., 2002). It was found that out of nine L. reuteri strains tested two could adhere to these receptor molecules. In addition, both strains successfully inhibited binding of H. pylori. Furthermore, it was shown that a protein with a molecular mass of 47 kDa, present in the cell surface extract, was responsible for this inhibition. Helicobacter pylori: clinical trials The results from in vitro and animal model experiments encouraged people to set up clinical trials to investigate the potential of Lactobacillus species or CFCS as biotherapeutics for treatment of H. pylori infections. Lactobacilli were particularly combined with the standard triple therapy applied to combat H. pylori to be able to improve cure and eradication rates. An overview of all clinical trials up to now is given in Table 17.3. Different studies mention that a Lactobacillus culture or its CFCS is never sufficient as a therapeutic, since eradication of H. pylori has never been achieved (Mrda et al., 1998; Michetti et al., 1999; Felley et al., 2001; Sakamoto et al., 2001; Wendakoon et al., 2002). However, when a probiotic is combined with the standard triple therapy, different results are found. A significant increase in eradication rate has been found with a heat-killed culture of L. acidophilus LB (Canducci et al., 2000). This indicates that the responsible agent was already present in the CFCS and that no de novo synthesis occurred. In vitro studies had already shown that CFCS of a culture of L. acidophilus LB displayed a strong inhibitory activity towards H. pylori (Coconnier et al., 1998). Similar findings have been made with the probiotic strain L. johnsonii La1. This strain also displayed good in vitro activity. Again, the supplementation of CFCS of a culture of L. johnsonii La1 was sufficient to have an in vivo suppressive effect

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435

(Michetti et al., 1999). Supplementation of L. johnsonii La1 also decreased urea breath test (UBT) values in asymptotic volunteers (Gotteland and Cruchet, 2003) and children (Cruchet et al., 2003). This led to the hypothesis that the effect was caused by an extracellular compound and that living cells were not necessary. In contrast, Cats et al. (2003) reported that for a full inhibitory effect living cells were needed. However, this was only shown in vitro and not in vivo. AB yoghurt involving a Lactobacillus and a Bifidobacterium strain showed no significant increase in eradication rate when combining the probiotic yoghurt with the standard triple therapy (Sheu et al., 2002). Also, fermented milk containing L. johnsonii La1 did not improve eradication rates of the triple therapy (Felley et al., 2001), although this strain showed improvement in H. pylori inflammation both in vitro and in humans when supplemented solely (Michetti et al., 1999; Gotteland and Cruchet, 2003). Lactobacillus rhamnosus GG did not improve eradication rates in combination with the triple therapy either (Armuzzi et al., 2000a; 2000b). Nevertheless, the strain had been shown to possess moderate anti-H. pylori activity in vitro (Coconnier et al., 1998). However, L. rhamnosus GG and the AB yoghurt reduced antibiotic-associated diarrhoea during H. pylori treatment (Armuzzi et al., 2000a; 2000b; Cremonini et al., 2002; Sheu et al., 2002), and hence may improve H. pylori eradication rates by a better drug compliance (Sheu et al., 2002). Also for L. paracasei subsp. paracasei F19, UBT values did not decrease upon ingestion of a fermented milk containing the probiotic when given to healthy elderly people that were seropositive for H. pylori (Sullivan et al., 2002). These data suggest that the effects on H. pylori are strain-dependent. Conflicting results have been reported with the strain L. gasseri OLL 2716. Sakamoto et al. (2001) reported on an effective reduction of H. pylori and gastric mucosal inflammation in adults infected with H. pylori. In addition, L. gasseri OLL 2716 had been proved effectively in reducing H. pylori numbers both in vitro and in mice (see above). However, L. gasseri OLL 2716 was not able significantly to reduce UBT values in infected children, although a higher dose of fermented milk was given (Shimizu et al., 2002). These differences might be explained by a difference in immunopathogenesis of H. pyloriassociated gastritis and gastric inflammation between children and adults. Escherichia coli Tables 17.4 and 17.5 give an overview of the in vitro studies conducted on bacterial interference of lactobacilli with E. coli. It has been reported that E. coli was inhibited by lactic acid produced by probiotic lactobacilli (Gilliland and Speck, 1977; Ogawa et al., 2001b; Jin et al., 2000, Fooks and Gibson, 2002, FernaÂndez et al., 2003), although E. coli possesses some acid resistance (Merrell and Camilli, 2002). It has been shown that undissociated lactic acid inhibits growth of E. coli at a concentration of about 2±11 mM and kills the cells at a concentration of 70 mM or higher (Merrell and Camilli, 2002; Ostling and Lindgren, 1993). Furthermore, lactic acid permeabilises the membrane of E. coli and Salmonella at a concentration of 5 mM at pH 4.0. This effect was stronger

Table 17.3 Clinical trials with probiotic lactobacilli towards Helicobacter pylori infections. The papers are listed chronologically Therapeutic

End point

Results

Reference

L. casei Shirota fermented milk Probiotic preparation containing L. johnsonii La1 or L. paracasei ST11, either living or heat-killed L. johnsonii La1 commercial product Different probiotic preparations in combination with triple therapy Yoghurt with living Lactobacillus and Bifidobacterium in combination with triple therapy Yoghurt containing L. gasseri OLL 2716 Fermented milk supplemented with Lactobacillus F19

UBT* UBT*

Slight, non-significant decrease of UBT values. A significant decrease was observed in children receiving live L. johnsonii La1.

Cats et al. (2003) Cruchet et al. (2003)

UBT*

A decrease in UBT values in asymptotic volunteers. Lower incidence of side-effects during treatment. No differences between the different probiotic preparations. Improvement of eradication rate. Faster restoration of Bifidobacterium levels in stools after therapy.

Gotteland and Cruchet (2003)

Anti-H. pylori therapy sideeffects Eradication rate

UBT*; serum pepsinogens; and faecal H. pylori antigens UBT*

A slight, non-significant decrease in UBT values in infected children. No statistical difference with the placebocontrolled group.

Cremonini et al. (2002) Sheu et al. (2002)

Shimizu et al. (2002) Sullivan et al. (2002)

2 L. acidophilus, 1 L. casei, 1 Suppression of H. pylori in commercial starter (combining vitro; UBT* in vivo 3 strains) L. rhamnosus GG combined Eradication and side-effects with a triple therapy L. rhamnosus GG combined with a triple therapy

Antibiotic-associated sideeffects and H. pylori eradication Fermented milk with living L. Bacterial density and gastric johnsonii La1 inflammation

Yoghurt containing live L. gasseri OLL 2716 Inactivated culture of L. acidophilus LB in combination with a triple therapy L. johnsonii whey-based culture supernatant L. acidophilus living cells *

UBT: [13C]urea breath test

UBT*; serum pepsinogens; and bacterial density Eradication rate UBT*; bacterial density Eradication rate

All strains inhibited H. pylori growth in vitro. No eradication in patients with a yoghurt combining all 6 strains. No differences in eradication were observed. Improvement of side-effects and treatment tolerance. No differences in eradication were observed between groups. Improvement of side-effects and treatment tolerance. A decrease in H. pylori density, inflammation and gastritis activity. H. pylori was not eradicated. No improvement in eradication when L. johnsonii was combined with clarithromycin. Decrease in H. pylori numbers, UBT values, and gastric mucosal inflammation. A significant increase in eradication rate.

Wendakoon et al. (2002)

A marked decrease in UBT values. Persistence of H. pylori infection. In 6 out of 14 patients H. pylori was eradicated.

Michetti et al. (1999)

Armuzzi et al. (2001a) Armuzzi et al. (2001b) Felley et al. (2001)

Sakamoto et al. (2001) Canducci et al. (2000)

Mrda et al. (1998)

Table 17.4 In vitro and animal model studies with probiotic lactobacilli towards Escherichia coli. The papers are listed chronologically Species

Model

Results

Reference

L. rhamnosus ATCC 53103

Adhesion to and invasion of Caco-2 cells.

Inhibition of invasion but not of adhesion, in spite of excellent adhesive properties of L. rhamnosus. Increased MUC3 mucin expression by L. plantarum 299v and L. rhamnosus GG. Decreased adherence of E. coli to HT-29 cells by the 2 same strains. Inhibition of adhesion and decrease of EPECinduced neutrophil migration. Living cells, prevented pathogen adhesion and invasion. Furthermore it counteracted several effects induced by E. coli. Also, probiotics alone increased TEER*. 2 L. paracasei were inhibitory, ascribed to bacteriocin-like compounds. Inhibition of adhesion for both L. crispatus cells and its S-layer protein. No antibacterial activity was observed. Decrease of viability of the pathogen by direct contact and when adherent on Caco-2 cells. Protection against brush border damage.

Hirano et al. (2003)

MUC3 mucin expression in L. plantarum 299v, L. rhamnosus GG, L. acidophilus HT-29 cells. Inhibition of DDS-1 attachment to HT-29 L. plantarum 299v L. acidophilus and S. thermophilus

Adhesion to T-84 cells; neutrophil migration Adhesion; invasion; physiological dysfunction by E. coli

20 LAB isolates from cheese

Well diffusion assay

L. crispatus JCM 5810

Adhesion to Matrigel or immobilised laminin

L. acidophilus LB

Contact with SCS** directly or on Caco-2 cells

Mack et al. (2003)

Michail and Abernathy (2003) Resta-Lenert and Barrett (2003) Caridi (2002) Horie et al. (2002) LieÂvin-Le Moal et al. (2002)

L. plantarum 299v

Passage of mannitol across the intestinal wall of rats

L. plantarum 299v L. rhamnosus HN001 (DR20)

Adhesion to Caco-2 cells; Isc***; and TEER*; direct contact Murine infection model

L. casei Shirota L. rhamnosus Lcr35

Murine model of UTI**** Adhesion to Caco-2 cells

L. rhamnosus HN001 (DR20), L. acidophilus HN017, L. johnsonii La1, L. bulgaricus LB L. rhamnosus GG L. casei Shirota

Well-diffusion assay; adhesion to or invasion of HT-29, Caco-2, and HT29MTX cells Caco-2 cells Pathogen-free infant rabbits

L. casei Shirota, L. acidophilus YIT0070, L. brevis YIT0076 15 hydrogen peroxideproducing Lactobacillus strains

Coculture batch fermentations Adhesion to vaginal epithelial cells

No effect of L. plantarum 299v on intestinal permeability when supplemented solely. One week pretreatment with L. plantarum 299v abolished the E. coli induced increase of intestinal permeability. No killing when direct contact. Inhibition of adhesion and decrease of the increased Isc*** when L. plantarum was preincubated. Reduced infection, translocation, and morbidity. Enhanced humoral and cellular immune response. Inhibition of E. coli growth in the urinary tract. Inhibition of adhesion in both pre-, co-, and post-incubation experiments. Antimicrobial activity for L. rhamnosus HN001, L. acidophilus HN017, and L. johnsonii La1. Inhibition of bacterial translocation. Decreased colonisation of gastrointestinal tract. Decreased level of Shiga toxins. Increased level of IgAs in the colon against Shiga toxins and E. coli cells. Inhibition due to undissociated lactic acid production. L. crispatus strains were the most inhibitory. L. crispatus 35 was able to block E. coli adhesion with 80, 54, and 21 % by exclusion, competition, and displacement, respectively.

Mangell et al. (2002)

Michail and Abernathy (2002) Shu et al. (2002) Asahara et al. (2001b) Forestier et al. (2001) Gopal et al. (2001)

Mattar et al. (2001) Ogawa et al. (2001a)

Ogawa et al. (2001b) Osset et al. (2001)

Table 17.4 Continued Species

Model

Results

Reference

14 adhering Lactobacillus strains isolated from porcine intestine L. plantarum 299v, L. rhamnosus GG

Adhesion to intestinal mucus from piglets

6 out of 14 Lactobacillus strains were inhibitory. This inhibition could be attributed to lactic acid and acetic acid production. Decrease of adhesion to HT-29 cells by L. plantarum 299v and L. rhamnosus GG. Increase of MUC2 and MUC3 mRNA levels upon incubation of HT-29 cells and L. plantarum 299v or its supernatant. 74 % of the strains tested produced a bacteriocin. None of them was active against E. coli. Inhibition of adhesion due to the production of an extracellular proteinaceous compound. Inhibition of adhesion by both living and heatkilled cells. Inhibitory activity for both strains probably due to an inhibitory substance with an estimated molecular mass of 12 to 14 kDa. Production of an inhibitory substance combined with a coaggregation ability of L. rhamnosus with E. coli. Inhibition of E. coli by SCS.

Jin et al. (2000)

Species of the L. acidophilus group isolated from human faeces L. fermentum 104R L. acidophilus LB L. rhamnosus GR-1, L. acidophilus 76 L. rhamnosus GR-1 L. rhamnosus GG

Inhibition of binding to HT29 and Hep-2 cells by probiotics and mucins Well-diffusion assay Adhesion to piglet ileal mucus Adhesion to Caco-2 cells Suppression of cell growth and survival of E. coli on plates or urine Inhibition of growth with an agar overlay Well-diffusion assay; viability after direct contact with SCS**

Mack et al. (1999)

Itoh et al. (1995) Blomberg et al. (1993) ChauvieÁre et al. (1992) McGroarty and Reid (1988) Reid et al. (1988) Silva et al. (1987)

*TEER: transepithelial electrical resistance; **SCS: spent culture supernatant; ***Isc: short circuit current; ****UTI: Urinary tract infection

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441

than that of hydrogen chloride. Lactic acid liberated lipopolysaccharides from the outer membrane of these bacteria, making them susceptible to other antimicrobials such as bacteriocins (Alakomi et al., 2000). Many investigators have reported on the inhibition of the adhesion of E. coli to gastrointestinal epithelial cells (ChauvieÁre et al., 1992; Coconnier et al., 1993; Bernet et al., 1994; Mack et al., 1999, 2003; Forestier et al., 2001; Gopal et al., 2001; LieÂvin-Le Moal et al., 2002; Michail and Abernathy, 2002; 2003; FernaÂndez et al., 2003; Resta-Lenert and Barrett, 2003) and of its translocation (Mattar et al., 2001) by using cell line models (cells of Caco-2, T-84, and HT29). Also, inhibition of adhesion to mucins (Jin et al., 2000; Tuomola et al., 1999; Blomberg et al., 1993) and human vaginal cells (Osset et al., 2001) has been reported. Hirano et al. (2003) reported on a L. rhamnosus strain that adhered very well to Caco-2 cells, but failed to inhibit the adhesion of E. coli. However, invasion of the human cell line by E. coli was inhibited. The authors suggested that a strong interaction between L. rhamnosus and the host cells triggered intracellular events that inhibited internalisation of E. coli. Horie et al. (2002) used a basement membrane as a model for bacterial adhesion. Salmonella and E. coli can bind to oligomannose chains of the laminin network in these basement membranes (Kukkonen, 1993). It was found that L. crispatus inhibited the adhesion of E. coli by means of its S-layer protein, CbsA. When this S-layer was added solely, adhesion of E. coli was inhibited (Toba et al., 1995; Horie et al., 2002). In addition, it was found that L. johnsonii La1 shared carbohydrate-binding specificities with enteropathogenic Salmonella and E. coli (Neeser et al., 2000). These studies supported the hypothesis of competitive exclusion as a possible explanation for the reported inhibition of adhesion (Lee et al., 2000). Moreover, L. fermentum 104R was able to inhibit adhesion of E. coli to mucus by the production of an extracellular proteinaceous compound (Blomberg et al., 1993). Also, L. casei Shirota, L. rhamnosus GG, L. rhamnosus LC-705 and L. johnsonii La1, as well as another L. rhamnosus strain isolated from human faeces, were able to reduce the S-fimbria-mediated adhesion of E. coli and the adhesion of Salmonella to human intestinal glycoproteins extracted from faeces, which constitute a mucin-like model (Tuomola et al., 1999). Michail and Abernathy (2002, 2003) reported on an inhibition effect of adhesion by L. plantarum 299v that was not related to a killing effect of the bacterium and that was independent of secreted compounds, since no activity was observed with CFCS of a culture of L. plantarum 299v. Similar results were obtained with L. rhamnosus Lcr35 (Forestier et al., 2001). Remarkably, this strain was able to inhibit adhesion to Caco-2 cells when added 30 min. before, together with, and 30 min. after a challenge with Salmonella. No direct killing effect was observed with CFCS. Alternatively, adhesion to intestinal epithelial cells by E. coli may be prevented by a direct killing effect on the bacterium. For instance, LieÂvin-Le Moal et al. (2002) found that a twofold concentrated CFCS of a culture of L. acidophilus LB decreased viable numbers of E. coli with 2.5 log units within three hours of incubation. When the same material was applied to E. coli

Table 17.5 In vitro and animal model studies with probiotic lactobacilli that interfere with both E. coli and Salmonella. The papers are listed chronologically Species

Model

Results

Reference

L. acidophilus UO 001, L. gasseri UO 002

Growth in mixed culture and adhesion to Caco-2 cells

FernaÂndez et al. (2003)

L. plantarum 0407

Co-culture experiments

Complete inhibition of the pathogen in a mixed population most probably due to lactic acid. Inhibition of adhesion of E. coli by L. gasseri. Antagonistic activity was observed against E. coli, Salmonella, and Clostridium, due to the production of organic acids. Inhibition of adhesion, probably by steric hindrance in the case of L. rhamnosus GG and by competition for binding sites of adhesins in the case L. casei Shirota. Lipoteichoic acids from Lactobacillus inhibited the proinflammatory immune response on E. coli and Salmonella lipopolysaccharides. High decrease of adhesive Enterococcus faecalis with L. crispatus JCM 8779, no decrease of E. coli or Salmonella. Killing effect on E. faecalis by L. crispatus SCS* due to the presence of a bacteriocin. Inhibition of both E. coli and Salmonella by overlaid colonies. Active compound in supernatant was only active against Grampositive strains, not against E. coli and Salmonella.

L. rhamnosus GG and L. casei Adhesion to Caco-2 cells Shirota L. johnsonii La1, L. acidophilus La10 L. crispatus JCM 8779, L. reuteri JCM 1081

L. salivarius UCC118

Production of proinflammatory cytokines in the presence of soluble CD14 Adhesion to Caco-2 cells; direct contact with the pathogen and SCS* Inhibition of growth by an agar overlay

Fooks and Gibson (2002) Lee and Puong (2002)

Vidal et al. (2002) Todoriki et al. (2001)

Dunne et al. (1999)

L. rhamnosus GG, L. S-fimbria-mediated adhesion rhamnosus LC-705, L. to human intestinal mucus rhamnosus new intestinal isolate, L. johnsonii La1, and L. casei Shirota L. acidophilus IBB 801 L. acidophilus LB L. paracasei B21060 and B21070, L. acidophilus B21190 L. johnsonii La1 103 Lactobacillus isolates L. acidophilus LB

*SCS: spent culture supernatant

Inhibition of growth by the agar overlay technique Time kill studies (contact between pathogen and SCS*) Co-culture and well-diffusion assay Adhesion to and invasion of Caco-2 cells Top agar overlay of the pathogen Adhesion to and invasion of Caco-2 cells

Slight reduction of adhesion of E. coli with the three L. rhamnosus strains. Significant inhibition of adhesion of Salmonella with L. johnsonii La1 and L. casei Shirota. Increased adhesion of Salmonella with L. rhamnosus GG and L. rhamnosus (intestinal isolate). SCS* inhibited both a Salmonella and an E. coli strain. An extracellular compound different from lactic acid inhibited a wide range of both Grampositive and Gram-negative pathogens. No inhibition in the well-diffusion assay. Strong inhibition in the coculture experiments.

Tuomola et al. (1999)

Inhibition of cell adhesion and invasion.

Bernet et al. (1994)

Around 70% of the strains were inhibitory to at least one of the tested pathogens (Listeria, Salmonella, and E. coli). The heat-killed L. acidophilus strain adhered well to Caco-2 cells and inhibited adhesion of several enteropathogens.

Chateau et al. (1993)

Zamfir et al. (1999) Coconnier et al. (1997) Drago et al. (1997)

Coconnier et al. (1993)

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attached to Caco-2 cells, a decrease of 3 log units was observed within the same time interval. Furthermore, Gopal et al. (2001) observed that 2.5 times concentrated CFCS of a culture of L. rhamnosus DR20, L. acidophilus HN017, and L. johnsonii La1 showed a strong inhibitory activity of E. coli. This inhibitory activity was partially lost upon enzyme treatment with lactate dehydrogenase, trypsin, and proteinase K. The authors concluded that the overall inhibition was due to the synergistic action of lactic acid and a proteinaceous factor. In addition, the concentrated CFCS of cultures of these strains contributed to an inhibitory effect of the adhesion to, and invasion of, gastrointestinal epithelial cells by E. coli. Todoriki et al. (2001) used two strains of Lactobacillus that strongly adhered to Caco-2 cells. However, when these two strains were investigated for inhibition of adhesion of E. coli or Salmonella, both strains performed poorly. In contrast, adhesion of Enterococcus faecalis was significantly inhibited by L. crispatus. Furthermore, this inhibition of adhesion was related to a proteinaceous antibacterial compound present in the CFCS. These results led the authors to suggest that competition for adherence sites or steric hindrance plays only a minor role in the inhibition of adherence and that antibacterial activity is of greater importance. It is sometimes difficult therefore to discriminate between a direct killing effect by a metabolite produced by the probiotic LAB strain and its subsequent loss of adhesion to gastrointestinal cells. Several investigators have observed a direct killing effect of Lactobacillus that could be ascribed to the production of bacteriocins or bacteriocin-like compounds (Silva et al., 1987; McGroarty and Reid, 1988; Reid et al., 1988; Chateau et al., 1993; Coconnier et al., 1997; Dunne et al., 1999; Gopal et al., 2001; Caridi, 2002; LieÂvin-Le Moal, 2002). The L. acidophilus LB strain has been proved to inhibit the adhesion of many enteropathogens to Caco-2 cells (Coconnier et al., 1993, ChauvieÁre et al., 1992). This inhibition is probably due to the secretion of an antimicrobial into the culture medium (Coconnier et al., 1997; LieÂvin-Le Moal et al., 2002). A strain with interesting properties is L. salivarius subsp. salivarius UCC118. This strain showed good adherence to Caco-2 cells and was antimicrobial towards Bacillus, Enterococcus, E. coli, Listeria, Pseudomonas, Salmonella, and Staphylococcus, but not towards Streptococcus and Lactobacillus species, with the exception of L. fermentum. Also, CFCS of a culture of the strain displayed antibacterial activity, although the activity against E. coli and Salmonella could not be retrieved in the supernatant (Dunne et al., 1999). Later, a bacteriocin (ABP-118), present in the CFCS, was purified and characterised to be responsible for inhibition of the Gram-positive indicators (Flynn et al., 2002). Although bacteriocins from lactobacilli and LAB in general are not active against Gram-negative bacteria (De Vuyst and Vandamme, 1994), it cannot be ruled out that bacteriocins from lactobacilli such as ABP-118 may play a role in the reported inhibition towards E. coli and Salmonella. For instance, it has been found that bacteriocins of LAB may antagonise Gram-negative bacteria under stressed conditions such as low pH or high salt concentrations (GaÈnzle et al., 1999; Alakomi et al., 2000). Lactic acid, next to its antimicrobial effect by

Probiotics, prebiotics and gut health

445

lowering the pH, also functions as a permeabiliser of the outer membrane of Gram-negative bacteria and may thus potentiate the effects of bacteriocins and other antimicrobial substances (Alakomi et al., 2000). In addition, it has been shown that some bacteriocins such as pediocin PA-1, sakacin P, and curvacin A, produced by Pediococcus acidilactici, Lactobacillus sakei, and Lactobacillus curvatus, respectively, displayed a strong synergy with the eukaryotic antimicrobial peptide pleurocidin in the inhibition of Gram-negative E. coli (LuÈders et al., 2003). Moreover, the dairy strain L. acidophilus IBB 801 produces a bacteriocin that is probably involved in the inhibition of both E. coli and Salmonella (Zamfir et al., 1999). Also, bacteriocin-like compounds produced by L. paracasei subsp. paracasei (Caridi, 2002), Enterococcus faecium (Pantev et al., 2002), and several other enterococci (Simonetta et al., 1997) have been reported to antagonise Gram-negative bacteria. Lactobacillus reuteri is able to produce reuterin and reutericyclin. Reuterin is a broadspectrum antimicrobial including both Gram-negative (i.e., E. coli and Salmonella) and Gram-positive (i.e., Clostridium and Staphylococcus) bacterial species that is synthesised from glycerol (Talarico et al., 1988). Reutericyclin is a tetrameric acid derivative that has a wide inhibitory spectrum against Grampositive bacteria (GaÈnzle et al., 2000; HoÈltzel et al., 2000). Finally, in the past several compounds have been described that display anti-E. coli activities. Examples are acidolin (Hamdan and Mikolajcik, 1974) and acidophilin (Shahani et al., 1977). However, these compounds were poorly characterised (De Vuyst and Vandamme, 1994). Another mechanism of action is the up-regulation of the natural epithelial barrier mechanisms by a probiotic. It has been shown that mucin gene expression can be stimulated by lactobacilli and that the increased mucus production contributes to the inhibition of the adhesion of E. coli (Mack et al., 1999; 2003). Resta-Lenert and Barrett (2003) described how a combination of living S. thermophilus and L. acidophilus cells increased the transepithelial resistance of Caco-2 monolayers. Furthermore, this probiotic preparation inhibited adhesion and invasion by an enteroinvasive E. coli when the probiotic strain was incubated prior to infection. Several physiological dysfunctions caused by E. coli such as a decrease in transepithelial resistance, an increase in epithelial permeability, a disruption of the cytoskeleton and tight junctional proteins, an ion transport dysfunction, and an inactivation of the epidermal growth factor receptor were also prevented. A full inhibitory effect could be obtained only with living cells of S. thermophilus and L. acidophilus. Inhibition of E. coli induced an increase of intestinal permeability and was also observed in rats pretreated with L. plantarum 299v. In addition, it was shown that L. rhamnosus HN001 protects mice from infection with E. coli by stimulation of the immune response (Shu and Gill, 2002). These authors found an increased level of anti-E. coli antibody IgA when the mice were fed with L. rhamnosus HN001. Also, mice treated with L. rhamnosus HN001 exhibited a higher phagocytic capacity. The inhibitory effect of L. casei Shirota has mainly been investigated in animal models (Ogawa et al., 2001a; Asahara et al., 2001b). In an infant rabbit

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model of a Shiga toxin-producing E. coli infection, it was observed that L. casei Shirota decreased the severity of diarrhoea, lowered E. coli colonisation levels, and decreased both the level of Shiga toxins and histological damage. On the other hand, specific antibodies (IgA) against E. coli and its two toxins were increased when the rabbits were treated with L. casei Shirota, suggesting that the improvements observed in the group treated with L. casei were caused by an enhancement of the immune response (Ogawa et al., 2001a). Similarly, it was found that L. casei Shirota suppressed infection with E. coli in a murine urinary tract infection model. A significant antimicrobial effect was seen for both living and heat-killed cells when supplied in a single dose as a pre-treatment or in a multiple daily treatment during the post-infection period. This effect was specific for L. casei Shirota and could not be attributed to the production of organic acids or a bacteriocin-like molecule (Asahara et al., 2001b). In addition, L. casei Shirota has been shown to adhere in vitro to Caco-2 cells and to inhibit adhesion of both E. coli and Salmonella spp. in competitive inhibition, exclusion, and displacement experiments. Probably, L. casei Shirota possesses two types of surface adhesins that can compete with the adhesins of Salmonella and E. coli for receptor sites (Lee and Puong, 2002). It has also been shown that the lipoteichoic acids from the cytoplasmic membrane of Gram-positive bacteria may temper a proinflammatory response. Vidal et al. (2002) observed that lipopolysaccharides from E. coli and Salmonella were able to induce a proinflammatory response of HT-29 cells in the presence of soluble CD14. This phenomenon was not observed with the lipoteichoic acids from L. johnsonii La1 and L. acidophilus La10. In contrast, the presence of these cell wall fragments down-regulated the stimulation of proinflammatory cytokines by the lipopolysaccharides from the enteropathogenic E. coli and Salmonella. Although many clinical studies dealt with the potential of probiotics to prevent or treat diarrhoea (Szajewska and Mrukowicz, 2001; Gismondo et al., 1999), none of these really focused on the effect on E. coli during probiotic treatment. Reid et al. (2003) studied the effect of oral supplementation of a combination of L. rhamnosus GR-1 and L. fermentum RC-14 on the vaginal flora of healthy women. The group taking the probiotic showed significantly higher counts of vaginal lactobacilli and lower amounts of coliforms than the control group. Salmonella Lactobacilli have been shown to inhibit the pathogenesis caused by Salmonella by interfering at various stages of the invasion process (Tables 17.5 and 17.6). As for H. pylori and E. coli, Salmonella has been shown to be sensitive towards the organic acids produced by lactobacilli (Juven et al., 1991; Lehto and Salminen, 1997; Fooks and Gibson, 2002; FernaÂndez et al., 2003). However, some lactobacilli produce also other metabolites with anti-Salmonella properties. L. acidophilus LB secretes a compound into the culture supernatant with a broad inhibitory spectrum including Salmonella (Coconnier et al., 1997). As a consequence, a L. acidophilus LB culture was able to inhibit adhesion to and invasion of human gastrointestinal epithelial cells in vitro (Coconnier et al.,

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1993; 2000). Similar experiments were carried out with L. johnsonii La1 with a comparable outcome (Bernet-Camard et al. 1997). The latter authors reported that L. johnsonii La1 produced an antibacterial substance active against several enteropathogens including Salmonella. This compound did not inhibit lactobacilli and bifidobacteria. The antibacterial activity was unrelated to lactic acid production and was relatively insensitive to proteolytic enzymes. Furthermore, L. johnsonii La1 was able to develop its anti-Salmonella activity in both conventional and germ-free mice. A significant decrease in Salmonella counts in faeces was observed in conventional mice that were given L. johnsonii La1. On the other hand, association of germ-free mice with L. johnsonii La1 was not sufficient to survive infection with Salmonella, although a significant prolongation of survival was noticed. Hudault et al. (1997) further showed that a culture of L. rhamnosus GG or its CFCS was able to inhibit the invasion by S. Typhimurium of Caco-2 cells without killing the bacteria. Furthermore, the supplementation of L. rhamnosus GG to conventional mice reduced the numbers of Salmonella shedding in the faeces and delayed mortality of infected germ-free mice. The viability of Salmonella was not influenced by contact with CFCS of a culture of L. rhamnosus GG, although Silva et al. (1987) reported earlier that L. rhamnosus GG produced an antimicrobial substance different from lactic acid that killed Salmonella. The fact that the viability of the strain was not modified but that the activity was abolished after the culture had been neutralised to pH 7 suggested a pH-dependent mechanism (Lehto and Salminen, 1997). Perhaps, L. rhamnosus GG inhibited the adhesion of Salmonella to gastrointestinal cells by steric hindrance (Lee and Puong, 2002). Gill et al. (2001) performed one of the most convincing animal studies. Ninety per cent of the conventional mice fed with L. rhamnosus HN001 survived a single dose Salmonella challenge whilst only 7 per cent of the control mice survived. Furthermore, the investigators showed that anti-Salmonella antibodies and leucocyte phagocytosis responses significantly increased, and counts of Salmonella translocating to visceral tissues decreased upon probiotic administration, suggesting that immunoenhancement may play an important role in improved survival. Valdez et al. (2001) started from a completely opposite point of view. They investigated how LAB could inhibit apoptosis in macrophages infected with Salmonella. The ability to survive vacuolation by macrophages and to induce their apoptosis may lead to the infection of deeper tissues. Hence, this ability may be an important feature in the pathogenesis of invading enteropathogens like Salmonella (Sansonetti and Phalipon, 1999; Valdez et al., 2001). Strains of S. thermophilus and L. delbrueckii subsp. bulgaricus reduced the induced apoptosis, L. delbrueckii subsp. bulgaricus being the most effective (Valdez et al., 2001). Furthermore, it was necessary that cells were viable since the dead cells had no effect. These observations might be mediated by an increase in the release of oxidant radicals, of which an increased production was noticed with L. delbrueckii subsp. bulgaricus.

Table 17.6 In vitro and animal model studies with probiotic Lactobacilli towards Salmonella. The papers are listed chronologically Probiotic

Model

Results

Reference

L. rhamnosus HN001

Infection of conventional mice

Gill et al. (2001)

VSL#3

Epithelial resistance and permeability of T84 cells; protection against invasion

Vitacanis, L. acidophilus

Infection of gnotobiotic mice; in vitro activity by agar overlay

L. delbrueckii subsp. bulgaricus, S. thermophilus

Apoptosis of infected macrophages

L. acidophilus LB

Invasion of Caco-2 cells

Increased survival rate (90% vs. 7%), decreased counts of translocated Salmonella, increased intestinal and serum antibodies, and higher leucocyte phagocytosis upon probiotic intake. Increased resistance and permeability due to the secretion of a soluble protein. Decreased adherence of Salmonella because of a proteinaceous soluble factor. In vitro activity against Salmonella. In vivo, Salmonella was established in similar levels as compared with the controls. Survival was longer. Inhibition of apoptosis induced by infection with Salmonella possibly by oxidant radical release. SCS*-treated Caco-2 cells were less invaded by Salmonella. SCS* reduced the numbers of intracellular Salmonella after invasion took place.

Madsen et al. (2001)

Maia et al. (2001)

Valdez et al. (2001) Coconnier et al. (2000)

L. acidophilus UFV-H2b20 together with Saccharomyces boulardii and E. coli L. johnsonii La1

L. rhamnosus GG

L. rhamnosus GG

*

SCS: spent culture supernatant

Mice mono-associated with the probiotic prior to infection Time kill studies of Salmonella with SCS; adhesion and invasion of Caco-2 cells; infection of conventional and germ-free mice Invasion of Caco-2 cells; infection of conventional and germ-free mice Adhesion to Caco-2 cells

No effect on colonisation of Salmonella by probiotic treatment.

Filho-Lima et al. (2000)

Antibacterial activity was observed against several pathogens in vitro. SCS* also inhibited adhesion and invasion by Salmonella to Caco-2 cells. Lactobacillus was shown to be active against Salmonella in vivo in germ-free and conventional mice. Inhibition of adhesion to Caco-2 cells by SCS* without modifying viability of Salmonella. Reduced faecal counts of Salmonella in conventional infected animals. Prolongation of survival in infected germ-free mice. No inhibition of adhesion by adhered Lactobacillus cells. Inhibition of adhesion with SCS most probably due to lactic acid.

Bernet-Camard et al. (1997)

Hudault et al. (1997)

Lehto and Salminen (1997)

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Madsen et al. (2001) studied changes in intestinal cytokine secretion, epithelial barrier function, and Salmonella invasion upon treatment with the probiotic preparation VSL#3 and IL-10 gene-deficient mice and T84 gastrointestinal epithelial cells. VSL#3 is a combination of three Bifidobacterium strains, four Lactobacillus strains, and one S. thermophilus strain. It was found that treating IL-10 gene-deficient mice with VSL#3 led to a normalised colonic physiological function and barrier integrity in conjunction with a reduced mucosal secretion of proinflammatory cytokines, interferon , and tumour necrosis factor . In vitro testing with T84 cells revealed that the epithelial barrier function and resistance to Salmonella invasion was enhanced by the probiotic treatment. This effect could be attributed to a proteinaceous soluble factor present in the culture medium. Almost all reported effects are very strain-dependent. Indeed, it cannot be guaranteed that a certain Lactobacillus strain that colonises the gut will develop an anti-Salmonella activity. Filho-Lima et al. (2000) observed an antibacterial activity in gnotobiotic mice, only when an E. coli strain was used and not with the tested L. acidophilus strain. Similarly, in the study of Maia et al. (2001) protection against S. enterica in gnotobiotic mice failed with L. acidophilus, although a slight increase in survival time was noticed. On the other hand, an E. faecium strain was more successful. With the latter strain, 82% of the mice survived while all mice died in the control group or in the group treated with L. acidophilus. Clinical trials focusing on Salmonella-associated diseases do not exist. However, a lot of research has been done on the effect of Lactobacillus preparations on Salmonella in poultry. Salmonella lives in the chicken's intestine in a non-pathological, commensal way, but is detrimental for human consumption. In 1973, it was shown that by giving the intestinal flora of a pathogen-free adult chicken to a newly hatched chick, both the chance to be colonised by Salmonella and the levels of Salmonella present in the faeces, if colonised, were reduced (Juven et al., 1991). Other pathogenic bacteria Pathogens other than H. pylori, E. coli, and Salmonella may be inhibited by probiotic Lactobacillus species. As it is out of the scope of this chapter, only one example will be given. Lactobacillus casei Shirota has the ability to reduce the numbers of L. monocytogenes in the stomach, caecum, faeces, spleen, and liver of pathogen-free mice, probably by an increased cellular immune response, as measured with the delayed-type hypersensitivity (DTH) response against heatkilled cells (de Waard et al., 2002). This same model has been used with the infectious parasite Trichinella spiralis. Again, DTH response was significantly enhanced, however, without a reduction of larval counts, increase in body weight, or change in histology (de Waard et al., 2001). These results led to the hypothesis that supplementation of L. casei Shirota can enhance the Th1 cellmediated immune response.

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Antibacterial activity of bifidobacteria In vitro experiments The in vitro inhibitory activity of bifidobacteria against pathogenic bacteria has been demonstrated in several reports and various mechanisms of action have been proposed (Table 17.7). Some reports suggested the production of organic acids, in particular acetic acid and lactic acid as the sole inhibitory factor. For example, growth inhibition experiments with E. coli were performed in the presence of both neutralised and non-neutralised CFCS of bifidobacteria cultures. The inhibitory activity of five Bifidobacterium strains, belonging to different species, was pH-dependent and could be reproduced by a mixture of organic acids (Ibrahim and Bezkorovainy, 1993). Further, three B. infantis strains that showed strong inhibitory activity against S. Typhimurium and E. coli ETEC, using paper disc assays, significantly decreased the pathogenic cell numbers when added to weaning foods. The production of acetic acid and lactic acid and the pH lowering were suggested as the underlying mechanisms of this inhibition (Rokiah et al., 2000). Recently, similar findings were reported with CFCS of Bifidobacterium cultures that inhibited the growth of eleven different pathogens in well-diffusion assays and growth assays in the presence of their CFCS. When the CFCS was neutralised there was no inhibitory activity (Bruno and Shah, 2002). Moreover, Fooks and Gibson (2002) reported the in vitro inhibition of Salmonella enteritidis, E. coli, and C. jejuni by B. animalis Bb12 in combination with prebiotic substrates. Plate assays with CFCS as well as coculture experiments were performed and it was demonstrated that B. animalis Bb12 strongly inhibited the three pathogenic strains and that the inhibitory effect was due to the production of acetic acid and lactic acid and not necessarily a result of the low pH. The production of compounds, different from organic acids, inhibiting the growth of pathogenic bacteria or exerting a killing effect towards pathogens has been reported as well. The CFCS of cultures of six Bifidobacterium strains showed strong inhibitory activity against Bacillus cereus, Salmonella typhosa, S. dysenteriae, E. coli, Micrococcus flavus, Staphylococcus aureus, and Pseudomonas fluorescens (Anand et al., 1984a). However, the intensity of the inhibition was strain-dependent. Bifidobacterium bifidum NCDO 1452 showed the highest activity. An inhibitory factor, termed bifidin, was partially purified by a methanol-acetone (M-A) extraction, and was heat-stable and active towards E. coli and M. flavus at pH values between 4.8 and 5.5 (Anand et al., 1984b). Likewise, it was demonstrated by plate assays, serum tube assays, and co-culture experiments that a B. infantis strain exerted antimicrobial activity against E. coli, C. perfringens, and Bact. fragilis, which could not be attributed either to competition for growth substrates or to an acidic environment (Gibson and Wang, 1993). The inhibitory factor(s) produced by B. infantis could be concentrated by a M-A extraction and further purification of the M-A extracts by gel filtration yielded a number of fractions that exhibited antibacterial activity. Further studies with eight Bifidobacterium strains, belonging to different species, showed that the M-A extracts of all the strains exerted antimicrobial

Table 17.7 Studies on the in vitro antibacterial activity of bifidobacteria. The papers are listed chronologically Microorganisms tested

Model

Several Bifidobacterium strains Growth experiments in the isolated from infants' stools presence of CFCS and agar spot assays with Cl. difficile, E. coli and St. aureus B. bifidum Bb12 Plate assays and co-culture experiments with S. enteritis, E. coli and Campylobacter jejuni B. lactis Well-diffusion assays, killing assays and adhesion to and/or invasion of Caco-2, HT-29 and HT29-MTX cell lines by E.coli Three B. infantis strains Paper disc assays with S. typhimurium and E. coli ETEC Five Bifidobacterium strains Well-diffusion assays and growth of different pathogenic bacteria in the presence of CFCS

Results

Reference

Bifidobacterium strain exerted antimicrobial activity against Cl. difficile and E. coli

Lee et al. (2003)

Strong inhibition of all the pathogenic strains

Fooks and Gibson (2002)

B. lactis strongly inhibited the growth of E. coli and had a significant killing effect against the pathogen. The anti-invasive effect was less distinct

Gopal et al. (2001)

Large inhibition zones produced by the three Bifidobacterium strains

Rokiah et al. (2000)

Inhibition of eleven pathogenic bacteria, including E. coli

Bruno and Shah (2000)

Thirteen Bifidobacterium strains from infant stools

Killing assays with several pathogens, adhesion to and/or invasion of Caco-2 cells by S. enterica ser. Typhimurium Ten Bifidobacterium strains Growth of H. pylori in the isolated from human faeces presence of CFCS and coculture experiments Two B. bifidum strains isolated Adhesion to and/or of from infants' stools invasion of Caco-2 cells by S. arizonae Twenty-seven Bifidobacterium Plates assays with several strains Gram-positive and Gramnegative indicators Fifteen Bifidobacterium strains Co-culture experiment with six Salmonella strains Three B. adolescentis and one Well diffusion assays with E. B. bifidum coli, S. typhosa, P. aeruginosa, St. aureus, B. cereus and Sh. sonnei Bifidobacterium spp. Co-culture experiment with Cl. perfringens Five Bifidobacterium bifidum Agar spot assays against strains several Gram-positive and Gram-negative indicators

Two strains showed strong killing effect and inhibited the adhesion and invasion to Caco-2 cells.

LieÂvin et al. (2000)

Five Bifidobacterium strains inhibited H. pylori growth and HP urease activity

Bae et al. (2000)

Both Bifidobacterium strains inhibited the invasion of S. arizonae to Caco-2 cells

Bibiloni et al. (1999)

Twelve strains produced large inhibition zones

O'Riordan and Fitzgerald (1998)

All bifidobacterium showed antagonistic activity towards the pathogen. Three Bifidobacterium exerted strong antimicrobial activity The three B. adolescentis strains exerted antimicrobial activity towards the pathogenic indicators

Bielecka et al. (1998)

Decreased growth rate of Cl. perfringens

Kullen et al. (1998)

B. bifidum NCFB 1454 inhibited several Grampositive indicators including Listeria spp.

Yildirim and Johnson (1998)

Khedkar et al. (1998)

Table 17.7 Continued Microorganisms tested

Model

Results

Reference

Eight Bifidobacterium strains of human origin B. bifidum

Adhesion of E. coli ETEC to human epithelial cells Plate assays with several pathogens Growth of E. coli in the presence of CFCS Plates assays, growth in the presence of CFCS, cocultures with E. coli, Cl. perfringens and B. fragilis Adhesion to and/or invasion of Caco-2 cells by different pathogens

All bifidobacteria inhibited the adhesion of E. coli to human epithelial cells Inhibition of different pathogens, including E. coli and Salmonella spp. Strong inhibition by all Bifidobacterium strains

Fujiwara et al. (1997)

Five Bifidobacterium strains B. infantis

Eight Bifidobacterium strains of human origin Thirteen Bifidobacterium strains B. longum B. bifidum NCDO 1452

Well-diffusion assays against several Gram-positive and Gram-negative indicators Plates assays with several pathogenic bacteria Well-diffusion assays

Inhibiton of all the pathogenic bacteria

All strains inhibited, in different exent, the adhesion and invasion of E. coli (ETEC, DAEC, EPEC), S. enterica ser. Typhimurium and Y. pseudotuberculosis to Caco-2 cells One Bifidobacterium strain was active towards various Gram-positive indicators including Clostridia spp. B. longum inhibited various pathogens including E. coli and Salmonella spp. Inhibition of E. coli and Micrococcus flavus

Kebary (1995) Ibrahim and Bezkovainy (1993) Gibson and Wang (1993)

Bernet et al. (1993)

Meghrous et al. (1990) Kang et al. (1989) Anand et al. (1984a,b)

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activity towards a wide range of pathogenic bacteria in plate assays. Further, a B. bifidum strain produced an antimicrobial substance active towards a wide range of Gram-negative indicators, including Salmonella spp. and E. coli (Kebary, 1995). The antimicrobial compound was concentrated and partially purified by M-A extraction and chromatography. It was proteinaceous, heat-stable, and active only at acidic pH. Similar results were obtained by LieÂvin et al. (2000) who isolated thirteen Bifidobacterium strains from infant stools, which were screened for antimicrobial activity towards pathogenic bacteria. Using killing assays, it was shown that the CFCS of the cultures of two strains exerted high antimicrobial activity against S. Typhimurium, E. coli, K. pneumoniae, P. aeruginosa and S. aureus. A lipophilic molecule, which could be extracted with a chloroform/ methanol mixture and with a molecular mass estimated at lower than 3500 Da, was suggested to be the responsible inhibitory factor. O'Riordan and Fitzgerald (1998) tested twenty-seven strains of bifidobacteria for production of antimicrobial compounds and found that the growth of several indicator strains, including Gram-positive and Gram-negative bacteria, was inhibited around colonies of twelve Bifidobacterium strains. The inhibitory effect could not be attributed only to low pH and organic acid production, as the inhibitory zones did not disappear when buffered media were used. Moreover, the inhibition of Pseudomonas species appeared to be unstable suggesting that the inhibitory compound was not produced as part of a constitutive metabolic process. Although the inhibitory factor could not be isolated from CFCS when two active Bifidobacterium strains were applied in a food system (cottage cheese), inhibitory activity towards Pseudomonas strains was found. Similarly, Bifidobacterium strains were found to exert antimicrobial activity towards Salmonella spp. and E. coli (Kang et al., 1989; Bielecka et al., 1998; Khedkar et al., 1998), Clostridium spp. (Meghrous et al., 1990; Kullen et al., 1998; Lee et al., 2003), and H. pylori (Bae et al., 2000), but also towards the water-borne protozoan parasite C. parvum (Foster et al., 2003). All reports mentioned above failed in the purification of the inhibitory substance to homogeneity. Only a bacteriocin, referred to as bifidocin B, produced by B. bifidum NCFB 1454, has been described up to now (Yildirim and Johnson, 1998). It was active against Listeria spp., Enterococcus spp., B. cereus, and Lactobacillus, Leuconostoc, Micrococcus, and Pediococcus species, using the agar spot assay. However, it was not active against any of the Gram-negative indicators tested. Bifidocin B preparations were heat-stable, stable at pH values between 2 and 12, and sensitive towards proteolytic enzymes. Bifidocin B belongs to the group of the class IIa bacteriocins of LAB (Yildirim et al., 1999). The in vitro antibacterial activity of bifidobacteria towards pathogens can also be attributed to the production of compounds that inhibit the adhesion to and/or the invasion of intestinal epithelial cells by pathogenic bacteria. This was clearly demonstrated for a B. longum strain. The CFCS of cultures of several Bifidobacterium strains of human origin (B. longum, B. breve, B. bifidum, and B. infantis) inhibited the adhesion of pathogenic E. coli strains to human epithelial

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cells. It was shown that the inhibitory factor was a proteinaceous molecule (Fujiwara et al., 1997). Further studies showed that B. longum BL2928 was producing a protein with a molecular mass of 104 kDa, designated BIF, which prevented the binding of E. coli (ETEC strain) to the glycolipid binding receptor gangliotetraosylceramide (Fujiwara et al., 1999). Moreover, it was found that this compound was also inhibiting the binding of an E. coli ETEC strain in a human intestinal epithelial HCT-8 cell line (Fujiwara et al., 2001b). Interestingly, similar results were obtained in other studies. For instance, it was found that bifidobacteria exerted inhibition of the adhesion and/or the invasion of different pathogenic bacteria, including E. coli DAEC strain, S. Typhimurium, and Yersinia pseudotuberculosis (Bernet et al., 1993, LieÂvin et al., 2000), Salmonella arizonae (Bibiloni et al., 1999), and E. coli ETEC strain (Gopal et al., 2001). However, the inhibitory substances were not characterised. Finally, one report demonstrated that CFCS of a B. longum culture stimulated mouse monocyte-macrophage cells (J774) to phagocytose S. typhimurium cells (Hatcher and Labrecht, 1993). Animal models The antibacterial activity of Bifidobacterium strains has been shown in animal models as well. Several studies demonstrated that administration of bifidobacteria to animals provided a preventive or therapeutic effect towards infections by pathogenic bacteria (Table 17.8). In many reports, administration of Bifidobacterium preparations to animals led to the overgrowth of bifidobacteria in the gut and suppression of pathogenic bacteria. Asahara et al. (2001a) reported a decrease of faecal S. Typhimurium LT-2 excretion levels in mice, when the B. breve Yakult strain or Bifidobacterium pseudocatenulatum DSM 20439 were administered orally. When bifidobacteria were administered together with TOS the effect on the levels of the pathogen in the faeces of the mice was more pronounced. The production of organic acids and the lowering of the pH by the administered bifidobacteria were the main inhibitory factors. Further, animals dosed with one or several selected Bifidobacterium strains, isolated from human stools, had lower levels of faecal Salmonella than animals of the control group (Henriksson and Conway, 2001). Also, LieÂvin et al. (2000) reported on the inhibition of Salmonella in mice. Administration of two Bifidobacterium strains, isolated from infant stools, to axenic mice infected with S. Typhimurium, resulted in colonisation of the GIT by the two Bifidobacterium strains and protection of the mice against lethal infection. Similar results were obtained for the inhibition of E. coli strains. Administration of fermented milk containing B. breve, B. bifidum, and L. acidophilus to mice inhibited the outgrowth of indigenous E. coli (Asahara et al., 2001c). The results showed that the underlying mechanism was the production of acetic acid by the bifidobacteria. Moreover, administration of a B. longum strain to germ-free rats provided protection against E. coli infection (Faure et al., 1984). Escherichia coli numbers in the lungs of two-week-old

Table 17.8 Animal model studies on the inhibition of pathogenic bacteria by bifidobacteria. The papers are listed chronologically Microorganisms tested

Animal Model

Results

Reference

B. breve, B. catenulatum, and B. longum B. breve Yakult, B. pseudocatenulatum DSM 20439, B. bifidum ATCC 15696, B. catenulatum ATCC 27539T Three Bifidobacterium strains of human origin B. breve, B. bifidum, and Lb. acidophilus

Germ-free mice associated with Bacteroides vulgatus Specific-pathogen-free mice with an antibiotic induced infection of S. enterica ser. Typhimurium

Protection against B. vulgatus infection

Setoyama et al. (2003)

Significant decrease of the number of Salmonela enterica ser. Typhimurium in the faeces of mice administered with B. breve and B. pseudocatenulatum Lower faecal S. typhimurium and protection against infection Inhibition of indigenous E .coli outgrowth

Ashahara et al. (2001a)

Asahara et al. (2001c)

Reduced severity of E. coli infection Lower severity of weanling diarrhoea

Shu and Gill (2001) Shu et al. (2001)

Colonisation of the gastrointestinal tract and protection against lethal infection Lower morbidity, reduced bacterial translocation

LieÂvin et al. (2000) Shu et al. (2000)

Increased survival rates in the conventional mice

Silva et al. (1999)

Decreased faecal clostridial spores and Bacteroides vulgatus numbers in Prolonged survival of the mice, decreased systemic dissemination and inhibition of Candida albicans in the alimentary tract Protection against E. coli infection

Romond et al. (1997a)

B. lactis B. lactis Two Bifidobacterium strains isolated from human stools B. lactis B. bifidum B. breve

Specific-pathogen-free mice infected with S. typhimurium Specific-pathogen-free mice with an antibiotic-induced infection of indigenous E. coli Mice infected with E. coli Piglet with weanling diarrhoea associated with rotavirus and E. coli infection Axenic mice infected with S. enterica ser. Typhimurium Mice infected with S. typhimurium Gnotobiotic and conventional mice infected with S. enteritis subsp. typhimurium Specific-pathogen-free mice

B. animalis

Immunodeficient mice with candidiasis

B. longum

Germ-free rats infected with E. coli

Henriksson and Conway (2001)

Wagner et al. (1997) Faure et al. (1984)

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chickens were decreased when CFCS of a Bifidobacterium thermophilum culture was administered orally (Kobayashi et al., 2002). Inhibition through the production of organic acids by Bifidobacterium strains was also demonstrated as the responsible mechanism for the preventive effect of gut inflammation in mice associated with Bact. vulgatus strains, isolated from patients with ulcerative colitis (Setoyama et al., 2003). It has been suggested that prevention resulted from a direct inhibition of the Bact. vulgatus strains by the bifidobacteria, through the production of particularly succinic acid in the caecum. Finally, protection against Clostridium infections through overgrowth of bifidobacteria has been reported in gnotobiotic quails (Butel et al., 1998) and gnotobiotic mice (Morishita et al., 2002). The antimicrobial activity of bifidobacteria in animal models is not attributed only to the overgrowth of bifidobacteria and the production of organic acids. Romond et al. (1997b) reported that oral administration to mice of cell-free whey, derived from milk fermented with a B. breve strain of human origin, stimulated intestinal bifidobacteria, repressed clostridial spores and decreased Bact. fragilis in the faeces of the mice, and decreased the pH in the intestines of the mice. It was shown that the inhibitory effect was independent of the overgrowth of bifidobacteria and pH decrease. When mice were dosed with 30± 100 kDa and 100±300 kDa cell-free whey fractions, obtained by ultrafiltration of the cell-free whey and analysed by SDS-PAGE, similar effects were observed, indicating that these cell-free whey fractions were responsible for the regulatory effect. They were primarily composed of glycoproteins and they showed a high lactosidase activity. Further studies were performed to investigate the nature of the active substances (Mullie et al., 2002). The cell-free whey produced from the B. breve strain mentioned above was compared with the inactive cell-free whey of another B. breve strain. No major differences were found regarding protein and sugar composition. However, the inactive cell-free whey exhibited poor lactosidase activity, whereas the active cell-free whey showed high activity of this enzyme. Moreover, heating or oxidation of the cell-free whey resulted in loss of the inhibitory effect in mice. A treatment with cysteine hydrochloride (reductive agent) resulted in the recovery of the activity. These findings suggested that the lactosidase enzyme was the real active substance. Immunomodulatory mechanisms have been proposed as well. Oral administration of B. lactis HN019 to mice infected with S. Typhimurium resulted in lower morbidity rates and reduced bacterial translocation compared with the controls (Shu et al., 2000). It was suggested that enhancement of the host immunity played an important role in this effect, as higher phagocytic capacity of both blood leukocytes and peritoneal macrophage preparations and increased intestinal IgA antibody responses were observed. Shu and Gill (2001) reported similar results with the same Bifidobacterium strain in mice infected with E. coli. Moreover, treatment of weanling diarrhoea, associated with rotavirus and E. coli infections, using B. lactis resulted in a protective effect in a piglet model. Compared with the controls, piglets that received B. lactis showed lower severity of weanling diarrhoea (Shu et al., 2001). Also, oral administration to

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conventional mice of milk fermented with a different Bifidobacterium strain increased the survival rate after an infection with S. Typhimurium (Silva et al., 1999). Host immunomodulation was suggested as the protective mechanism, as the numbers of pathogenic bacteria were not reduced. Similarly, concentrated culture of a B. longum strain inhibited the bacterial translocation, when administered orally to specific-pathogen-free and germ-free mice monoassociated with E.coli (Suzuki et al., 1997). Finally, Wagner et al. (1997) reported that a B. animalis strain displayed a therapeutic effect on candidiasis in immunodeficient mice. Oral administration of B. animalis resulted in prolonged survival of the mice, decreased systemic dissemination, inhibition of Candida albicans in the alimentary tract, stimulation of antibodies and cell-mediated immunity, and decreased severity of orogastric candidiasis. The in vivo suppression of C. albicans suggested that the production of inhibitory compounds by the Bifidobacterium strain participated in the biotherapeutic effect. However, this effect was not always correlated with the inhibition of C. albicans in the alimentary tract. Thus, a stimulation of host defence mechanisms was suggested as the most important factor in the therapy of candidiasis in mice. Clinical trials Although the inhibitory activity of bifidobacteria against pathogenic bacteria has been well demonstrated in vitro and in animal models, only a few studies investigated this probiotic effect in humans (Table 17.9). In most of the human studies strains of the species B. longum were used. They are found in high numbers in the gut of healthy humans. Ballongue et al. (1993) reported the decrease of the Clostridium, Bacteroides and coliform flora in adults who consumed milk fermented with B. longum BB536. It was also shown that this strain was able to colonise and grow in the large intestine. When a B. animalis strain was used, there was no effect on the pathogenic bacteria. Oral administration of B. longum SBT2928 resulted in a high persistence of this strain in the GIT of healthy volunteers, and a significant decrease in the number of both enterobacteriaceae and Clostridium spp. in the faeces (Fujiwara et al., 2001c). The anti-infectious effect was specific and independent of the production of organic acids. It was previously shown that this strain produces a proteinaceous factor that inhibits the binding of pathogenic E. coli to human intestinal epithelial cells (Fujiwara et al., 1997). Similarly, a decrease of coliform bacteria in the faeces of healthy volunteers was found after ingestion of yoghurt containing bifidobacteria (Chen et al., 1999). A significant drop in the numbers of clostridia was observed in the faeces of healthy volunteers after administration of B. longum in lyophilised form (Benno and Mitsuoka, 1992). In some studies it was observed that administration of B. longum strains, together with antibiotics, provided a protective effect towards the increase of some infectious bacteria due to the application of the antibiotic. Colombel et al. (1987) reported that oral absorption of yoghurt, containing B. longum, reduced erythromycin-induced gastrointestinal effects, and decreased the level of faecal

Table 17.9 Clinical trials on the inhibition of pathogenic bacteria by bifidobacteria Probiotic preparation

Trial

Results

Reference

Lyophilised powder B. longum SBT2928 Fermented milk containing B. longum BB536, Lb. acidophilus NCFB 1748, and oligofructose Yogurt containing bifidobacteria Cell-free whey from B. breve

Oral administration to healthy volunteers Oral administration to healthy volunteers receiving an antibiotic

Significant decrease of faecal Clostridium spp. and enterobacteriaceae Reduced number of Cl. difficile

Fujiwara et al. (2001a) Orrhage et al. (2000)

Oral administration to healthy volunteers Oral administration to healthy volunteers Oral administration to healthy volunteers

Decrease of coliform bacteria in the faeces

Chen et al. (1999)

Decreases faecal clostridial spores, Cl. perfringens and Bacteroides fragilis Decreased Clostridium spp., Bacteroides spp. and coliform flora in the faeces after consumption of milk fermented with B. longum BB536 Decreased Clostridium spp. in the faeces

Romond et al. (1997a)

Decreased level of clostridial spores in the faeces

Colombel et al. (1987)

Milk fermented with B. longum BB536 or B. animalis B. longum in lyophilised form Yogurt containing B. longum

Oral administration to healthy volunteers Oral administration to patients receiving an antibiotic therapy

Ballongue et al. (1993)

Benno and Mitsuoka (1992)

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clostridial spores. Further, it was observed that supplementation with B. longum BB536, L. acidophilus NCFB 1748, and oligofructose, during administration of cefrodoxime to healthy volunteers, resulted in a decrease of the numbers of C. difficile in the faeces, compared with the subjects that were administered with the antibiotic only (Orrhage et al., 2000). Interestingly, positive antibacterial effects were obtained when cell-free whey from B. breve was administered to healthy volunteers, indicating that the presence of viable bifidobacteria is not necessarily required in probiotic preparations. Romond et al. (1997a) reported that administration to ten healthy volunteers of cell-free whey from B. breve, led to an increase in bifidobacteria and a significant decrease in clostridial spores, and in cell numbers of C. perfringens and of Bact. fragilis in the faeces. In addition, the pH was lowered. In further studies, the effect of concentrated cell-free whey of milk fermented with B. breve was compared with the effect of commercial milk fermented with B. breve and S. thermophilus (Romond et al., 1998). This again resulted in an increase of bifidobacteria in the faeces, together with a reduction of C. perfringens and Bact. fragilis. The decrease of the two pathogenic bacteria occurred also when fermented milk was administered, but there was no increase of the bifidobacteria. These findings indicated that the inhibitory effect was probably due to active compounds present in the cell-free whey and that the presence of S. thermophilus could change the metabolism of bifidobacteria in milk.

17.4 Improving the effectiveness of probiotics and prebiotics in optimising gut health Realising the complexity of the present market situation and the fact that the success of one strain of a certain LAB species in a certain application does not imply that all related strains of this species will be capable of producing a comparable health response, it is clear that the identification of microorganisms at only the species level would no longer provide the transparency required by the consumer, industry, legislative bodies, and responsible scientists. Strains selected for their particular functional properties have to be clearly characterised below the species level. Molecular fingerprinting methods provide reliable and highly discriminatory solutions to these challenges (Holzapfel et al., 2001). Further, knowing that the human GIT is a complex and hostile environment, it is more likely that gut health effects with probiotics will require the introduction of a consortium of strains, e.g., several lactobacilli and/or bifidobacteria, instead of a single probiotic bacterial strain, that performs effectively in the GIT through in vitro selection criteria (Dunne et al., 2001). In this case it should be known if interactions (antagonistic or synergistic) will take place. Concerning prebiotics, most of the current generation products have not been rationally developed with specific targets or functions in mind (Rastall and Maitin, 2002). For instance, existing prebiotics and natural dietary components already

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tend to increase selectively the numbers of certain species of genera other than those belonging to the lactic acid bacteria, e.g., Eubacterium, Roseburia, or Ruminococcus species (Duncan et al., 2003). Hence, the generation of prebiotics that acts at the species rather than the genus level offers further refinement to the approach (Rabiu et al., 2001). Further, prebiotics with novel structures, e.g., NDOs with different degrees of polymerisation, branching, or solubilisation, as well as algal polysaccharides, and bacterial exopolysaccharides, may act at different sites in the gut and may yield different end-products or a different ratio of short-chain fatty acids through fermentation. For instance, it has been shown that long chains of oligosaccharides are metabolised slower than short chains, so that the rapid saccharolytic fermentation in the proximal colon may be extended towards distal regions of the colon, where many chronic gut disorders (ulcerative colitis and colorectal cancer) are primarily located (Roberfroid, 1997, 1998; Van der Meulen et al., 2004). Likewise, the levels of propionate and butyrate may differ considerably. Such prebiotics may be found through extensive screening of a wide range of novel substrates or constructed through synthesis with (probiotic) enzymes from mono- and/or oligosaccharides (Rastall and Maitin, 2002). Also, prebiotics with enhanced persistence are a desirable novel type of prebiotics. In this respect, the potential of biomass-derived oligosaccharides, in particular plant cell wall polysaccharides, as sources of novel prebiotics, has started to receive some attention (Van Laere et al., 2000). Further, several targets can be envisaged such as infants, the elderly, certain individuals, and probiotics (Rastall and Maitin, 2002). Such specific oligosaccharides may be used in combination with proved probiotics to enhance their survival, i.e., as synbiotics. Finally, prebiotics could be targeted towards anti-adhesive activity. Oligosaccharide receptor mimics as (part of) the prebiotic may selectively prevent adhesion of certain pathogenic species. The use of oligosaccharides to prevent pathogen binding has received interest in a therapeutic context during the last decade (Zopf and Roth, 1996). However, very little is known about the structure-function relationships of prebiotic oligosaccharides, although there has been some interest in this area recently (Kaplan and Hutkins, 2000, 2003; Van Laere et al., 2000; Gopal et al., 2001; Perrin et al., 2001; Rastall and Maitin, 2002). Also, the further development of synbiotics may improve the effectiveness of both probiotic strains and appropriate health-stimulating substrates, in particular reaching an increased number of ingested bacteria reaching the colon in a viable form. However, less is known about the interaction between different combinations of pro- and prebiotics, although this is necessary to have a rationale for selecting different pro- and prebiotics and developing efficacious synbiotics. Moreover, it is essential that these components be developed as the active ingredients of the food products, that are ultimately intended for human consumption, in particular when the probiotic, prebiotic, or synbiotic is considered a food ingredient or food supplement.

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17.5

463

Future trends

As the number of antibiotic-resistant bacteria is rising quickly, probiotics are now being considered as alternatives to antibiotics and may become an important issue in near future medicine. However, the validity of such a concept has to be proved. Moreover, tools are necessary to monitor and to quantify the in vivo performance and efficacy of probiotics, prebiotics, and synbiotics. Since it appears that cell components, enzymes, or metabolic/fermentation products derived from probiotic LAB may contribute to or even mediate specific beneficial health effects, non-viable probiotics may become of importance, in particular as biotherapeutic. For instance, once certain components have been found to be responsible for a particular pathogen inhibition or inactivation these might be used as therapeutic agents. Further, several studies have shown that non-viable probiotics can have beneficial effects such as immune modulation and carcinogen binding in the gut of the host (Ouwehand and Salminen, 1998; Salminen et al., 1999; Mercenier et al., 2003). In contrast, upon oral administration, viable cells seem to be more effective than killed ones. However, emerging data suggest that while viable organisms may be most effective/essential for specific effects, abiotics, non-viable probiotic organisms or cellular components (cell wall components, lysates) or cellular metabolites (bacteriocins, nutraceuticals) thereof, may be efficacious in specific situations (Salminen et al., 1999). It is likely that the abiotic concept ± if accepted ± will further broaden the health and therapeutic potential of probiotics in the future. Moreover, the use of non-viable preparations would offer both economic and social advantages, for instance, facilitating their use in developing countries. Hence, in the future it may become possible to address certain gastrointestinal complaints prophylactically through the selective use of probiotics and/or the selective targeting of gut bacteria with prebiotics, or biotherapeutics. Therefore, extensive research has to be carried out to elucidate the mechanism(s) of action of particular probiotics against particular pathogens. It may well be that different regions of the GIT require different probiotic bacteria (disease-specific strains), for instance with respect to rotavirus diarrhoea and gastritis caused by H. pylori. In addition, the same probiotic may inhibit different pathogens by different mechanisms. Alternatively, genetic engineering may enhance the activity of probiotics, for instance by bringing together the ability to survive in the GIT with the ability to produce metabolites that are responsible for the probiotic effect, hence enhancing the traits considered important for their functional and in vivo roles. To conclude, it should always be remembered that in vitro and animal studies are frequently not transferable to humans. The in vitro or animal effect is not necessarily observed in vivo in humans. Therefore, any unclear and/or unresolved issue can be answered only by well-designed and well-controlled clinical trials (randomised, placebo-controlled, double-blind, established endpoints, multi-centre).

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17.6

Sources of further information and advice

For further information on probiotic research the reader is referred to the website of the PROEUHEALTH cluster http://proeuhealth.vtt.fi.

17.7

Acknowledgements

The authors' research on pro- and prebiotics was financially supported by the European Commission (grant Quality of Life QLK1±CT-2001-01179), the Flemish Institute for the Promotion of Innovation by Science and Technology in Flanders (IWT) (grant GBOU-10054), the Fund for Scientific Research ± Flanders (FWO-Vlaanderen), the Research Council of the Vrije Universiteit Brussel (VUB), and several national and international food companies.

17.8

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and MENTIS, A. (2001), `Inhibition of H. pylori colonisation and associated gastritis in the HpSS1 C57BL/6 mouse model via administration of the probiotic Lactobacillus casei ACA-DC6002', Gut, 49, 816. SHAHANI, K., VAKIL J.R. and KILARA, A. (1977), `Natural antibiotic activity of Lactobacil1us acidophilus and bulgaricus. II. Isolation of acidophilin from L. acidophilus', Cult. Dairy Prod. J., 12, 8. SHEU, B.S., WU, J.J., LO, C.Y., WU, H.W., CHEN, J.H., LIN, Y.S. and LIN, M.D. (2002), `Impact of supplement with Lactobacillus- and Bifidobacterium-containing yogurt on triple therapy for Helicobacter pylori eradication', Aliment. Pharmacol. Ther., 16, 1669± 1675. SHIMIZU, T., HARUNA, H., HISADA, K. and YAMASHIRO, Y. (2002), `Effects of Lactobacillus gasseri OLL 2716 (LG21) on Helicobacter pylori infection in children', J. Antimicrob. Chemoth., 50, 617±618. SHU, Q. and GILL, H.S. (2001), `A dietary probiotic (Bifidobacterium lactis HN019) reduces the severity of Escherichia coli O157:H7 infection in mice', Med. Microbiol. Immun., 189, 147±152. SHU, Q. and GILL, H.S. (2002), `Immune protection mediated by the probiotic Lactobacillus rhamnosus HN001 (DR20 (TM)) against Escherichia coli O157:H7 infection in mice', FEMS Immunol. Med. Microbiol., 34, 59±64. SHU, Q., LIN, H., RUTHERFURD, K.J., FENWICK, S.G., PRASAD, J., GOPAL, P.K. and GILL, H.S. (2000), `Dietary Bifidobacterium lactis (HN019) enhances resistance to oral Salmonella typhimurium infection in mice', Microbiol. Immunol., 44, 213±222. SHU, Q., QU, F. and GILL, H.S. (2001), `Probiotic treatment using Bifidobacterium lactis HN019 reduces weanling diarrhea associated with rotavirus and Escherichia coli infection in a piglet model', J. Pediatr. Gastr. Nutr., 33, 171±177. SILVA, M., JACOBUS, N.V., DENEKE, C. and GORBACH, S.L. (1987), `Antimicrobial substance from a human Lactobacillus strain', Antimicrob. Agents Chemother., 31, 1231± 1233. SILVA, A.M., BAMBIRRA, E.A., OLIVEIRA, A.L., SOUZA, P.P., GOMES, D.A., VIEIRA, E.C. and NICOLI, J.R. (1999), `Protective effect of bifidus milk on the experimental infection with Salmonella enteritidis subsp. typhimurium in conventional and gnotobiotic mice', J. Appl. Microbiol., 86, 331±336. SILVI, S., RUMNEY, C.J., CRESCI, A. and ROWLAND, I.R. (1999), `Resistant starch modifies gut microflora and microbial metabolism in human flora-associated rats inoculated with faeces from Italian and UK donors', J. Appl. Microbiol., 86, 521±530. SIMONETTA, A.C., DEVELASCO, L.G.M. and FRISON, L.N. (1997), `Antibacterial activity of enterococci strains against Vibrio cholerae', Lett. Appl. Microbiol., 24, 139±143. SPERTI, G. (1971), Probiotics, West Point, Avi Publishing. SREEKUMAR, O. and HOSONO, A. (1998), `Antimutagenicity and the influence of physical factors in binding Lactobacillus gasseri and Bifidobacterium longum cells to amino acid pyrolysates', J. Dairy Sci., 81, 1508±1516. SULLIVAN, A., BENNET, R., VIITANEN, M., PALMGREN, A.-C. and NORD, C. E. (2002), `Influence of Lactobacillus F19 on intestinal microflora in children and elderly persons and impact on Helicobacter pylori infections', Microbial Ecol. in Health and Dis., S3, 17±21. SUZUKI, T., ITO, K., KANEKO, T. and SUZUKI, H. (1997), `Inhibition of bacterial translocation from the gastrointestinal tract of mice by oral administration of a culture condensate of Bifidobacterium longum', J. Vet. Med. Sci., 59, 665±669.

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18 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

Reactive oxygen species (ROS), produced either by normal physiological processes or by exogenous factors, and the ensuing cumulative damage catalyzed by oxidative stress resulting from increased levels of ROS, have been identified as a major initiator of a pathological process that translates into cellular aging, degenerative diseases, cell death, shortened life expectancy, and death. The role of the mitochondrion, the major source of oxygen consumption for ATP production and reactive oxygen species generation, is discussed in light of its involvement in the onset and progression of degenerative diseases. The biochemical characterization of potential events and steps involved in the onset and or progression of degenerative diseases is discussed as well as the role of endogenous and exogenous stimulators of apoptosis. The connection between oxidative phosphorylation (OXPHOS), apoptosis, and angiogenesis in health and disease is presented. The range of and mechanisms by which functional foods for disease prevention may prevent angiogenesis are provided. Future trends in anti-angiogenic functional foods are also discussed. 18.1.1 Mechanisms of degenerative diseases Chronic degenerative diseases involve pathological conditions characterized by the loss of homeostatic balance, a long latency period, the occurrence of cellular degenerative phenomena, and include age-related neoplastic and non-neoplastic pathologies such as cancer, cardiovascular diseases, diabetes, macular degeneration, Alzheimer's and Parkinson's diseases, HIV infection, ischemiareperfusion, multiple sclerosis, chronic inflammation, arthritis, liver and

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respiratory diseases, and many others (Izzotti, 2003). Chronic degenerative diseases carry enormous medical and social burdens and have a multi-factorial origin. Each disease is associated with a network of genetic/endogenous and environmental/exogenous risk factors.

18.2

Genetic/endogenous risk factors

18.2.1 The mitochondria and mitochondrial dysfunction The mitochondrion performs multiple cellular functions, among which (i) the exclusive ability to produce ATP, through the process of oxidative phosphorylation (OXPHOS), for the cell's biological activity and (ii) the regulation of apoptosis are the major roles (Read and Calna, 2000). Although a variety of pathways of aerobic metabolism generate ROS, the mitochondrion through OXPHOS, is quantitatively the most important physiological site for oxygen consumption and ROS (O2ÿ , H2O2, and OH ) generation in the cell as toxic by-products of respiration. The major sites of ROS production in the respiratory chain are the respiratory enzyme complex I and the proton motive Q cycle operating in Complex III (Wei et al., 1998). The mitochondrial electron transfer chain constantly receives electrons from NADH or FADH2 (flavoprotein-linked dehydrogenases) and reduces oxygen to water, through a concerted catalytic activity of endogenous antioxidants such as Mn2+-dependent superoxide dismutase (MnSOD), copper/zinc SOD, glutathione peroxidase (GPx), glutathione reductase (GR), catalase (CAT) which sequentially catalyze the conversion of the ROS into hydrogen peroxide and the latter is transformed into water by CAT. A small fraction (2±4%) of oxygen consumed during the electron transport phenomenon constantly produces a small amount of ROS which locally regulate many cellular functions (including cytokine secretion, growth, differentiation, and defense against invaders) and serves as a secondary messenger to activate transcription factors such as NF-B and AP-1 (Dalton et al., 1999; Linnane et al., 1989). The mitochondrial DNA (mtDNA) is not protected by histones or DNA-binding proteins, and like the mitochondrial membrane lipids and proteins, is openly exposed to the damaging effects of ROS and free radicals generated during OXPHOS. To maintain redox homeostasis under physiological conditions, cells use a battery of biochemical antioxidants such as gluthatione, pyridine nucleotides, ascorbate, retinoic acid, tocopherols, CoQ10, SOD, CAT, and nitric oxide to scavenge and repair the damaging effects of radicals. However, over time, chronic generation of ROS and free radicals may deplete and escape the cell defense mechanism and induce a broad spectrum of oxidative damage to biomolecules such as enzymes, lipids, and proteins in the mitochondria and create an accumulation of somatic mutations in and oxidative damage to mtDNA (Fig. 18.1). Melov et al. (1999) used Mn-SOD-knockout mice to demonstrate that increased levels of ROS in the mitochondria leads to a decline in the activity of

Anti-angiogenic functional food, degenerative disease and cancer 487

Fig. 18.1

Mechanisms in degenerative diseases. The role of mitochondria (from Wei and Lee, 2002; with permission).

complex I and complex III in skeletal muscle and heart. The accumulation of defective bioactive molecules has been identified as the early molecular event associated with the onset and progression of degenerative diseases, such as chronic inflammation, artherosclerosis, hypoxia/reperfusion and some forms of neoplasms and aging through decline in mitochondrial respiratory function. Mutations of the mitochondrial genome and depletion of endogenous repair, the base excision repair (BER) system, impair the function of the respiratory and oxidative phosphorylation systems by degrading proteins and enzymes such as thiol proteases. The respiratory enzymes containing mutated and defective mtDNA exhibit impaired electron transport capability and enhance OH formation and accumulation of toxic compounds such as 8-OH-dG and deletions

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in mtDNA cause a loss of genes encoding tRNA and mRNA that are essential for the proper functioning of mitochondria (Wei and Lee, 2002). The vicious cycle operates in various tissue cells at different rates leading to differential accumulation of oxidatively modified and mutant mtDNAs in different organs and tissues during aging. Oxidatively damaged or mutated DNA is transcribed and translated to generate defective protein subunits that generate defective ETC which in turn is less efficient and associated with the production of more ROS. As the body tissues age, there is a progressive decline in the bioenergetics function of the aging cells. These changes can facilitate lipid peroxidation, the dissipation of mitochondrial electrochemical potential, and the opening of a nonspecific mitochondrial inner membrane, also known as mitochondrial permeability transition pores (mtPtP). The opening of the mtPtP provides a passage through which substances from within the mitochondrion such as cytochrome c, second mitochondria-derived activator of caspases/direct IAP binding protein (Smac/ Diablo), apoptosis inducing factor (AIF), apoptosis protease activating factor (apaf 1), pro-caspases, and small molecules of less than 2000 daltons leak outside the mitochondrion (Fig. 18.2). As a result, the mitochondrion swells, loses its membrane potential, and initiates apoptosis (Parone et al., 2002; Esposito et al., 1999; Wallace, 2000; Wallace et al., 1995). Mitochondrial DNA is transmitted only by the mother to the children and by the daughters to the next generation because during egg fertilization, the sperm contributes only its DNA to the zygote. Affected mothers pass along dysfunctional mtDNA while affected fathers do not. Mitochondrial dysfunction

Fig. 18.2 Mechanisms in apoptosis. In response to various stimuli, such as the release of cytochrome c by mitochondria, the cytochrome c binds to the protein apaf-1 to form a complex that activates pro-caspase 9 to caspase-9 which in turn cleaves and activates effector caspase 3 and others to execute apoptosis (from Parone et al., 2002; with permission).

Anti-angiogenic functional food, degenerative disease and cancer 489 is exaggerated in aging human tissues and severely impaired in individuals with mitochondrial diseases. ROS also cause somatically acquired mutations such as deletion in mtDNA. Point mutations, either maternally inherited or somatically acquired and with clinical symptoms ranging from asymptomatic to incapacitating, accelerate oxidative damage and mtDNA lesions which are expressed as premature aging in patients with degenerative diseases (Ozawa, 1999). Age-related accumulation of oxidative damage and mtDNA deletion result in loss of mitochondrial ETP activities, cell apoptosis, functional decline in organs which may lead to age-related neoplastic and non-neoplastic diseases (Mandavilli et al., 2002; Lee and Wei, 2001; Ozawa, 1999). Coenzyme Q10 Coenzyme Q10 (ubuquinone or CoQ10), a vitamin-like coenzyme nutrient is a key part of the inner mitochondrial enzyme complexes associated with OXPHOS. The fundamental properties of CoQ10 include (i) its role to accept electrons generated by the oxidation of NADH, FADH2 from complex I (NADH-CoQ oxidoreductase) and the oxidation of succinate from complex II (succinate-CoQ oxidoreductase) and transfer these electrons to complex III (the CoQH2-cytochrome c reductase complex) of the electron transfer chain; (ii) its unique nature as the only known naturally occurring liposoluble antioxidant that can be regenerated by the body enzyme systems; (iii) the ability to improve membrane fluidity, to regenerate the reduced active form of -tocopherol and lower the peroxidizability of LDL better than vitamin E, and ability to exert both intra- and extra-cellular antioxidative activity; (iv) a blood pressure lowering effect and ability to protect tissue during ischemia and reperfusion; and (v) its ability to boost the immune system, elevate the level of TNF- and reduce the levels of IL-6 (Hodges et al., 1999). Increased CoQ10 concentration in the inner mitochondrial membrane was reported to increase the efficiency of OXPHOS (Lenaz et al., 1994, 2000). The role of CoQ10 in the etiology of degenerative diseases such as congestive heart failure, PD, AD, and cancer has been associated with a highly significant correlation between low level of mitochondrial CoQ10 and reduced activity of complexes I and II/III of the electron transport chain in patients suffering from these diseases. CoQ10 levels also decreases with aging in humans as well as in other mammals. Coenzyme Q10 at dose up to 1200 mg/day was shown to slow the progressive deterioration of neurological disorders in PD (Shults et al., 2002). 18.2.2 Apoptosis Apoptosis, also known as genetically programmed cell death or controlled cell elimination, provides a controlled cell deletion to balance cell proliferation and therefore plays a key role in tissue homeostasis. Apoptosis selectively eliminates senescent, preneoplastic, or superfluous cells that can negatively affect normal cell functioning and stimulate cell transformation (James et al., 1998). Apoptosis is morphologically and biochemically different from necrosis. The

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apoptotic process is initiated by signaling pathways that include pro- and active caspases, regulated by protein kinases and phosphatases, executed by proteolysis, the processes of its progression are irreversibly regulated by myc, fos, jun, and p53, and modulated by the bcl-2 gene family which regulate the release of cytochrome c (Parone et al., 2002). Regulators of apoptosis include both intrinsic and extrinsic signals. Intrinsic pathway of apoptosis can be induced by a variety of stimuli such as oxidative stress, DNA damaging agents and kinase inhibitors. Fas-ligand is an example of extrinsic signal that triggers apoptosis. Abnormality in apoptosis can lead to accelerated aging, a variety of degenerative diseases, and reduce life span. In cells, caspases exist as procaspases. However, in response to various stimuli, such as the release of cytochrome c by mitochondria, the cytochrome c binds to the protein apaf-1 to form a complex that activates procaspase 9 to caspase-9 which in turn cleaves and activates effector caspases 3, 6, and 7 to execute apoptosis (Parone et al., 2002) (Fig. 18.2). Stimulators of apoptosis may inhibit the integrity of chromosomes and the appearance and progression of neoplastic diseases, but may stimulate the onset and progression of degenerative diseases such as PD, whereas inhibitors of apoptosis may enhance chronic degenerative diseases such as cancer (Thompson, 1995). In AD and Huntington's diseases, neuronal cells die by apoptosis (Lee and Wei, 2001). 18.2.3 Aging The onset of many degenerative diseases is closely associated with aging because aging involves chronic exposure to (i) the accumulation of ROS, oxidative stress, oxidative damage to mtDNA, potentially mutagenic lesions such as 8-hydroxy-20 -deoxyguanosine (OH8dG) and defective or mutated p53, and/or other defective homeostatic regulators, (ii) large mtDNA deletions and higher rates of DNA fragmentation, (iii) increase in point mutations in the replication control region of human mtDNA, and (iv) increase in oxidized mitochondrial and cellular lipoproteins, proteins, lipids, the levels of various reactive radicals, and subsequent release of apoptogenic proteins into the cytosol (Lin et al., 2002; Sastre et al., 2000). Aging is also associated with a decrease in (i) DNA repair system and mitochondrial OXPHOS system including low level of CoQ10 and enzyme activities, (ii) ATP synthesis, and (iii) immune function (Kapasi and Singhal, 1999). All these factors increase the likehood of apoptosis, shortened cell life, and susceptibility to, and inability to combat, diseases. 18.2.4 Oxidative induction of telomere shortening The maintenance of intact and protected chromosomes ends or telomeres is one of the requirements for cell proliferation and survival. Telomeres are specialized nucleoproteins which cap and protect the ends of linear chromosomes from degradation and associated with end-to-end fusion or the activation of DNA

Anti-angiogenic functional food, degenerative disease and cancer 491 damage. In mammals, telomeres are characterized by the tandem repeats of (TTAGGG)n sequence (Meyne et al., 1989; Greider, 1994). Free radicals generated by the OXPHOS cause widespread cellular destruction ranging from membrane and protein damage, DNA degradation, and premature removal of telomeres from chromosome tips (Bree et al., 2001). Telomere shortening accelerates with aging, can induce cell cycle arrest and apoptosis, is significant in hypercholesterolemic and diabetes mellitus patients with cardiovascular diseases, and may be involved in the mechanism that promotes coronary diseases (Obana et al., 2003). While telomerase prevents telomere shortening, the enzyme is associated with the prevalence of cancer and has been suggested as a target for therapy (Cherif et al., 2003). 18.2.5 Chronic inflammation Chronic inflammation such as gastritis, schistosomiasis, chronic cholecystitis, inflammatory bowel diseases, gingivitis, bronchitis, and cystitis caused by factors such as bacteria, viral, and parasitic infections, chemical irritants, or nondigestible particles, increases the risk of degenerative diseases because chronic exposure to these inflammatory mediators stimulates the formation of new blood vessels, increased risk of neoplastic transformation, cell proliferation, mutagenesis, oncogene activation, and proliferation of cells devoid of normal growth control. Chronic inflammation is closely associated to abnormal cell proliferation because granulation tissues require an extended vascular supply and macrophages, platelets, and fibroblasts are major sources of pro-angiogenic factors such as VEGF, bFGF, TNF, and others (Jackson et al., 1997). Peroxynitrite and its hydrogenated form peroxynitrous acid, generated by acute and chronic inflammation, are potent oxidants and react with proteins, lipids, and can enter the cell and bring about damage such DNA single strand fragmentation, DNA base modifications, DNA mutations, the formation of 8nitroguanine and 8-oxoguanine, activation of the nuclear enzyme poly(ADPribose)synthetase (PARS), apoptosis, and malignancies (Grisham et al., 2000; SzaboÂ and Ohshima, 1997). Cyclooxygenase-2 (COX-2) and prostaglandins synthesized in monocytes, macrophages, fibroblasts, epithelial and endothelial cells are expressed during inflammation and are associated with the incidence and progression of colon cancer and adenocarcinoma, chronic inflammatory diseases and retinopathies (InÄiguez et al., 2003; Shacter and Weitzman, 2002). Inflammation is the hallmark of almost all chronic diseases. Inhibitors of COX-2 also restrict angiogenesis and downregulate the production of pro-angiogenic factors VEGF and bFGF. This makes COX-2 a target not only for degenerative disease diagnosis but also for disease prevention and/or therapy. 18.2.6 Hypoxia, anoxia, and hyperoxia Hypoxia, the withdrawal of oxygen to an insufficient level for normal growth, decreases the net formation of oxygen free radicals and does not increase oxidized

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lipid, protein or DNA. However, chronic hypoxia can induce apoptosis in cells with 0±0.5% oxygen levels. Cells exposed to hypoxia tend to maintain homeostasis by up regulating angiogenesis and the expression of transcription factors such as hypoxia-inducible factor (HIF-1), p53, AP-1, nuclear factor-B (NFB), and the expression of genes such as c-jun, insulin-like growth factor-2 (IGF-2), IGFbinding protein 1 and 3 (IGFBP-1 and IGFBP-3), transforming growth factor (TGF- ), placenta growth factor (P1GF), urokinase receptor, tyrosine hydroxylase (TH), p27Kip1 and p21Waf1 (Maxwell, 2002; Wouters et al., 2002). HIF-1 influences the transcription of genes involved in apoptosis and induces the expression of genes involved in angiogenesis (Brunelle and Chandel, 2002). Hypoxia creates a unique environment conducive to the onset and development of genomic instability, apoptosis, angiogenesis, and metastasis (Wouters et al., 2002). The micro-environment created by hypoxia reduces the sensitivity of tumor cells to treatments and has been clinically demonstrated to be associated with poor prognosis. VEFGF, a major stimulator of angiogenesis is upregulated under hypoxic conditions. Hypoxia also upregulates the expression of nitric oxide synthase, platelet derived growth factor, angiopoietin2 (Ang2), matrix metalloproteinases (MMPs), urokinase plasminogen activator (uPA), and others which all promote the angiogenic signal. Hypoxia is a biomarker of several poorprognosis human malignancies. Anoxia causes the release of large amounts of calcium from the mitochondria and subsequent increase of cytosolic calcium to a pathological level (Hatanaka et al., 1995). Anoxia causes acute apoptosis by enhancing the release of cytolosic enzymes from mitochondrial content (Jones, 1995). Hyperoxic conditions in absence of sufficient antioxidant defense system may increase the level of reactive oxygen species (ROS) and lead to oxidative stress-induced cellular damage such as decreased enzyme antioxidant levels and activity, mtDNA fragmentation and apoptosis (Singhal and Jain, 2000; Amicarelli et al., 1999; Yoneda et al., 1995). Ischemia/reperfusion, which involves hypoxia and reoxygenation, stimulates angiogenesis (Ben-Yosef et al., 2002). Prolonged hypoxia/reoxygenation mediated MMP-2 upregulation both transcriptionally and post-translationally. Prolonged hypoxia, of more than 24 h, enhanced the production and secretion of MMP-2 by suppressing MT1-MMP and TIMP-2 mRNA, while reoxygenation enhanced the secretion of MMP-2 by upregulating MMP-2 and MT1-MMP mRNA expression (Ben Yosef et al., 2002). Hypoxia has been recognized as a stimulator of angiogenesis for maintaining homeostasis in muscle capillaries. 18.2.7 Defects in signaling transduction Oxidative stress activates various signaling pathways in mammalian cells. Defects in proteins involved in signal transduction, by extraneous agents or through mutations in the genes that encode them, can lead to degenerative diseases. ROS regulate the activity of preexisting proteins, affect the expression of many genes, and disrupt the regulation of transcription factor families such as

Anti-angiogenic functional food, degenerative disease and cancer 493 the activating protein-1 (AP-1), nuclear factor-kappaB (NF-B), and activating protein-2 (AP-2) that have a crucial function in proliferation, differentiation, and morphogenesis (Dalton et al., 1999). The activity of many transcription factors such as AP-1, mitogen activated factor (MAF), neural retina leucine zipper (NRL), and nuclear factor-interleukin-6 (NF-IL6) is regulated by redox cycling of cysteinyl residues. However, proteins such as protein kinase C (PKC), collagenases, and SRC tyrosine kinase, which are important in signal transduction and carcinogenesis, also contain cysteinyl residues whose redox status affect protein reactivity. NO, a highly reactive free radical species is an efficient radical scavenger and a messenger molecule involved in the regulation of blood flow and cellular signaling in the central nervous system. However, in the presence of insufficient antioxidants, NO can generate peroxynitrite (ONOOÿ), a highly reactive molecule which inhibits mitochondrial proteins. As a result, there is a decrease in either ATP synthesis or DNA fragmentation leading to apoptosis (Nucci et al., 2003).

18.3

Environmental/exogenous risk factors

18.3.1 Diet Caloric restriction, which provides only essential nutrients while lowering dietary energy intake, may prolong healthy lifespan by attenuating oxidative stress and slowing aging, which in turn delays the onset and/or progression of degenerative diseases in animal models of longevity (Shimokawa et al., 2003; Urao et al., 1995; Sohal and Weindruch, 1996). B6C3F1 murine models of liver tumors fed NIH-31 open formula diet showed a higher incidence of spontaneous hepatoma over a three-year period than similar animal models fed only 60% of the same diet. Dietary restriction may be the most effective intervention, among many others, to decrease oxidative stress in vivo, achieve a long and disease-free life span, probably owing to a better protection against mitochondria-linked oxygen stress (Miquel, 2001; Frame et al., 1998). Endurance exercise has been suggested to serve to some extent as a substitute for caloric restriction in humans. The phenotypes of laboratory animals with reduced insulin growth factor (IGF) resemble those induced by caloric restriction. Lower levels of IGF were measured in caloric restriction animals and an inverse relationship has been established with life span (Shimokawa et al., 2003; Dunn et al., 1997). Caloric consumption may be an important risk factor associated with a spectrum of human degenerative diseases when genetic and environmental factors are taken into consideration (Lutz and Schlatter, 1992). A positive correlation between increased body mass index (BMI), in both men and women, and chronic diseases such as cardiovascular disease and cancer has been established through epidemiological observations. However, a three-way relationship between caloric consumption, body weight, and morbidity in humans is so far difficult to establish whereas the relationship has been readily established in genetically similar and environmentally controlled laboratory animals.

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Hyperglycemia can cause glucose to undergo autooxidation to generate intermediates that lead to the formation of ROS, NO., proxynitrite (ONOO-), advanced glycation end products (AGE) which all have damaging effects such as DNA point mutation and double-strand breaks, caspase activation, and apoptosis (Allen et al., 2003). 18.3.2 Environmental toxicants Environmental factors and gene-environmental interaction may play a large role in the pathogenesis of degenerative diseases. Exposure to environmental factors known to increase ROS production, such as ultraviolet and ionizing radiation, cigarette smoke, benzoyl peroxide, organic peroxides, heavy metals, redox active chemicals, hypoxia, anoxia, and hyperoxia, and other environmental pollutants may also increase the free radical burden. 18.3.3 Light Solar UV-B ( 280±320 nm) and UV-A ( 320±400 nm) cause oxidative stress that generates singlet oxygen, lipid hydroperoxides, and oxidized sulfhydryls which damage proteins and lipids (Dalton et al., 1999). Singlet oxygen-derived from UV-A radiation affects the transcriptional expression of several genes in mammalian cells, such as those coding for intercellular adhesion molecule-1 (ICAM-1), IL-1, IL-6, IL-8, and many others (GretherBeck et al., 1997; Scharffetter-Kochanek et al., 1997). 18.3.4 Stress In today's society, stressors of various types such as psychological, physical, and biological abound and occur in food, medicine, the working place, and the environment and are suspected to play a role in degenerative disease development. The impact of stressors on cells is associated with protein denaturation. Stress gene and proteins, also known as heat shock genes and proteins, mediate the impact of stressors on cells. A cell lacking an antistress mechanism (chaperones), including a complete and functional set of chaperones, may undergo protein unfolding and misfolding which lead to irreversible protein damage and promote protein abnormalities which result in age-related degenerative disorders whereby protein aggregates deposit in various tissues, most importantly in the brain where the deposits are associated with neuronal degeneration (Macario and Conway de Macario, 2000). Chaperones are critical for protein correct folding and stabilization at the cell locales where the protein performs its physiological function. Mitochondrial dysfunction may be both a cause and a consequence of degenerative diseases. Mutations in mtDNA or depletion of endogenous scavengers of ROS can also result in degenerative diseases. Conversely, some degenerative diseases show decreased OXPHOS and apoptosis and/or mtDNA

Anti-angiogenic functional food, degenerative disease and cancer 495 mutations as secondary phenomena (Quigley et al., 2000; Jarreta et al., 2000). Mitochondria may also function as sites of oxygen sensing because it may initiate signaling cascades involved in adaptive response to hypoxia (Chandel and Schumacker, 2000). Mitochondrial respiratory chain deficiency may cause some degenerative diseases by upregulating the expression of VEGF and angiopoietin, two major stimulators of angiogenesis (Tham et al., 2002).

18.4

Angiogenesis, body function, and degenerative disease

The building and remodeling of blood vessels is a critical event in the formation of every organ, and the relationship between the blood vessels and the tissues they serve is tightly balanced between stasis and growth, and regression (CancerProtocol.com). Angiogenesis ± defined as the formation of new blood vessels from preexisting vasculature ± is important in both physiological and pathological conditions. In the healthy body, angiogenesis is important for reproduction, development of a child in a mother's womb, for connective tissue matrix formation, maintenance of vascular integrity during wound healing, the repair process of damaged tissue, bone growth and bone fracture repair, and maintenance of appropriate oxygen delivery to working muscle with increased metabolic activities (Drixler et al., 2002; Gerber and Ferrara, 2000; Haas, 2002; Schilephake, 2002). Physiological angiogenesis is transient and of very short duration, tightly controlled by a network of stimulators and inhibitors which work in balance with each other, and occurs predominantly during embryo development where vasculature needed in adult life is formed from endothelial cell precursors known as angioblasts by the process of vasculogenesis or from existing blood vessels by the process of angiogenesis. Vasculogenesis and angiogenesis are mediated by paracrine growth factors. Vascular endothelial growth factor (VEGF) is a major regulator of vasculogenesis, and both physiological and pathological angiogenesis. The steps of vasculogenesis involve the binding of VEGF to one of its two receptors, the VEGF-R2 (Flk1) protein, followed by the formation of the capillary tube when VEGF binds to its second receptor, VEGF-R1 (Flt1). Then the endothelial blood vessels interact with mesodermal cells, and the angiopoietin-1 factor binds to the Tie2 receptor tyrosine kinase and allows the blood vessel to recruit the peri-endothelial cells that will surround it. The recruited cells become the pericytes and smooth muscle tissue of the blood vessel, and play an important role in maintaining the stability of the blood vessels. Mice lacking Flk1 genes lacked endothelial cells and died at embryonic day 8; and mice lacking Flt1 failed to form blood vessels and died at embryonic day 8 (CancerProtocol.com). In a healthy body, the vasculature is quiescent, and in most healthy adult tissues growth is almost non-existent (Hobson and Denekamp, 1984). In adulthood, physiological angiogenesis occurs during wound healing, ovarian

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cycle, and although endothelial cells are surrounded by many proangiogenic stimulators, only about 0.01% of adult endothelial cells undergo the process of angiogenesis at any given time (Drixler et al., 2002; Hobson and Denekamp, 1984). The mechanism of physiological angiogenesis involves several steps which include (i) the removal of pericyte from the endothelium and angiopoietin-2 (Ang2)-catalyzed transformation of endothelial cells from a stable to a proliferative phenotype; (ii) growth factors, specifically VEGF and VE cadherin catalyzed vessel hyperpermeability and matrix remodeling by the action of serine and matrix metalloproteinases; (iii) VEGF, FGF, and EGF catalyzed endothelial cell proliferation; (iv) endothelial cell migration catalyzed by v 3 integrin, VEGF, and FGF; (v) VE cadherin and ephrin B2/ephrin B4 catalyzed cell-cell contact; (vi) FGF, PDGF, TNF-, and Eph-2A catalyzed tube formation as blood conduits; (vii) PDGF-and Ang1/Tie2±catalyzed proliferation and migration of mesenchymal cells along the new vessels and TGF- catalyzed pericyte differentiation into mature pericytes; and finally (viii) Ang1/Tie2, PDGF, VE cadherins and TGF- catalyzed vessel stabilization (Papetti and Herman, 2002). Ang2 is indispensable for embryonic development and required for postnatal vascular remodeling (Gale et al., 2002). The vascular endothelium plays multiple homeostatic roles including (i) nutrient and gas exchange between tissues and blood, and (ii) inductive effect on liver and pancreas development before the establishment of blood flow (LeCouter et al., 2003). PDGF, a dimeric 30 kDa glycoprotein ± with four different isoforms ± is actively involved in the growth and development of many tissues and organs such as the CNS and messengial cells of kidney glomeruli. Blockade of PDGF signaling was associated with perinatal lethality (Schilephake, 2002). IGF is a circulating mediator of growth hormone with autocrine and paracrine activity. PDGT, TGF-beta, and IGFs are important mediators in general growth, bone regeneration, and maintenance of the body skeleton. Under normal physiological conditions, there is a balance between metalloproteinases and their endogenous inhibitors and as a result, minimal proteolysis catalyzed by the enzymes occurs (Haas, 2002). Other stimulators of physiological angiogenesis include the physical effect of increased muscle blood flow or of muscle contraction, muscle stretch/overload, metabolite such as adenosine released by exercising muscle, limited proteolysis, and bioactive compounds such as NO that are known as angiogenic activators (Haas, 2002; Wagner, 2001). Different angiogenic molecules, such as VEGF/VPF, bFGF, or IL-8, differentially regulate distinct steps in the process of angiogenesis, however, any given molecule may be necessary but in itself insufficient for establishment of a viable vasculature (Kumar et al., 1998). There are two types of pathological angiogenesis; excessive and insufficient angiogenesis. Excessive angiogensis occurs when angiogenic stimulators outbalance inhibitors and insufficient angiogenesis occurs when stimulators are deficient. Table 18.1 lists the stimulators of physiological and pathological angiogenesis. Vascular endothelial growth factor (VEGF) has been recognized

Anti-angiogenic functional food, degenerative disease and cancer 497 Table 18.1

Stimulators of physiological and pathological angiogenesis

Physiological angiogenesis

Pathological angiogenesis*

1.

Growth factors VEGF family, aFGF, bFGF, PDGF, TGF- , TNF-, EGF, TGF-, BMP

1.

Growth factors VEGF family, aFGF, bFGF, PDGF, TGF- , TNF-, EGF, TGF-

2.

Hormones IGF-1,LH, estrogen, progesterone Androgens

2.

Hormones IGF-1-prolactin, placental Lactogen

3.

Enzymes Aspartic proteinases (cathepsin D) Serine proteinases (urokinase) Cysteine proteinases (cathepsin B) Metalloproteinases Signal transduction enzymes

3.

Enzymes Aspartic proteinases (cathepsin D) Serine proteinases (urokinase) Cysteine proteinases (caspases) Metalloproteinases (MMP-2, -9, . . .) Cyclooxygenase-2 Lipoxygenase-1 and -2 Heparanase, PKC Thymidine phosphorylase Proteasome Signal transduction enzymes

4.

Other proteins Angiogenin Angiopoietin-1 Angiopoietin-2

5. Other proteins Angiogenin Angiopoietin-2

5.

Cell adhesion molecules v 3-integrin v 5-integrin 5 1-integrin

6.

6.

Membrane bound proteins VE cadherin Ephrin (A1, 2A, B2, 4B)

7.

Other mediators Prostaglandin Factor V Nicotinamide Shear stress Muscle stretch Adenosine

7. Mediators of inflammatory reaction TNF- IL-8 IL-1 Prostaglandin

Cell adhesion molecules E-selectin v 3-integrins

8. Environmental factors Copper Hypoxia 9. Oncogenes c-myc c-jun c-fos H-ras

Adapted from: Papetti and Herman (2002), Han et al., (2003), Cid et al. (2002), Jojovic et al. (1998), Jaggers et al. (1996), Lloyd et al. (2003), and Losso, (2003). Abbreviations: VEGF vascular endothelial growth factor, aFGF acidic fibroblast growth factor, bFGF basic fibroblast growth factor, PDGF plasminogen derived growth factor, TGF- transforming growth factor-alpha, TGF- transforming growth factor-beta, TNF- tumor necrosis factor-alpha, EGF epidermal growth factor, BMP bone morphogenetic protein, IGF-1 insulin growth factor-1, LH luteinizing hormone.

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as a major stimulator of both physiological and pathological angiogenesis and plays multiple roles in a variety of pathological angiogenesis. VEGF stimulates blood vessel growth and increases vascular permeability. Other important growth factors that complement the action of VEGF include the fibroblast growth factor (FGF) which stimulates endothelial cell proliferation, induces neovascularization in vivo, and is important for tumor survival and maintenance (Folkman et al., 1971; Folkman, 1995). FGF includes the acidic and basic growth factor (aFGF and bFGF). bFGF mediates the activity of heparanase which promotes angiogenesis by (i) stimulating endothelial cell invasion and vascular sprouting, (ii) releasing heparin-sulfate-bound bFGF from the endothelial cell matrix (ECM), and (iii) rendering nonmetastatic cell lines metastatic in the presence of overexpressed heparanase (Vlodavsky et al., 2000). Ang2, which also plays an important role in physiological angiogenesis in cooperation with proteinases, mediates the dissolution of the existing basement membrane and the interstitial matrix and induces blood vessel growth in cooperation with VEGF. Excessive or insufficient angiogenesis is associated with numerous non-neoplastic diseases (Table 18.2). In non-neoplastic angiogenesis-dependent diseases, vessels do not grow but abnormally remodel. Chronic inflammation and hypoxia are also mediators in non-neoplastic angiogenesis-dependent diseases. The mechanism of excessive pathological angiogenesis involves: (i) retraction of pericytes from the abluminal surface of the capillary, (ii) release of proteases from the activated endothelial cells, (iii) protease-catalyzed dissolution of the basement membrane surrounding the pre-existing vessels, (iv) endothelial cell migration toward an angiogenic stimulus and their proliferation, (v) formation of tube-like structures, (vi) fusion of the formed vessels, and (vii) initiation of blood flow (Losso, 2003; Fig. 18.3). Plasma of cancer patients showed high levels of five cytokines, VEGF, HGF, bFGF, TGF- , and TNF- compared to healthy individuals (Fuhrmann-Benzakein et al., 2000). Insufficient angiogenesis is characterized by (i) insufficient vascularization, (ii) delayed formation of granulation tissue, (iii) decreased collagen content, (iv) low breaking strength compared to normal tissue, loss of vascular tone, (v) higher content of oxidized lipids, (vi) absence of microtubular structures, (vii) impaired collateral vessel formation, and (viii) impaired signal transduction pathways. The similarity between physiological and pathological angiogenesis includes (i) both processes are conduits to supply blood and nutrients to and remove metabolic wastes from living tissues; (ii) both processes require proximity to a vascular supply. However, the vasculature that fulfils the metabolic requirements for tissue and cells (physiological angiogenesis) is generated by vasculogenesis and angiogenesis. Pathological angiogenesis may use circulating endothelial precursors and induce abnormal vessels lacking functional pericytes to fulfil their metabolic requirements; (iii) stimulators of physiological angiogenesis are also stimulators of pathological angiogenesis but not vice versa, and (iv) VEGF is considered as the major soluble mediator of both normal and pathological angiogenesis and acts through its receptors kinase, kinase insert

Anti-angiogenic functional food, degenerative disease and cancer 499 Table 18.2

A partial list of human diseases associated with pathological angiogenesis Pathological angiogenesis

Insufficient · Delayed wound healing · Ischemia (myocardial, peripheral, cerebral) · Stroke · Heart disease · Scleroderma · Infertility · Diabetic neuropathy · Systemic sclerosis · Coronary heart disease · Placental insufficiency · Impaired healing of fracture · Pulmonary and systemic hypertension · Vascular dementia · Lymphoedema · Liver regeneration · Impaired collateral vessel formation · Chronic non-healing ulcer

· · · · · · · · · · · · · · · · · · · · · · · · · · ·

Excessive Rheumatoid arthritis Multiple sclerosis Age-related macular degeneration Diabetes retinopathy AIDS complications Kaposi sarcoma Tumor growth and metastasis Osteoporosis Alzheimer's Parkinson's Obesity Psoriasis Hepatitis Asthma Thyroid enlargement Ocular neovascularization Retinopathy of prematurity Synovitis Osteomyelitis Nasal polyps Liver regeneration Panus growth Bone/cartilage destruction Endometriosis Hematological malignancies Hemangioma Adipose tissue

domain-containing receptor/fetal liver kinase 1(KDR/Flk-1) and fms-like tyrosine kinase 1 (Flt-1). The major differences between physiological and pathological angiogenesis involve (i) the ultrastructural differences between pathological angiogenesisinduced vessels and physiological ones; (ii) vessels generated by tumor cells are mostly devoid of pericytes, tortuous, dilated, extremely fenestrated, disorganized microvascular networks, leaky because of unusually large gaps between adjacent endothelial cells, devoid of functional lymphatic system, and may be made up of both endothelial cells and tumor cells (Papetti and Herman, 2002); and (iii) the mechanism of tumor angiogenesis involves in addition to stimulators of normal angiogenesis, other stimulators such as heparanase, IL-8, and MMP-2 and MMP-9 (Losso, 2003; Drixler et al., 2002; Papetii and Herman, 2002). Table 18.3 summarizes the stimulators of normal and pathological angiogenesis.

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Fig. 18.3 The key steps in tumor angiogenesis include (a) the degradation of the basement membrane by proteases secreted by activated endothelial cells, (b) migration, proliferation of endothelial cells toward angiogenic stimulus, (c) formation of solid endothelial cell sprouts into the stromal space, and (d) formation of vascular loop that supplies oxygen and blood to the tumor, and development of capillary tubes and new basement membrane. Photograph from Wernert et al. (1999). Copyright permission from the publisher.

Table 18.3 Molecular mechanism associated with pathological angiogenesis Insufficient angiogenesis

Excessive angiogenesis

· · · · · · · · · ·

· · · · · · ·

Impaired collateral vessel formation Impaired collagen metabolism Impaired signal transduction Impaired integrin(s) Elevated levels of MMP Low level of PKC Excessive ROS Reduced expression of cytokines Insufficient growth of collateral vessels Reduced angiogenic growth factor levels · Improper basement membrane degradation

Excessive levels of VEGF Excessive levels of MMPs Elevated levels of inflammatory stimuli Elevated levels of HIF and IGF Excessive levels of chemokines Elevated levels of COX-2 Elevated level of ODC

Anti-angiogenic functional food, degenerative disease and cancer 501

18.5

Anti-angiogenic functional food compounds

The promise of angiogenesis in phase III clinicals has not yet been realized as expected (Sweeney et al., 2003, McCarty, 2003). One potential reason has been that patients enrolled in clinical trials of anti-angiogenic compounds are often those at an advanced stage of the disease while preclinical data are often collected using animals with relatively small tumors (McCarty, 2003). Other factors involved in resistance to anti-angiogenic therapy include (i) endothelial cell heterogeneity, (ii) tumor cell heterogeneity, (iii) impact of tumor microenvironment, (iv) compensatory response to treatment, and (v) tumor regrowth that is independent of angiogenesis (Sweeney et al., 2003). Scappaticci (2002) and Losso (2003) suggested that the effectiveness of anti-angiogenic compounds may be realized at early stages of the disease when few stimulators may be more manageable than at late stages when myriads of regulators complicate matters. Throughout history, humans have heavily relied on food to prevent or fight diseases. Functional foods or Nutraceuticals do not undermine medical advances accomplished over the last two centuries for chronic diseases. However, disease prevention ± made possible by good eating habits and knowledge of biological activities of food components ± has helped medical interventions to save more lives. Foods in general, and functional foods in particular, are being considered as good anti- or pro-angiogenic candidates for disease prevention because these compounds can be safe, effective, reversible inhibitors, ingested appropriately over a life span without severe toxicity, amenable to clinical trials, and possibly become low cost budget prescriptions (Losso, 2003). Cao et al. (2002) reported that 60 to 80% of antibiotics and anti-cancer drugs approved between 1983 and 1994 and almost 50% of the best-selling drugs in 1999 were natural products, some of them were of raw food origin. 18.5.1 The range and mechanism of action of anti-angiogenic compounds in foods Anti-angiogenic compounds in foods encompass compounds in all macro- as well as micronutrients classes. Anti-angiogenic compounds in foods may be endogenous or a result of food-processing operations. Most peptides are products of enzymatic hydrolysis and therefore classify as products of foodprocessing operations. The major mechanism by which functional foods inhibit angiogenesis involves the inhibition of enzyme(s) associated with either the onset and/or the progression of the angiogenic process (Losso, 2003). Therefore, enzyme inhibition may attenuate angiogenesis. Growth factors, hormones, enzymes, metals, signal transduction enzymes, cytokines, and endogenous modulators are stimulators of angiogenesis and represent attractive targets for anti-angiogenic functional foods. Apotosis is a regulated cellular process while angiogenesis is a deregulated process. Apoptosis offers opportunities for the inhibition of uncontrolled cell proliferation associated with angiogenesis.

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Neovastat (AE-941), a shark cartilage derived anti-angiogeneic compound, was found to be proapoptotic (Boivin et al., 2002). Quercetin and resveratrol have been reported to induce apoptosis in acute myelogenous leukemia cell lines (Liesveld et al., 2003; Gautam et al., 2000). However, apoptosis also offers a challenge for cell death that characterizes degenerative diseases such as AD and PD. The following paragraphs describe the range of anti-angiogenic functional foods and provide the inhibition target and the mechanism(s) by which the inhibition or the stimulation occurs. Angiogenesis is a multistep process that involves the catalysis of different enzymes at different steps. Each of the steps of pathological angiogenesis may represent a target for the inhibition of the progression of the angiogenic process. 18.5.2 Proteins and peptides Shark cartilage, a proteoglycan of 10,000 daltons, is undergoing Phase III clinical trials for effectiveness against metalloproteinases and VEGF, and its ability to stimulate the release of precaspase-3, -8, and -9 from the mitochondria and their activation through the release of cytochrome c (Boivin et al., 2002). SCAIF-I, an 18 kDa protein from shark cartilage, inhibited endothelial cell proliferation and movement in a CAM assay, and suppressed 87% tumor growth in C57BL/6 models of Lewis lung carcinoma (Shen et al., 2001). Liang and Wong (2000) isolated a 10 kDa heat stable sulfated proteoglycan antiangiogenic inhibitor from shark cartilage. Neovastat (AE-941), a bioavailable anti-angiogenic proteoglycan isolated from cartilage caused a 71% decrease in the growth of blood vessels in a CAM assay compared to bFGF treated embryos (Falardeau et al., 2001). Oral administration of AE-941 dose-dependently inhibited bFGF-catalyzed angiogenesis in a Matrigel mouse model and decreased metastasis in animal models of lung carcinoma. Neovastat, in combination with cisplatinum, protected against cisplatin-induced body weight loss and myelosuppression. High-molecular-weight hyaluronic acid inhibits angiogenesis in tumors whereas degradation products of hyaluronic acid by hyaluronidase are angiogenic (Temple et al., 2000). Troponin I, a subunit of the troponin complex, at a dose between 2.5 and 20 g/ml, inhibited endothelial cell proliferation in vitro and halted angiogenesis and metastasis in vivo possibly by competition between TnI and bFGF receptor (Feldman and Rouleau, 2002). The 2-macroglobulin (2M), a 718 kDa protein present in the human plasma, is a general proteinase and cytokine inhibitor which inactivates all classes of enzymes and whose concentration is inversely related to pathological state. The ability of 2M to bind and inactivate VEGF is inhibited by bFGF, heparin, and heparan sulfate (Soker et al., 1993). Asplin et al. (2001) suggested that the ability of 2M to regulate the activities of FGF depends on the ability of 2M to inactivate proteinase which in turn may permit 2M to localize and regulate the activity of cytokines and growth factors. Since many types of carcinoma release heparin from degranulating mast cells, bioactive compounds inhibitors of heparin coagulant effect can cause

Anti-angiogenic functional food, degenerative disease and cancer 503 thrombosis in tumor cells. Protamine or its thermolysin hydrolyzates maintained heparin neutralization function and may inhibit angiogenesis by binding to heparin (Liang et al., 2003). Low molecular weight protamine fragments have the advantage of retaining anti-heparin activity without the immunogenicity of the native protamine (Liang et al., 2003). Arietta et al. (1998) indicated that the anti-angiogenic effect of non-toxic doses of protamine against glial tumor growth was similar to that produced by toxic doses of suramin. Protamine is also a potent antagonist of angiogenin-ribonuclease interaction and therefore a potential regulator of angiogenin-triggered angiogenesis (Moenner et al., 1999). Protamine abolished the anticoagulant action of heparin and potentiated the ulcer-healing effect of heparin, gastric mucosal proliferation, angiogenesis, and constitutive NOS activity (Li et al., 1999). The soybean Bowman-Birk inhibitor (BBI) has been extensively studied as an anti-carcinogen (Malykh and Larionova, 2002; Kennedy, 1998). BBI inhibits chymases, tryptases, cathepsin G, and elastase. BBI at concentrations as low as 0.02 mg/ml inhibited the activation of pro-MMP-1 and pro-MMP-9 which are precursors of angiogenesis catalysts MMP-1 and MMP-9 (Losso, 2003). Sharma et al. (2001) indicated that BBI at 10 g/ml was effective against angiogenesis in a CAM assay. Fenugreek contains a Bowman-Birk inhibitor type whose effectiveness against in vitro angiogenesis has not been evaluated. Rice Bowman-Birk inhibitor was effective against the activation of pro-MMP-1 in vitro (Bawadi et al., 2004). A hyaluronan-binding complex isolated from bovine cartilage inhibited the growth of TSU human prostate cancer cells growing on the chicken chorioallantoic membrane, the migration and proliferation of cultured endothelial cells and VEGF-induced angiogenesis on the chorioallantoic membrane (Liu et al., 2001). 18.5.3 Carbohydrates Chitosan, a deacetylated or partially deacetylated chitin, has been reported to accelerate wound healing (Ueno et al., 1999; Okamoto et al., 1995). Chou et al. (2003) used chitosan of 50,000 daltons to demonstrate that chitosan may enhance platelet aggregation in rabbit platelet suspension by increasing calcium mobilization and enhancing the expression of GPIIb/IIIa complex on platelet membrane surface. However, pretreatment of human astrocyte cell line CCFSTTG1 cells with 10 g/ml of high molecular weight chitosan of about 300,000 daltons inhibited serum starvation-induced DNA fragmentation and protected against apoptosis (Koo et al., 2002). Apoptosis has been implicated in neuronal death in neurological diseases such as Alzheimer's. Muzzarelli et al. (1999) reported chitin and chitosan to possess antiinflammatory activities, possibly due to the inhibition of cyclooxygenase. Sulfated -cyclodextrin at 200 ng stimulated angiogenesis in mice, but at 2000 ng reduced vessel formation in a subcutaneous plastic sponge model in mice (Strauss et al., 2002). Lentinan, a polysaccharide present in Shiitake mushroom

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was associated with the stimulation of the host defense mechanism, cytotoxicity against endothelial cells, decreased IL-6 production, and enhanced apoptosis (Sano et al., 2002). An additive effect against angiogenesis was observed when lentinan at 2 mg/kg was combined with a synthetic analog of fumigallin, TNP470, a well known inhibitor of angiogenesis. Polysaccharide-K (polysaccharideKureha; PSK), also known as krestin, has been tested in vitro and clinically and may act as an antioxidant, a metalloproteinase inhibitor, and a biological response modifier (Fisher and Lang, 2002). Balb-c mice challenged with human colon-25 tumor and treated with modified citrus pectin (1.6 mg/ml) showed a 70% reduction in tumor size (Hayashi et al., 2000). Garlic oligosaccharides have inhibitory activity against adenosine deaminase and cyclic AMP phosphodiesterase (Agarwal, 1996). pH-modified citrus pectin (MCP) at doses of 0.8±1.6 mg/ml inhibited prostate cancer and melanoma metastasis in mice and reduced tumor by as much as 70% (Hayashi et al., 2000). 18.5.4 Lipids and liposoluble compounds Oleic acid at 5 10ÿ5 M reduced the activity of MMP-2 released by oncogenetransformed human bronchial epithelial cells by 50% (Polette et al., 1999). Serum levels of vascular endothelial growth factor (VEGF) and whole mammary gland levels of VEGF and its receptor Flk-1 decreased when CD2/ F(1) mice were placed on synthetic diets containing 0, 1, or 2% of cis-9, trans11 or trans-10, cis-12 CLA isomers CLA for six weeks, before angiogenic challenge by s.c. injection with an angiogenic gel substrate known as Matrigel pellet assay (Masso-Welch et al., 2002). Valproic acid inhibited cell adhesion and reduced cell ability to penetrate the human endothelium (Cinatl et al., 2002). CLA in mice diet also decreased initial cellular recruitment of stromal vascular precursors into the EHS-RBM and decreased local expression of VEGF protein within the mammary gland. Omega-3-rich fish oils may inhibit angiogenesis by inhibiting lipoxygenase which is a stimulator of chronic inflammation and angiogenesis and have shown in vitro ability to inhibit endothelial cell proliferation (Kanayasu et al., 1991). The ability of EPA to inhibit the production of Flk-1 by cultured endothelial cells was reported by McCarty (2003). Conjugated linoleic acid (CLA) which does not inhibit endothelial cell migration may inhibit angiogenesis by decreasing bFGF-induced endothelial cell proliferation and DNA synthesis in a dose-dependent manner. Milk fat is the best-known source of CLA (Parodi, 2001). CLA showed significant inhibitory effects against animal mammary carcinogenosis even at concentrations less or equal to 1%, alone or in combination with 20% saturated or unsaturated fat, in the diet (Ip et al., 1999). Dietary sphingolipids are intracellular second messengers that stimulate apoptosis by inducing the release of cytochrome c from the mitochondria (Spiegel and Merrill, 1996). Milkfat components, specifically the ceramide, sphingosine, and butyric acid have been studied as potential inhibitors of tumor

Anti-angiogenic functional food, degenerative disease and cancer 505 growth in cell cultures and animal models of cancer because these compounds can suppress tumor growth when added to cultures or tumor environment. The particular characteristic of milkfat (mostly buttermilk and whey lipids) lies in its content of several highly desirable biologically active compounds having considerable potential for use in the food and pharmaceutical industries, particularly relative to developments in the area of angiogenesis. Milkfat contains, among others, ceramides (glucosylceramide and lactosylceramide), sphingosine, sphingomyelin, and short chain fatty acids C4:0 to C10:0 (butyric, capric, caprylic, and caproic acid) (Kester and Kolesnick, 2003; Radin, 2003; Berra et al., 2002). Ceramides and sphingosine are the backbone moieties of sphingolipids. Sphingosine can be enzymatically converted by sphingosine kinase to sphingosine-1-phosphate. Milk sphingolipids and short chain fatty acids are not required for growth. However, these micronutrients have been identified as functional components of food because studies with experimental animals showed that they are anticarcinogens, cytotoxic to tumor cells, reduce serum low density lipoprotein, and elevate high-density lipoproteins (Vesper et al., 1999). Ceramides lead to cell growth arrest and apoptosis and increased ceramide enables Fas killing and induction of apoptosis (Cremesti et al., 2001). Glycosphingolipids (21.24 Ô 2.88 g/g in lyophilized milk and 84.40 g/g in fresh cheese) in bovine milk fat include glucosylceramide (39%), lactosylceramide (51%), GM3 (hematoside), GM2, GM1, GD3 (disialohematoside), GD2, and GD1b (Berra et al., 2002; Fox and McSweeney, 1998). Dietary sphingolipids are intracellular second messengers that stimulate apoptosis by inducing the release of cytochrome c from the mitochondria (Spiegel and Merrill, 1996). Sphingolipids and glycosphingolipids appear to have obligatory functions in cell proliferation, transformation, and tumor progression. Caspases, which are intracellular cysteine-containing proteases that cleave their substrates after aspartate residue in a tetrapeptide sequencespecific manner, are believed to be the terminal executors of apoptosis and their activation is mediated either through the mitochondria or through cell surface death receptors. Schmelz et al. (2000) administered bovine glucosylceramide, lactosylceramide, and ganglioside GD3 to 1,2-dimethylhydrazinetreated CF1 mice at 0.025 or 0.1 g/100 g of diet for four weeks and showed that these sphingolipids were hydrolyzed to ceramide by colonic enzymes and caused a 40% reduction of aberrant crypt focci which are early markers of colon cancer. However, Duan et al. (2003) demonstrated that the expression and the activity of human sphingomyelinases, the colonic enzymes that convert sphingolipid to ceramide, were decreased in parallel in human colon cancer tissues compared with the adjacent normal tissue. Sawada et al. (2000) demonstrated that increased ceramide level in C6 glioma cells was associated with induction of apoptosis, disruption of the mitochondrial respiratory chain, activation of c-Jun N-terminal kinase (JNK), and inactivation of Bcl-2. Most anticancer regimens, including taxol, antiestrogens, and alkaloid promote endogenous ceramide accumulation to induce tumor apoptosis (Sridhar

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et al., 2000). The absence of sphyngomyelinase in human colon cancer cells was correlated with the low level of ceramide in colon cancer tissues and the progression of the disease (Berra et al., 2002). As a result, the anti-apoptotic activity of ceramide is not realized in human colon cancer cells. The mechanism by which ceramide triggers apoptosis is not well known (Berra et al., 2002). Ceramides Studies with experimental animals have shown that feeding the lipid backbone of mammalian GluCer (sphingosine and ceramide) to carcinogen treated mice induced cell death (apoptosis) and inhibited the appearance of early markers of colon carcinogenesis as well as the appearance of adenocarcinoma (Schmelz et al., 2000; Vesper et al., 1999). It has been shown that in human colorectal carcinoma the activity of sphingomyelinase is reduced or lost (Hertervig et al., 1997). Short chain ceramides, sphingosine, and sphinganine bypass a sphingolipid signalling defect that is important in cancer, are easily transported through the mucosa and appear in systemic circulation (Berra et al., 2002). Ceramides lead to cell growth arrest and apoptosis and increased Cer level enables Fas killing and induction of apoptosis (Cremesti et al., 2001). Garlic oil Cyclooxygenase-2 (COX-2) has been implicated in the pathogenesis of several inflammatory diseases and in cancer, especially the gastrointestinal type (Dirsch and Vollmar, 2001). At 100 M diallyl disulfide was cytostatic to human colon (HCT15), lung (A549), and skin (SK MEL-2) cancer cell lines (Sundaram and Milner, 1996). Surh et al. (1995) indicated that garlic oil suppresses vinyl carbamate and N-nitrosodimethylamine (NDMA) induced mutagenesis or tumorigenesis. Diallyl sulfide administered to ehrlich ascites (EA) tumor bearing Swiss albino mice suppressed angiogenesis and increased life span by about 25% (Shukla et al., 2002). Capsaicin Capsaicin (trans-8-methyl-N-vanillyl-6-nonenamide), a major lipophilic ingredient of hot chilli peppers is a potent anti-inflammatory bioactive compound. Capsaicin and dihydrocapsaicin (trans-8-methyl-N-vanillyl-6-nonanamide) bound irreversibly to cytochrome P-450 HE1 and other P-450 isoforms and cause enzyme inactivation (Suhr et al., 1995). The antiangiogenic activity of capsaicin and its analog involves the inhibition of microsomal monooxygenases and cytochrome P450 II E1 isoforms, respectively. Subcutaneous administration of capsaicin (100 mg/kg) enhanced gastric wound healing by decreasing gastric mucosa cell proliferation and angiogenesis in the granulation tissue (Ma et al., 2000). Plant sterols Phytosterols are bioactive compounds with striking similarities to cholesterol and are found in oils and fats. The major phytosterols reported from oils and fats

Anti-angiogenic functional food, degenerative disease and cancer 507 include campesterol, stigmastero, -sitosterol, 5-avennasterol, and 7stigmasterol. Phytosterols are effective against inflammation, dermatitis, psoriasis and find several applications in cosmetics (Wachter et al., 1995). Ergosterol Ergosterol isolated from the lipid from Agaricus blazei Murill and administered i.p. to C57BL/6 mice at doses of 5±20 mg/kg for five consecutive days inhibited angiogenesis associated with neovascularization in sarcoma 180- and Lewis Lung carcinoma-bearing mice (Takaku et al., 2001). Ergosterol at 800 mg/L prevented neovascularization in an in vivo model of C57BL/6 mice subcutaneously injected with Matrigel containing acidic fibroblast growth factor, heparin and 400 to 800 mg ergosterol per ml (Takaku et al., 2001). 18.5.5 Short chain fatty acids Short chain fatty acids, such as the C 4:0 to C10:0 released by the action of lipases in milk or dairy products and which make about 6.2% of total fatty acids, contribute to the desirable flavor of certain cheeses such as Blue, Romano, and Parmesan (Fox and McSweeney, 1998). Most short chain fatty acids, from C4:0 to C8:0, are water soluble and bioavailable. Butyrate produced by the fermentation of dietary fiber, at 2mM, inhibited the angiogenic properties of Caco-2 cells possibly by inhibition of HIF-1 nuclear translocation and subsequent inhibition of VEGF (Zgouras et al., 2003). Data collected by Buda et al. (2003) indicated that butyrate induced apoptosis in colorectal cancer cells by downregulating the expression and functional activity of alpha(2)beta(1) integrin. Treatment of human glioblastoma U251MG and U87MG cells with 5 mM sodium butyrate suppressed the expression of VEGF-D mRNA in U251MG cells and the expression of VEGF-B mRNA in U87MG cells (Sawa et al., 2002). Sodium butyrate at concentrations less than 2 M can modulate the expression of vascular endothelial growth factor (VEGF), the most potent angiogenic factor, and hypoxia-inducible factor (HIF)-1alpha, the main transcription activator of the VEGF gene. Both factors are constitutively expressed at high levels in HT29 colon carcinoma cell lines and in normoxic conditions (Pellizzaro et al., 2002). 18.5.6 Other water soluble minor bioactive compounds Squalamine Squalamine (7,24-dihydroxylated 24-sulfated cholestane steroid conjugated to spermidine at C-3), a naturally occurring aminosterol, from tissues of the dogfish shark, inhibits angiogenesis and tumor growth induced by VEGF and a wide range of growth factors in vivo in animal models of brain, breast, and lung cancer by blocking VEGF-induced activation of MAP kinase and endothelial cell proliferation (Li et al., 2002; Teicher et al., 1998). Squalamine enhanced the cytotoxic effect of cisplatin chemotherapy for ovarian cancer xenographs treatments (Li et al., 2002).

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Minerals In vitro assays of angiogenesis have associated selenium, at concentration found in the plasma of average US consumers, with 50% inhibition of the expression of VEGF, MMP-2, and cancer epithelial expression of vascular endothelial growth factor (Lu and Jiang, 2001). Ingestion of selenium as Se-enriched garlic, after implantation and establishment of mammary carcinoma, led to a significant reduction of the carcinoma intra-tumoral microvessel density (Jiang et al., 1999). The authors also reported that in cell cultures, Se inhibited angiogenesis by apoptosis. Methyl selenol, a metabolite of selenium was suggested to upregulate apoptosis and suppress VEGF production by cancer cells (McCarty, 2003). Metal chelators Copper is a powerful cofactor of some angiogenic enzymes. Angiogenic growth factors and copper-binding molecules such as ceruloplasmin and heparin are efficient stimulators in the presence of copper and cancer patients appear to have higher plasma levels of copper than healthy individuals (Cancer Protocol.com). Copper, at 500 M as CuSO4, stimulates the proliferation of HUVEC (Hu, 1998). Copper chelation using high-dose zinc supplementation, tea polyphenols, glycine, can achieve copper reduction in the blood (McCarty, 2003). Carnosine, a copper chelator may also reduce amyloid beta in AD (Kang et al., 2002; Gnjec et al., 2002). 18.5.7 Vitamins The anti-angiogenic mechanism of vitamin E involves the inhibition of PKC in endothelial cells and platelets. Vitamin E, dose-dependently inhibited lysophospholipid induced endothelial dysfunction and lysophospholipid induced platelet activation while preserving the release of nitric oxide (Murohata et al., 2002).Vitamin E succinate prevents angiogenesis by suppressing the expression of VEGF, VEGF receptor 1, and VEGF receptor 2 in melanoma tumors (Malafa et al., 2002). Vitamins B3 (Nicotinamide and Nicotinic acid) inhibited angiogenesis in the lesion of autoimmune disorders by reducing the interferon-gamma (IFN gamma)-induced intercellular adhesion molecule-1 (ICAM-1) and HLA-DR antigen expression on endothelial cell and endothelial cell proliferation (Hiromatsu et al., 1992). Vitamin D3 derivative 1,25-dihydroxyvitamin D3 (1,25[OH]2D3) alone or in combination with retinoids can significantly inhibit angiogenesis in mice models of angiogenesis (Majewski et al., 1996). Vitamin D3 at 0.05 M inhibited corneal angiogenesis (Shokravi et al., 1995) and has shown in vitro effectiveness against MCF-7 breast carcinoma cells overexpressing VEGF (Iseki et al., 1999). The mechanism by which vitamin B6 may inhibit angiogenesis is not well established (Komatsu et al., 2003). However, it was reported that vitamin B6 supplemented to mice for 22 weeks along with a weekly injection of

Anti-angiogenic functional food, degenerative disease and cancer 509 azoxymethane for the first ten weeks reduced cell proliferation, NO production, and expression of c-myc and c-fos proteins. 18.5.8 Phenolics Silymarin, at 50±100 g/ml dose-dependently decreased the secretion of MMP2 expression in HUVEC cells and inhibited HUVEC tube formation on a reconstituted extracellular matrix (Jiang et al., 2000). The same authors showed that the secretion of VEGF by DU 145 prostate, MCF-7, and MDA-MB-486 breast cancer cells decreased following exposure of cells to increasing concentration of silymarin. The anti-angiogenic properties of phenolic compounds such as epigallocatechin gallate (EGCG), epicatechin gallate (ECG), genistein, resveratrol, daidzein, fisetin, myricetin, luteolin, apigenin, hesperetin, theaflavin, and many others have been reported (Cao et al., 2002). Luteolin was shown to inhibit endothelial cell proliferation and migration, and a mixture of luteolin and apigenin was considered as the most potent anti-tumor proliferative flavonoid which also showed ability to inhibit the secretion of MMP-2 and MMP-9 by tumor cells (Huang et al., 1999). Other anti-angiogenic properties of luteolin may be associated with inhibition of FGF-2 induced corneal neovascularization in rabbits (Joussen et al., 2000), upregulation of p21 expression in tumor cells, decreased c-myc protein levels and apoptosis (Yin et al., 1999a). Apigenin and luteolin, at concentrations between 21.7 M and 32.1 M were also effective in reducing 50% of the activity of human thyroid carcinoma cell lines, UCLA NPA-87-1 (NPA) (papillary carcinoma), UCLA RO-82W-1 (WRO) (follicular carcinoma), and UCLA RO-81A-1 (ARO) (anaplastic carcinoma) (Yin et al., 1999b). Caffeic acid phenethyl ester (CAPE), an active component of propolis, is antiangiogenic and anti-inflammatory, and was reported to inhibit angiogenesis in the CAM assay (Song et al., 2002). Curcumin, a bioactive compound present in turmeric was shown to inhibit the transcription levels of two major angiogenic simulators VEGF and bFGF in estrogen receptor negative MDA-MB-231 cells through down regulation of MMP-2 and up regulation of TIMP-1 (Shao et al., 2002). Anti-inflammatory activity and inhibition of cyclooxygenase, lipoxygenase, and MMP-9 (by targeting the FGF-2 angiogenic signaling pathway) have also been ascribed to curcumin (Suhr, 2002; Mohan et al., 2000). At 2,000 ppm, curcumin significantly inhibited the ornithine decarboxylase (ODC) and tyrosine protein kinase activities in the liver and colonic mucosa of azoxymethane (AOM) treated rats (Rao et al., 1993). Genistein and daidzein were effective in suppressing the expression of the angiogenic factors, VEGF and bFGF in renal cell carcinoma (Sasamura et al., 2002). Daidzein at 0.2±1.0% of the AIN-76 diet reduced angiogenesis and inhibited cell proliferation in mice transplanted with 5 104 murine (MB49 and MBT-2) or human (HT-1376, UM-UC-3, RT-4, J82, and TCCSUP) bladder carcinoma cells (Zhou et al., 1998).

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The anti-angiogenic activities of resveratrol, (3,5,40 -trihydroxystilbene), include the inhibition of MAP kinase phosphorylations, 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced AP-1 activation, c-Src, and COX-2 activity, the accumulation of p53 in endothelial cells, induction of apoptosis, and cell cycle G1/S-phase arrest (Cao et al., 2002; Gautam et al., 2000). The mechanism of anti-angiogenic activities of several polymethoxylated flavones (tangeretin, sinesetin, quercetin, and their ether derivatives) from citrus fruits have been reported (Losso, 2003). 18.5.9 Terpenes and saponins Ganoderic acid F, an acid soluble triterpenoid from the fruit bodies of Ganoderma lucidum, at 800 g/mL, inhibited tumor metastasis by inhibiting VEGF and heparin catalyzed angiogenesis induced in Matrigel (Kimura et al., 2002). Humulone, a bitter acid from beer hop and inhibitor of the transcription of the COX-2 gene, dose-dependently inhibited in vitro tube formation of vascular endothelial cells, suppressed the proliferation of endothelial cells and the production of VEGF, and prevented in vivo angiogenesis in a CAM assay with an IC50 of 1.5 g/CAM (Shimamura et al., 2001). In vitro and in vivo experiments with rat models of human cancers showed that the cannabinoid terpene JWH-133 at 50 g/day inhibited angiogenesis by blocking vascular endothelial cell migration, suppressing VEGF, Ang2, and MMP-2 expression, and inducing apoptotic death of the cancer cells (Blazquez et al., 2003). Similar results were obtained using cannabinoid terpenes on mice models of human skin malignat cells (Casanova et al., 2003). Shibata (2001) reviewed the health-enhancing activities of Panax ginseng C.A. Meyet (Araliaceae) and reported that deglycosylated cyclic saponins or sapogenins from ginseng were more anti-angiogenic than their glycosylated saponins counterparts. The main component of essential oil from bay-leaves (Laurus nobilis L) is 1,8-cineole. The compound, time- and dose-dependently, induced apoptosis in human leukemia Molt 4B and HL-60 cells but not in human stomach cancer KATO III cells (Moteki et al., 2002). Monoterpenes such as limonene, perillic acid, perillyl alcohol, menthol, and pinene (at 0.25±2.5 mM) inhibited the growth of viral Ha-ras-transformed rat liver epithelial cells (WBras cells) (Ruch and Sigler, 1994). Trans retinoic acids and 9-cis retinoic acid, at concentrations as low as 10ÿ7 M, reduced bFGF-induced ETS-1 overexpression in endothelial cells and downregulated angiogenesis by suppressing the expression of MMP-1, MMP-3, MMP-9, u-PA and integrin beta3 in endothelial cells (Igarashi et al., 2001). All trans retinoic acid therapy inhibited VEGF production and suppressed angiogenesis in oral squamous cell carcinoma and in acute promyelocytic leukemia (APL), a distinct form of leukemia characterized by retinoic acid receptor (RAR) gene on chromosome 17 in all patients (Kini et al., 2001).

Anti-angiogenic functional food, degenerative disease and cancer 511 18.5.10 Dietary restriction Dietary restriction, although not a functional food, has been reported to promote apoptosis and reduce angiogenesis in brain tumors (Mukherjee et al., 2002). Dietary antioxidants with ability to modulate oxidative stress have shown efficacy to delay the aging process in animal models (Meydani, 2002). Angiogenesis in adipose tissue has been associated with overexpression of VEGF and rebound weight gain after a restricted diet was indicative of the possible correlation between angiogenesis and food intake (Morimura et al., 2001). Platz (2002) suggested that when energy intake is greater than demand, cellular proliferation, reduced apoptosis, and angiogenesis are enhanced. As a result energy imbalance where intake is greater than demand tends to favor increased levels of angiogenic stimulators such as IGF-I which has been associated with several tumors such as prostate cancer. None of the LW-rats fed a restricted diet and challenged with methylnitrosourea developed prostateseminal vehicle tumors while 20% of the full-fed controls developed P-SV tumors about one year later (Pollard, 1999).

18.6

Conclusion

Angiogenesis is a cascade of enzymatic reactions and enzyme inhibition appears to be the major molecular basis of anti-angiogenic functional foods. Understanding the biology and biochemistry of angiogenesis and identifying specific molecular targets of functional foods will assist in identifying functional foods with inhibitory activities to delay the onset and/or progression of lifethreatening diseases, and hopefully to specifically contribute to healthier life. The potential for anti-angiogenic functional foods as emerging preventive and/or therapeutic targets is clearly validated.

18.7

Future trends

The physiological and pathological events that form the basis for health and disease are, at their core, protein-driven processes (Aebersold and Cravatt, 2002). We are only beginning to appreciate the enormous potential of functional foods in preventing or delaying the onset and/or progression of degenerative diseases. In the postgenomic era which involves mostly proteomics, new strategies for assessing the effects of functional foods on angiogenesis will be needed. To date, the use of proteomics in functional foods research focuses on the five major enzyme classes including serine, cysteine, aspartic, and metalloproteases, and the proteasome. However, in the near future, interest will also focus on other classes of enzymes including (i) kinases and ATP-utilizing enzymes such as ATPase, G proteins, ligases, (ii) caspases that are critical in apoptosis process, (iii) signal transduction enzymes that are critical inside the cell. This will assist

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in studying the effects of functional foods on early stages of many diseases such as neurological, neoplastic, and infectious diseases at the protein translational level. Protein profiles to a given functional food or effective signature pattern will need to be established for various functional foods to assist in future studies of functional foods. Proteome analysis will be an essential part of functional food research because proteins influence eventual normal or diseased phenotypes, and therefore are the usual targets for disease prevention as opposed to mRNA. Research in anti-angiogenic functional foods in this new millennium will need to give priority to studies that seek to understand the basic molecular and genetic mechanism by which bioactive compounds in foods influence the various steps of the angiogenic process whether it is the excessive or the insufficient angiogenesis. A well-coordinated, multidisciplinary effort among scientists including nutritionists, food scientists, molecular biologists, biochemists, plant scientists, bioinformaticians, biologists, medical doctors, and clinical oncologists will be required to advance a molecular approach to anti-angiogenic functional foods (Losso, 2002). Anti-angiogenic functional foods present enormous challenges and opportunities, some of which will be overcome with current and future technological advances and some others may have to wait for a long period of time (Losso, 2003). Deciding the end point, in the absence of standard biomarkers, by which the effectiveness of functional foods can be measured, will be a challenge. Identification of molecular targets of functional foods, through in vitro and in vivo experiments, will assist in small clinical trials where the effects of functional foods will be correlated with appropriate biomarkers In vivo phage display technique has been touted to localize novel vasculature markers of degenerative diseases such as cancer, chronic inflammatory diseases, and others that can be used as targets for anti-angiogenic functional food delivery (Pasqualini et al., 2002). Using animal models of human degenerative diseases may help to achieve the in vivo phage display hypothesis. Consumption of functional foods, in appropriate amounts and on a regular basis, may be considered as a metronomic dosing approach with the objective of preventing the onset and/or progression of chronic diseases in healthy individuals. Metronomic dosing of functional foods may also be good for individuals with early stages of chronic diseases where few enzymes are active and may be more easily inhibited than when a full-blown pathological event is associated with a myriad of enzymes. The advent of anti-angiogenic functional foods is exciting and has beckoned in a new era of functional food research. An understanding of the biology of angiogenesis will assist food scientists and nutritionists in better utilizing anti-angiogenic functional foods in routine functional food formulations.

Anti-angiogenic functional food, degenerative disease and cancer 513

18.8

Sources of further information and advice

At the present time, the best sources of information appear to be MEDLINE database, Google.com, msn.com, and The Functional Foods Division of the IFT. The Journal Angiogenesis (Kluwer Academic Publishers, Dordrecht, The Netherlands) is an international peer-reviewed journal for rapid communication of top-quality papers on innovative experimental studies covering in vitro, animal model systems, and clinical investigations of angiogenic disease research including anti- and pro-angiogenic compounds and therapeutic approaches, new markers, and techniques for disease diagnosis and prognosis. The weekly journal Angiogenesis Weekly (NewsRx Publisher, Atlanta, GA , USA. ISSN: 1531-6416 or http://www.newsrx.com) is a weekly publication of the most recent research on Angiogenesis. The publisher provides a reader's digest and the reference to the original paper. This is so far the best link to the latest on Angiogenesis research.

18.9

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(2002), `Hypoxia as a target for combined modality treatments', Eur J Cancer, 38(2), 240±257. YIN F, GIULIANO AE, VAN HERLE AJ (1999a), `Signal pathways involved in apigenin inhibition of growth and induction of apoptosis of human anaplastic thyroid cancer cells (ARO)', Anticancer Res, 19(5B), 4297±4303. YIN F, GIULIANO AE, VAN HERLE AJ (1999b), `Growth inhibitory effects of flavonoids in human thyroid cancer cell lines', Thyroid, 9(4), 369±376. YONEDA M, KATSUMATA K, HAYAKAWA M, TANAKA M, OZAWA T (1995), `Oxygen stress induces an apoptotic cell death associated with fragmentation of mitochondrial genome', Biochem Biophys Res Commun., 209(2), 723±729 ZGOURAS D, WACHTERSHAUSER A, FRINGS D, STEIN J (2003), `Butyrate impairs intestinal tumor cell-induced angiogenesis by inhibiting HIF-1alpha nuclear translocation', Biochem Biophys Res Commun, 300(4), 832±838. ZHOU, JR, MUKHERJEE P, GUGGER ET, TANAKA T, BLACKBURN G L, CLINTON S K (1998), `Inhibition of murine bladder tumorigenesis by soy isoflavones via alterations in the cell cycle, apoptosis, and angiogenesis', Cancer Res., 58, 5231±5238. P

19 Synbiotics and colon cancer M. F. Bennett, Y. E. Clune, F. Shanahan, G. O'Sullivan, J. K. Collins, University College Cork, Ireland

19.1

Introduction: probiotics, prebiotics and synbiotics

Colorectal cancer is the second commonest cause of death from cancer in developed countries. Despite advances in current therapies there has been very little increase in survival in patients with advanced disease. This has led to increased research in tumour biology and genetics and the search for new treatment modalities. In recent years there has been much interest in the influence of the normal gastrointestinal (GI) microflora on host health and in particular the role it plays in cancer initiation and progression. Modulation of the GI flora is a potential mechanism for influencing the process of carcinogenesis. Therefore the study of the potential use of prebiotics, probiotics and synbiotics in cancer prevention is a rapidly developing area. An added advantage of this approach is the absence of severe side effects, the ease of administration and the attractiveness of a more natural prevention strategy to subjects at risk. The aim of this chapter is to discuss the history and development of prebiotics and probiotics, and the more recent development of synbiotics, to describe the distribution and the functions of the normal flora of the gastrointestinal tract and methods employed in their study. We discuss the aetiology of colon cancer including genetic and environmental factors and the clinical management of this disease. We also discuss the composition and functions of the gut microflora and the effect of pre-, pro- and synbiotic consumption on this and suggest possible uses of pre, pro- and synbiotics in the prevention and treatment of colon cancer. A food can be said to be functional if it contains a component (which may or may not be a nutrient) that affects one or a limited number of functions in the body in a targeted way so as to have positive effects on health, or it has a physiologic or psychologic effect

Synbiotics and colon cancer 525 beyond the traditional nutritional effect. As such pre-, pro- and synbiotics are considered functional foods as they have been shown to exert positive health effects (Roberfroid, 2000). 19.1.1 Probiotics It was at the beginning of the 20th century that Metchnikoff first suggested the concept of what we now know as probiotics (Metchnikoff, 1907). His hypothesis was that the complex microbiota of the colon was having an adverse effect on the host through what he termed the `autointoxication effect'. Based on his observation that Bulgarian peasants who ingested large quantities of sour milk exhibited longevity of life, he advocated the ingestion of sour milk or fermented products in order to modify the activity of the colonic microflora suppressing the putrefactive-type fermentation of the intestinal flora. In the early 1900s there was much interest in the health benefits of consumption of fermented dairy products. In fact, it was during this period that some of the companies still associated with the production of yoghurt and fermented milk products (Danone and Yakult) were founded. (www.yakult.co.uk, www.danoneinstitute.org) The word probiotic is derived from the Greek meaning `for life'. The term was first used in 1965 by Lily and Stilwell, to describe substances secreted by one protozoan to stimulate the growth of another (Lily and Stilwell, 1965). In 1989 Fuller defined a probiotic as a `live microbial feed supplement, which beneficially affects the host animal by improving its intestinal microbial balance' (Fuller, 1989). The definition was again revised in 1992 by Havenaar and Huis in 't Veld (1992) who defined a probiotic as `a mono- or mixed-culture of live microorganisms which when applied to man or animal affects beneficially the host by improving the properties of the indigenous microflora'. However, the most accurate definition of a probiotic, was proposed by Salminen et al. (1999): `Probiotics are microbial cell preparations or components of microbial cells that have a beneficial effect on the health and well-being of the host.' This definition was based on research that showed that probiotics need not be viable in order to exert an effect and also that some beneficial probiotic effects were non-microflora mediated. Participants involved in the Lactic Acid Bacteria Industrial Platform (LABIP) workshop on probiotics concluded that `probiotics may be consumed either as a food component or as a non-food preparation'. Criteria, outlined by others, for use in the assessment of potential probiotics were summarised by Dunne et al. (1999) and a definitive set of selection criteria for probiotics was put forward. Probiotic micro-organisms should: · be of human origin · demonstrate non-pathogenic behaviour · exhibit resistance to technological processes (i.e., viability and activity in delivery vehicles) · prove resistant to gastric acid and bile

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adhere to gut epithelial tissue be able to persist, albeit for short periods, in the gastrointestinal tract produce antimicrobial substances modulate immune responses have the ability to influence metabolic activities (e.g., cholesterol assimilation, lactase activity, vitamin production).

Probiotics are generally lactic acid producing bacteria (LAB), and are primarily of the genus Lactobacillus and Bifidobacterium. However, some non-LAB bacteria and yeasts are considered as having probiotic properties, Table 19.1. Probiotic bacteria have been shown to lower the frequency and duration of diarrhoea associated with antibiotic therapy (Pochapin, 2000; D'Souza et al., 2002), rotavirus infection (Majamaa et al., 1995; Guandalini et al., 2000), chemotherapy, and, to a lesser extent, traveller's diarrhoea (Katelaris et al., 1995; Hilton et al., 1997). Probiotics have also demonstrated the ability to stimulate the host immune system (Schiffrin et al., 1997; Erickson and Hubbard, 2000) to improve gut barrier function (Madsen et al., 2001; Isolauri et al., 1993), alleviate symptoms of lactose intolerance (Montes et al., 1995; Shermak et al., Table 19.1 Microorganisms considered as probiotics (adapted from Holzapfel et al., 2001) Lactobacillus species Bifidobacterium species

Other lactic acid bacteria

Nonlactic acid bacteria

L. acidophilus

B. adolescentis

L. amylovorus

B. animalis

L. casei

B. bifidum

Enterococcus faecalis Enterococcus faecium Lactococcus lactis3

L. crispatus

B. breve

L. delbrueckii

B. infantis

subsp. bulgaricus3

B. lactis4

Bacillus cereus var. toyoi1,2 Escherichia coli strain nissle Propionibacterium freudenreichii1,2 Saccharomyces cerevisiae2 Saccharomyces boulardii2

L. gallinarum1

B. longum

L. L. L. L. L. L. 1 2 3 4

gasseri johnsonii paracasei plantarum reuteri rhamnosus

Leuconstoc mesenteroides Pediococcus acidilactici3 Sporolactobacillus inulinus1 Streptococcus thermophilus3

Main application for animals. Applied mainly as pharmaceutical preparations. There is either little known about the probiotic properties or the microorganism is nonprobiotic. Probably synonymous with B. animalis

Synbiotics and colon cancer 527 1995), reduce blood pressure in hypertensive subjects, reduce cholesterol and triacylglycerol plasma concentrations, (Taranto et al., 1998; Kiessling et al., 2002) and, as covered in this review, play a part in cancer prevention, (Wollowski et al., 2001; Rafter, 2002; Gallaher and Khil, 1999). 19.1.2 Prebiotics Delzenne and Roberfroid (1994) proposed the term prebiotics for the nondigestible oligosaccharides (NDO) used as food ingredients to modify the composition of endogenous gut flora. The definition was further developed by Gibson and Roberfroid (1995b) who defined a prebiotic as `a non-digestible food ingredient that benefits the host by selectively stimulating the growth or activity of one or a limited number of bacteria in the colon'. Any food ingredient that enters the large intestine may be considered a candidate prebiotic, however it is the selectivity of the fermentation in the presence of the diverse microflora that is the defining feature. Collins and Gibson (1999) stated that in order to classify a food ingredient as a prebiotic, it must be: · neither hydrolysed nor absorbed in the upper part of the gastrointestinal tract · a selective substrate for one or a limited number of potentially beneficial commensal bacteria in the colon, thus stimulating the bacteria to grow, become metabolically activated, or both · able as a consequence to alter the colonic microflora toward a healthier composition. Examples of prebiotics are specific oligosaccharides, starch fractions (resistant starch), pectins, lactose, lactulose, lactitol, sorbitol and xylitol. Prebiotics exhibit a dietary fibre-like action. Fermentation of prebiotics lowers the intestinal pH, leading to a decrease in the activity of the enzyme 7-hydroxylase responsible for the formation of secondary bile acids. Secondary bile acids comprise over 80% of faecal bile acids and are thought to play an important role in colon carcinogenesis by acting as promoters in tumour formation (Nagengast et al., 1995). One of the most interesting aspects of prebiotics, is that they enhance the growth of bifidobacteria and other Gram-positive bacteria but are not fermented by the majority of Gram negative bacteria in the colon. It is thought that prebiotics have laxative properties similar to carbohydrates. Carbohydrates that reach the large intestine, such as non-starchy polysaccharides and resistant starch, are known to have a laxative effect on bowel function. Stimulation of microbial growth leads to increased bacterial mass and bowel content, which leads to stimulation of peristalsis, decreased transit times and prevention of constipation. 19.1.3 Synbiotics It was previously thought that in order for a probiotic to exert a beneficial effect on the host, the bacterium must be metabolically active in the gut. However, in recent

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years it has been found that it is not always necessary for the probiotic to be metabolically active, although in some cases this property may be desirable. Within the colon the supply of fermentable carbohydrate is a growth-limiting factor for bacteria. The bacterial species which occur in the greatest numbers are those that can utilise the available nutrients most effectively. As discussed previously, prebiotics resist digestion in the upper gastrointestinal tract and reach the colon intact, where they are available as nutrients for the colonic flora. Current research indicates that consumption of prebiotics with probiotic strains enhances the growth of the introduced strain in the gut. This is known as the synbiotic effect and the term synbiotic is applied to a product, which contains both prebiotics and probiotics. In defining a synbiotic, Schrezenmeir and de Vrese (2001) stated, `because the word alludes to synergism, this term should be reserved for products in which the prebiotic compound selectively favours the probiotic compound.' In the strictest sense, therefore, a product containing oligofructose as a prebiotic and bifidobacteria as a probiotic would fit the definition of a synbiotic but a product containing oligofructose and a lactobacillus strain would not. However, it may be argued that promotion of the growth of indigenous bifidobacteria in vivo, combined with the introduction of a lactobacillus strain and subsequent beneficial effects on the health of the host fulfils the definition of synergism. One of the principal benefits of synbiotics is believed to be increased persistence of the probiotic in the GIT. Consumption of a prebiotic in a synbiotic compound provides a readily fermentable carbohydrate source for the introduced probiotic strain, giving the probiotic a competitive advantage over the indigenous flora. This advantage increases the persistence of the probiotic in the GIT increasing the time period in which the probiotic strain can exert its influence.

19.2

Gut microflora

The human gastrointestinal tract consists of the oral cavity, oesophagus, stomach, small intestine (duodenum, jejunum, ileum), large intestine (caecum, colon) and the rectum. The mucosal surface of the adult human gastrointestinal tract measures up to 300 m2, making it the largest body area in contact with the external environment. In comparison the skin surface measures only 2 m2. The large surface area in the gastrointestinal tract is achieved by folding of the mucosal surface. Circular folds give a threefold increase in surface, folding of the epithelium (villi), cause a seven to tenfold increase and the formation of microvilli in the enterocyte resorptive membrane a further 15±40fold increase (Holzapfel et al., 1998). This large surface area provides the required space for the interactions of the digestive process and allows bacterial adhesion to the mucosal wall and colonisation. Savage (1977) described the normal flora as being composed of the autochthonous and allochthonous flora. The autochthonous flora refers to microorganisms that natively colonise a particular habitat or environmental niche and are present in all communities of a particular animal species. The allochthonous

Synbiotics and colon cancer 529 flora refers to microorganisms that cannot colonise a particular habitat, except under abnormal circumstances. However these terms are little used today and the terms normal and indigenous flora are used interchangeably to describe the microorganisms that normally inhabit the gastrointestinal tract. 19.2.1 Development of the gastrointestinal flora Sterile at birth, colonisation of the GIT of newborns begins immediately after birth. Certain microorganisms colonise particular intestinal habitats at various times following birth that are characteristic of that particular habitat and host. This is a process known as bacterial succession. Factors that influence the composition of the flora in newborns are (a) delivery method (passage through the birth canal versus caesarean section); (b) diet (breast fed versus formula fed). Bacterial populations develop during the first day of life. Cooperstock and Zedd (1983) divided the development of the intestinal microflora in infants into four phases. Phase Phase Phase Phase

1 2 3 4

weeks 1±2, initial acquisition breast-feeding alone introduction of supplementation and cessation of breast-feeding cessation of breast-feeding and conversion to adult-like flora.

Not surprisingly, facultative anaerobic strains such as E.coli and streptococci initially exist in the highest numbers. It is thought that these bacteria create a reduced environment allowing the establishment of anaerobic bacteria such as bacteroides, bifidobacteria and clostridia generally by day 4±7 (Stark and Lee, 1982). In breast-fed infants there is a sharp increase in the numbers of bifidobacterium with a concomitant decrease in the numbers of E.coli and streptococci, with clostridial numbers being low or absent. In formula-fed infants this change does not occur and the composition of the GI flora becomes rather complex with high numbers of bacteroides, clostridium and streptococcus. Bifidobacteria are present but in much lower numbers than in breast-fed infants. During phases 3 and 4, after the introduction of solid food and weaning, the flora becomes similar to the normal adult flora, and the composition of the flora remains relatively stable throughout life but is distinct in different individuals, (Holzapfel et al., 1998). Breast-fed infants have been shown to have a lower risk of gastrointestinal infections than formula-fed infants. Infants delivered abdominally have far fewer lactobacilli in the early stages of life than those delivered vaginally (Collins and Gibson, 1999). 19.2.2 Microbial distribution in the gastrointestinal tract Within the gastrointestinal tract of an adult human the numbers and types of bacteria vary in different compartments (see Fig. 19.1). The oral cavity contains an indigenous flora of about 200 species. Saliva contains transient bacteria shed from the oral surfaces such as tongue and cheek and numbers may exceed 109 cells/ml. However, most of these are washed into the stomach with saliva and

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Fig. 19.1 Gut microflora (adapted from Berg, 1996).

most research of GIT flora centres around the stomach, small and large intestines and faecal matter. The human stomach contains between 101 and 103 cfu/ml of contents. The low numbers in this habitat are due principally to the low pH of the stomach (pH 2.5 to pH 3.5) which, after the more or less neutral environment of the oral cavity is destructive to most microorganisms and may also be due to the swift flow (peristalsis) through the stomach and small bowel. However numbers in this region may increase temporarily 100 to 1000fold following a meal due to increases in pH (Lambert and Hull, 1996). Acid tolerant Gram-positive species such as streptococci and lactobacilli predominate in the stomach and duodenum. Helicobacter pylori a significant causative agent of gastritis in adults, thrives on the mucosa of the stomach (Berg, 1996). The duodenum is also subject to short transit times which in addition to biliary and pancreatic secretions give rise to a hostile environment for transiting microorganisms. It is therefore not until the distal small intestine (Ileum) that the numbers and diversity of microorganisms increase significantly. Bacterial numbers of 108 cfu/ml of intestinal contents may be found in this zone, mainly species of streptococci, lactobacilli, bifidobacteria, bacteroides, fusobacteria and enterobacteriaceae (Nielsen et al., 1994). Below the ileocecal valve, the large intestine (colon) is the primary site of microbial colonisation in humans and animals, possibly because of the low

Synbiotics and colon cancer 531 oxidation-reduction potentials and the slow intestinal motility in this area (transit times up to 60 hr) (Tannock, 1995). Here bacterial numbers exceed 1010± 1012 cfu/g and represent 40±55% of luminal contents. It is thought that greater than 400 different species inhabit the colon, however a considerable number of these remain unculturable using existing culture techniques. It is also important to note that 99.9% of the indigenous flora of the large intestine, are obligate anaerobes. Genera such as Bacteroides, Eubacterium, Bifidobacterium and Peptostreptococcus dominate at levels of approx. 1010±1011/g. Enterobacteriaceae, streptococci and lactobacilli are also present at levels of 108cfu/ g, (Naidu et al., 1999). Faecal samples are most often used to study the colonic flora. However, the bacteria found in faecal samples represent the luminal environment and may differ greatly from the flora of the mucosa. The faecal flora may indicate the bacteria present in the distal colon but is not a true indication of the flora of the transverse and ascending colon and certainly not of the flora of the small intestine. The typical vertical distribution of the indigenous flora throughout the gastrointestinal tract is described above, however there is also a characteristic horizontal distribution of indigenous species from the GI lumen to the mucosal epithelium. Four microhabitats in the GI tract have been outlined by Freter (1992): 1. 2. 3. 4.

the the the the

surface of the cells of the epithelium crypts of the ileum, caecum and colon mucus gel that overlays the epithelium lumen of the intestine.

Based on this hypothesis certain bacteria live freely in the lumen, others colonise the crypts, while others interact with epithelial cell surfaces. The obligate anaerobes associate intimately with the gut wall to form layers on the mucosal epithelium (Berg, 1996). 19.2.3 Unculturables As mentioned earlier, there are more than 400 different species of bacteria inhabiting the colon, of which a considerable number are unculturable using existing culture techniques. Comparison of results from electron microscopic investigation of the gastrointestinal flora and traditional culture counts demonstrated that culture methods yielded between 50 to 80% of the total microscopic counts. Molecular methods have indicated that 60±80% of the organisms in the total human microflora have not been cultivated (Langendijk et al., 1995). Traditional culture techniques depend on the use of selective media for the enumeration of bacterial genera or species of interest. However, such techniques provide a limited view of the diversity of the flora. Advances in molecular biology have produced the tools required to study the diversity of complex bacterial environments without the use of traditional culture methods. 16S ribosomal RNA (16S rRNA) gene sequences contain regions conserved across

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all bacterial species interspersed with regions in which sequences are variable among bacterial types, (Stackebrandt and Rainey, 1995). Molecular methods based on amplification by the polymerase chain reaction (PCR) of the 16S rRNA gene sequences from microbial DNA extracted from faecal samples and the gastrointestinal tract are contributing to our understanding of the diversity of the gastrointestinal ecosystem. Two such methods are denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE). DGGE is based on electrophoresis of PCR-amplified 16S rDNA fragments in polyacrylamide gels containing linearly increasing gradients of denaturants allowing DNA fragments of the same length but with different base-pair sequences to separate, (Muyzer et al., 1993). TGGE separates rDNA fragments in polyacrylamide gels with a temperature gradient. With both DGGE and TGGE specific DNA fragments can be excised from the gel and sequenced to identify the particular bacterial species identified by the particular band. Fluorescent in situ hybridisation (FISH) is also based on the 16S rRNA region. rRNA targeted oligonucleotide probes specific for phylogenetic groups are used for the in situ identification of individual bacterial cells in a complex environment (Amann et al., 1995). Suau et al. (1999) investigated the bacterial diversity within an adult-male faecal sample using comparative analysis of cloned 16S rRNA gene sequences. They found that the majority of the generated rDNA sequences (76%) did not correspond to known organisms. 19.2.4 Functions of the gut microflora With more bacterial cells in the gut than eukaryotic cells in the entire body, the GIT microflora has often been described as the forgotten organ. The collective metabolic activity of the normal flora would rival that of the activity of the liver (Bocci, 1992; Berg, 1996). The functions of the intestinal microflora contribute to the health of the host in many ways. The important effects that the gut microflora exerts on its host have been revealed through the comparative studies of animals bred under germ-free conditions and conventional animals with a normal indigenous flora. Such studies have shown that the microflora has an influence on the physiological and immunological characteristics of the host. The beneficial activities of the normal gut flora include vitamin synthesis (Hill, 1997), and improved absorption of minerals by the host, e.g., calcium, magnesium and iron (Miyazawa et al., 1996; Younes et al., 2001). The flora is also involved in the conversion of pro-drugs to their active form and the metabolism of carcinogens ingested in the diet to both active and inactive compounds. It is thought that the composition of the flora influences whether the carcinogen is metabolised to its active or inactive form. A major source of energy within the colon is the fermentation of carbohydrates by the colonic flora the endpoint of which is the production of short-chain fatty acids (SCFA). Acetate, propionate and butyrate are the principal SCFA produced in the colon. Briefly, acetate traverses the liver and enters the peripheral circulation. Propionate is extracted by the liver and effects

Synbiotics and colon cancer 533 lipid metabolism (Bridges et al., 1992). Butyrate appears to have health promoting effects for colonocytes and only a portion of butyrate leaves the colon for extraction by the liver. Butyrate has been associated with many biological properties in the colon and is thought to directly enhance cell proliferation in normal cells and suppress proliferation in transformed cells. In addition, apoptosis may be increased in transformed cells but inhibited in normal cells when butyrate is present (Hague et al., 1995; Marchetti et al., 1997; Hass et al., 1997). Butyrate is known to reduce risk factors involved in the aetiology of colon cancer and adenoma development (Smith et al., 1998). The normal flora plays an important role as a barrier against pathogenic and opportunistic infection. This barrier effect is best demonstrated when the composition of the normal flora is altered by broad-spectrum antibiotic therapy. In such cases bacterial overgrowth particularly by Clostridium difficile occurs causing diarrhoea and other symptoms.

19.3

Colon cancer

Colorectal cancer is the second commonest cause of death from cancer in developed countries. The incidence of colorectal cancer increases steeply after the age of 40 and exponentially thereafter. An individual's lifetime risk of developing colorectal cancer is estimated at 5% and individuals with first-degree relatives diagnosed with colon cancer or adenomatous polyps, have a risk two to three times greater than this. In 1998 the age-standardised incidence of colorectal cancer in the European Union was 29.82 cases per 100,000 (as standardised to world figures), UK rates were 27.61 per 100,000 and Irish figures were 34.12 per 100,000. Mortality rates for the same year were 13.63 per 100,000 for the E.U., 13.52 per 100,000 for the U.K. and 15.22 per 100,000 for Ireland (EUCAN, 1999). Rates of colorectal cancer in developing countries are much lower than in developed countries. The incidence for males in 1998 in more developed countries is 37.3 per 100,000 people, compared to 9.91 per 100,000 in less developed countries (Globocan, 2001). Several hypotheses exist to explain this dramatic difference, including dietary intake and infectious challenges in childhood. The interaction of bacterial endotoxins and enterotoxins with the intestinal epithelium in early life is thought to influence colon carcinogenesis, reducing the risk in later life and this may explain the differences between these populations (Pitari et al., 2003). Genetics, experimental and epidemiological studies suggest that colorectal cancer results from a complex interaction between inherited susceptibility and environmental factors. 19.3.1 Fearon±Vogelstein cascade The adenoma-carcinoma sequence was first postulated in the 1980s by Fearon and Vogelstein and is thought to account for up to 90% of cases of colorectal

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Fig. 19.2

Genetic model for colorectal tumourigenesis (adapted from Fearon and Vogelstein, 1990; Gill and Rowland, 2002).

cancer (Vogelstein et al., 1988). This cascade refers to the multi-step progression from normal epithelium to hyperproliferative epithelium, to focally dysplastic crypts, to tubular adenoma, to dysplastic or villous adenoma and finally onto invasive carcinoma and metastasis (see Fig. 19.2). Evidence supporting this sequence includes the common finding of foci of cancer in adenomatous polyps, the similar colonic distribution of adenomas and cancer, the high correlation between the incidence of cancer and the prevalence of adenomas, and the reduction in the incidence of cancer seen following adenoma removal (US National Polyp Study) (Winawer et al., 1993). The multigene clonal evolution and selection model of the initiation and progression of colon cancer proposed by Fearon and Vogelstein originally identified the adenomatous polyposis coli (APC) gene, genes on 18q, and p53 as the genes in which mutations or epigenetic dysregulation contributes to evolution of colon cancer (Fearon and Vogelstein, 1990). This multistep model involves biallelic inactivation mutations in the APC tumour suppressor gene as an early event leading to the development of polyps, followed by inactivating mutations in the oncogene K-ras in the advancing adenoma, and biallelic inactivating mutations of p53 during the transition to malignancy in most colorectal cancers (Fearon, 1990; Vogelstein et al., 1988). The types of genomic alterations included point mutations, and allelic loss events known as loss of heterozygosity (LOH). This cascade is the classical sequence of events, however, not all mutations are necessary for tumour formation.

Synbiotics and colon cancer 535 19.3.2 Genetics The aetiology of colorectal cancer is heterogeneous, with environment or genetics playing varying key roles in different patients (Potter, 1999). About 80% of patients with colorectal cancer seem to have sporadic disease with no evidence of having inherited the disorder. In the remaining 20%, there seems to be a potentially definable genetic component. In the past decade, germline genetic mutations conferring a high lifetime risk of colorectal cancer in carriers have been found, accounting for 5±6% of all colorectal cancer cases. Other gene mutations, some with lower lifetime risks are continuing to be characterised. 19.3.3 Familial adenomatous polyposis (FAP) FAP is an autosomal dominant syndrome caused by germline mutation of APC (adenomatous polyposis coli) gene on chromosome 5q21 (Nishisho et al., 1991; Kinzler et al., 1991; Groden et al., 1991). The APC gene is a tumour suppressor or `gate keeping' gene that encodes a protein considered important in cell adhesion, signal transduction, and transcription activation. Beta catenin and c-myc have been identified as downstream targets (Kinzler and Vogelstein, 1998). Mutations of APC in FAP include insertions, deletions, and nonsense mutations that lead to frame shifts or premature stop codons, resulting in truncation of the APC gene product. Multiple colonic adenomas occur at an early age in patients with FAP, occasionally during the preteen years, and proliferate throughout the colon, with malignant degeneration in most patients by the age of 40 to 50 years. As the disease advances prophylactic subtotal colectomy is recommended. Chemoprevention with selective Cox-2 inhibitors has been shown to reduce the number of colonic polyps in FAP patients, but these agents should not replace screening. However, they may have a role in delaying the need for prophylactic colectomy (Lynch et al., 1995). 19.3.4 Hereditary nonpolyposis colorectal cancer (HNPCC) In 1966, Dr Henry Lynch and colleagues described familial aggregation of colorectal cancer with stomach and endometrial tumours in two extended kindreds and termed it Cancer Family Syndrome (Lynch et al., 1966), later, authors designated this constellation Lynch syndrome. More recently, this condition has been called HNPCC. The original definitions of the condition were based on clinical and pedigree criteria ± the Amsterdam Criteria. In HNPCC, unlike FAP, colorectal cancer usually arises from a single colorectal lesion in the absence of polyposis (Vasen et al., 1995; Lynch et al., 1993; Boland, 1998a). HNPCC is an autosomal dominant disorder accounting for about 3±5% of all colorectal cancer cases caused by mutation of one of the DNA mismatch repair genes (Mecklin, 1987; Rodriguez-Bigas et al., 1997; Aaltonen et al., 1998). Individuals with a HNPCC gene mutation have approximately a 70±80% lifetime risk of developing colon or rectal cancer (Scapoli et al., 1994; Vasen et al., 1996).

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Tumours are often poorly differentiated, with excess of mucoid and signetcell features and have a Crohn's-like reaction with infiltrating lymphocytes within the tumour (Smyrk et al., 2001; Alexander et al., 2001; Jass et al., 1998; Jass and Stewart, 1992). Accelerated carcinogenesis occurs where a colonic adenoma may emerge as a carcinoma within two to three years, as opposed to eight to ten years (Lynch and de la Chapelle, 1999; Jass and Stewart, 1992). Molecular screening is now both feasible and desirable in this condition. 19.3.5 Microsatellite instability (MSI) Exogenous and endogenous reagents constantly modify the genomic DNA of all organisms (Friedberg et al., 1995). In addition, some pathways of DNA metabolism such as DNA replication also modify the genetic material by introducing errors into the newly synthesised strands. The cells of all organisms have evolved highly sophisticated and efficient mechanisms that maintain the integrity of the genomes, these include; base excision repair, nucleotide excision repair, mismatch repair or recombination repair. Malfunction of mismatch repair in humans was first identified in 1993, in tumours of the colon, endometrium, ovary and other organs targeted by the hereditary non-polyposis syndrome (Aaltonen et al., 1993). Microsatellite instability is the hallmark of mismatch repair deficiency. A microsatellite is a repeated DNA sequence in which the length of the core sequence that is repeated is small (one to five nucleotides). Microsatellite instability describes the emergence of new microsatellite alleles within a tumour, of lengths that differ from the host's two parent (i.e. normal tissue) alleles for that microsatellite. Since MSI is found in virtually all hereditary nonpolyposis colorectal tumours (Aaltonen et al., 1994), it is unnecessary to search for germ-line mutations in mismatch repair genes in patients whose tumours do not have MSI. An exception is found in families with the MSH6 (mutS homolog 6) mutation, in which MSI may or may not be present (Miyaki et al., 1997; Wijnen et al., 1999). Most microsatellites occur in non-coding DNA; therefore, contractions or elongations are believed to have little or no effect on protein function. However, there are genes that have microsatellites in their coding regions, and MSI will thus lead to altered proteins. Recently, investigators found that MSI, as a marker for the mismatch repair pathway of colorectal neoplasia, is associated with improved survival, stage-forstage, in those sporadic patients with MSI-positive tumours (Gryfe et al., 2000; Halling et al., 1999). In addition, one study reported that adjuvant chemotherapy resulted in greater survival for patients with MSI-positive tumours (Elsaleh et al., 2000). MSI can be subdivided according to levels of instability in specific areas. These areas consist of a panel of five microsatellite loci that contain two mononucleotide repeats and three dinucleotide repeats. Cancers can then be separated into groups: MSI-High demonstrate instability in >30±40% of loci investigated; whereas MSI-Low demonstrate instability in <30±40% of loci (Boland et al., 1998). MSI-High is found in over 90% of HNPCC and 15% of sporadic cancers (Aaltonen et al., 1993; Liu et al., 1996). Due to the prevalence

Synbiotics and colon cancer 537 of sporadic colorectal malignancies, the majority of MSI positive cancers are not from patients with HNPCC. MSI positive colorectal cancers from HNPCC or sporadic cases have similar histological and biological features (Bubb et al., 1996). They present at an earlier stage than MSI-Low cancers and have a lower incidence of nodal disease or distant metastasis (Watson et al., 1998; Guillem et al., 1999; Percesepe et al., 1997). MSI-H cancers occur more frequently in the right side of the colon with around 66% of HNPCC and up to 90% of sporadically occurring MSI positive cancers being detected proximal to the splenic flexure (Jass, 2000; Kim et al., 1994a; Forster et al., 1998). They are more likely to be poorly differentiated, have a mucinous component or demonstrate a signet ring cell configuration (Mecklin and Jarvinen, 1986; Mecklin et al., 1986; Myrhoj et al., 1997; Lynch et al., 1993; Giacherro et al., 1985). MSI positive cancers often exhibit extensive infiltration by lymphocytes (TIL), predominantly of cytotoxic T cells (Dolcetti et al., 1999) and an accumulation of B and T cells around the tumour, similar to that seen in Crohn's disease (Jass, 2000). 19.3.6 Sporadic cancer Colorectal cancers involving the distal colon are more likely to have aneuploid DNA, harbour mutations in the APC, p53, K-ras genes and behave more aggressively; proximal colorectal cancers are more likely to have diploid DNA, possess microsatellite instability, harbour mutations in mismatch repair (MMR) genes, and behave less aggressively, as in HNCC, whereas FAP and sporadic cancer represents the former class of distal tumours (Lynch and de la Chapelle, 1999). Genes involved in the cascade initially proposed by Fearon and Vogelstein have been confirmed to be affected in sporadic cancers. But mismatch repair genes have also been implicated. A mutation in one of the three main MMR proteins occurs, which promotes mutations in some of the more traditional genes (Lynch and de la Chapelle, 2003). Genes in these cancers with coding microsatellites accumulate frame shift mutations and lose function, further affecting, and perhaps speeding up the evolution of cancer. 19.3.7 p53 p53 was first identified in 1979 as a protein that complexes with viral oncogenes such as SV40 large T-antigen, adenovirus E1B and papilloma virus E6, (Levine, 1990). p53 or part of its regulatory circuit is functionally inactivated in almost all cancers highlighting its importance in preventing tumourigenesis (Jin and Levine, 2001), and p53 is referred to as the `guardian of the genome' because of its role in checkpoint control following DNA damage (Lane, 1992). DNA may be damaged by UV radiation, (sunlight) or chemical carcinogens (e.g. diet). Such damage causes p53 induction and the levels of p53 protein in the cell increases. Increased p53 halts cell division in the late G1 phase, allowing the cell time to repair its DNA, and to ensure that a correct copy of each gene is present

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before allowing the cell cycle to continue (Kuerbitz et al., 1992). Increased p53 has been shown to cause apoptosis in some cells, however, the reason for this is not yet understood (Yonish-Rouach et al., 1991). p53 therefore ensures that the correct genetic information is present in the cell before allowing the cell to replicate. The role of p53 in tumourigenesis is highlighted in the Li-Fraumeni Syndrome (LFS), which will be discussed later (section 19.3.8). p53 itself is not mutated in all cancers, however, mutations may be present in genes involved in the p53 regulatory circuit. p53 regulates its own expression through feedback loops. One such regulatory mechanism works through a gene called mdm2. Following DNA repair p53 activates mdm2, and the mdm2 protein product binds to and inhibits p53 allowing the cell cycle to continue, (Wu et al., 1993). To induce apoptosis p53 transactivates the bax gene. There are many genes involved in the p53 functional circuit, impairment of whose function may play a part in tumourigenesis (Miyashita and Reed, 1995). 19.3.8 Li-Fraumeni Syndrome (LFS) LFS was first identified by, Drs Fred Li and Joseph F.Fraumeni Jr at the National Cancer Institute in 1969, (Li and Fraumeni, 1969). It is a rare familial dominantly inherited cancer syndrome characterised by a wide spectrum of neoplasms occurring in children and young adults. By definition it is the diagnosis of a sarcoma in an individual before the age of 45 who has at least two first-degree relatives who have cancer of any type diagnosed before the age of 45, (Li and Fraumeni, 1969; Li, 1988). The syndrome is unusual in that cancer may develop at a very young age, individuals often develop multiple primary tumours, and there is a high frequency of cancer within the family. Soft-tissue sarcoma, osteosarcoma, brain tumours, adrenocortical carcinoma, leukaemia and early onset breast cancer are the most common cancers in LFS. In 1990 the major cause of the predisposition to cancer in LFS families was identified as germline mutations in the p53 gene, (Srivastava et al., 1990). Although inherited p53 mutations are present in all somatic cells of affected individuals of LFS families, tumours form only in some organs or tissues. This is because loss of function of both alleles of p53 is necessary before a tumour develops. Also mutations in p53 alone will not produce a tumour, as the development of cancer involves the accumulation of multiple genetic alterations. The rate at which these changes accumulate in different tissues in addition to the inherited p53 mutation may explain the distribution of tumours in different tissues as seen in LFS. In LFS it is rare that colon cancers are the first tumour to develop, however, they may occur as second or third cancers. 19.3.9 Hamartomatous polyposis syndromes Peutz-Jeghers syndrome consists of perioral pigmentation, upper and lower gastrointestinal hamartomatous lesions, small bowel and pancreatic cancers, colorectal cancer and sex-cord tumours of the ovary. The mutant gene in this

Synbiotics and colon cancer 539 syndrome is LKB1 (Hemminki et al., 1998). Several other syndromes of this type exist including familial juvenile polyposis, Cowden's disease, and Bannayan-Ruvalcaba-Riley syndrome. 19.3.10 Multiple drug resistance (MDR) Efflux mechanisms have become the subject of considerable interest in recent studies of gut mucosal defence and pharmacokinetics of drugs. The efflux mechanism in the intestinal epithelium is mediated by the multidrug resistance pump (MDR1), also known as P-glycoprotein 170. The MDR 1 gene may have an important role in the tumourigenesis of colorectal cancer. It is hypothesised that overexpression of MDR1 may influence apoptotic pathways by secreting endogenous or xenobiotic toxic substances into the lumen or by mediating a more generic antiapoptotic function. The MDR 1 gene product, P-glycoprotein, protects the cell against a wide variety of caspase-dependent death stimuli including fas ligand, tumour necrosis factor, and ultraviolet radiation (Smyth et al., 1999). In colorectal tumours expression of P- glycoprotein correlated with pathological grading of tumours, being most intense in the well-differentiated tumours and low in the poorly differentiated ones (Potocnik et al., 2002). 19.3.11 Colonic crypt homeostasis Homeostasis of the normal colonic mucosa relies on a balance between proliferation at the base and apoptosis at the surface epithelium. Normal colonic epithelium is organised in crypts where cell proliferation, migration, differentiation and apoptosis are topographically organised in a linear fashion along the crypt axis. Normal colonic crypts are composed of stem cells at the base, a proliferation and a differentiation zone in the lower third of the crypt, a migration zone in the upper two thirds, and the surface epithelium where senescent cells are eliminated by apoptosis. Colonic epithelial cells migrate from the base of the crypt to the surface epithelium in six to seven days. The stem cells at the base of the crypt are susceptible to somatic mutations and these mutations persist during migration along the crypt axis but will be eliminated by apoptosis at the surface epithelium. However, genes directly involved in the normal apoptotic process can themselves be mutated causing a malfunction in the apoptotic pathways (Abdel-Rahman et al., 1999). Dysregulation of apoptosis in colorectal adenomas and carcinomas has been correlated with abnormal Bak expression (Partik et al., 1998). Studies have also shown dysregulation and mutations in the caspase family in colon cancer, especially caspases 3 and 5 (Leonardos et al., 1999; Schwartz et al., 1999). As a result, cells with mutations in the apoptotic machinery will accumulate at the top of the crypt and surface epithelium, be unable to detach and die, and contribute to neoplastic transformation. Alternatively, genes encoding for cell-cell and cell matrix adhesion proteins can also be over- or underexpressed, or undergo mutations causing resistance to

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anoikis (detachment of epithelial cells from the basement membrane). As a result of these alterations, cells will be unable to undergo the timely detachment and apoptotic process normally found at the surface colonic epithelium (Kim et al., 1994b). 19.3.12 Mucins Mucins are high molecular weight glycoproteins that represent the major secreted products of the gastrointestinal tract. They are a major component of the mucus layer, which guards the epithelium of the gastrointestinal tract from mechanical, chemical, enzymatic, and microbial damage. Mucins are secreted in large quantities by colon cancer cells, and alterations in mucin expression and glycosylation occur during tumourigenesis and tumour progression in the colon (Bresalier et al., 1991; Chang et al., 1994; Bresalier et al., 1996; Hanski et al., 1997; Bresalier et al., 1998). MUC 2 mucin is the major product of the goblet cells in the small and large intestine in man and is the main mucin produced by many colon cancers. Previous studies have shown that mucins are frequently altered in colon cancers. Such alterations include differences in mucin-associated antigen expression between normal colon, preneoplastic adenomas and dysplasia, primary colon cancers, and metastatic disease (Itzkowitz et al., 1990; Hanisch et al., 1992; Nakamori et al., 1993; Bresalier et al., 1996). A study in athymic mice showed inhibition of mucin glycosylation by chemical inhibitors (Bresalier et al., 1991) and specific inhibition of MUC 2 mucin synthesis by antisense technology (Sternberg et al., 1999) in cultured colon cancer cells which inhibited colon cancer metastasis and liver colonisation by human colon cancer cells. Also, alterations in cellular behaviour such as proliferation, apoptosis, and cell migration occur with loss of MUC2 mucin and this could play a part in the early stages of carcinogenesis in the intestine (Velcich et al., 2002). 19.3.13 Immunogenicity of colon cancer Colorectal carcinoma, like most epithelial solid tumours, has long been considered poorly immunogenic and has never been an obvious target for immunotherapy. Indirect data has shown in epidemiological studies a lack of spontaneous regression of colon cancer; also a lack of activity of tumour infiltrating lymphocytes (TIL) in in vitro studies, and the first generation of clinical trials of immunotherapy in colorectal cancer patients have shown it to be substantially refractory to therapy. In some tumours, such as melanoma, primary lesions can sporadically undergo spontaneous regression associated with tumour infiltration by immune effectors (3±7% of cases). In colorectal carcinoma spontaneous regression is only rarely observed, and does not appear to be associated with immune response (Serpick, 1976; Papac, 1996; Francis, 1997). In vitro studies performed with bulk TIL cultures purified from colorectal carcinomas failed to demonstrate substantial lytic activity against autologous

Synbiotics and colon cancer 541 cancer cells (Balch et al., 1990; Rosenberg, 1992). Also, standard immunotherapies, known to be active against melanoma, such as systemic administration of cytokines (interleukin-2, interferon-) or adoptive transfer of autologous lymphocyte effectors (lymphokine-activated killer cells, LAK; TIL), have proved ineffective against colorectal tumours (Rosenberg, 1992; Hawkins et al., 1994; Greco et al., 1996; Wolmark et al., 1998). However, during the last decade, continuous progress in the molecular characterisation of T cell-defined tumour associated antigens (TAA) and in methods allowing detection of antigen-specific T cell responses have slowly modified the scientific community's perspective on this issue. Indeed, several different lines of evidence indicate today that epithelial solid tumours may express TAA recognised by T cells, and that anti-tumour immune responses indeed may take place in colorectal cancer patients, influencing patient prognosis and shaping the tumour immunological profile. Recent data have provided some support for the opinion that the immune system can affect colorectal cancer growth and metastatic dissemination. Among the most convincing lines of evidence supporting this hypothesis is the finding that CD8+ T cell intraepithelial infiltration of primary tumours represents a strong favourable prognostic factor, as well as the frequent loss of class I HLA by tumour cells. Despite the limited success of immunotherapy in clinical trials, there is a growing knowledge of the immune system cell circuitry and of the molecular mechanisms of T cell activation. These advances, together with the clinical experience collected in clinical trials exploring anti-tumour vaccination in melanoma patients, should allow the design of a new generation of more effective anti-tumour vaccines, based on new immunological adjuvants (dendritic cells, heat shock proteins) as well as on the chemical modification of antigenic peptides to increase their immunogenicity. The full deployment of T cell immunotherapy as a treatment option against colorectal cancer, however, is still hampered by the lack of molecularly defined T cell TAA that can be reliably considered as tumour-specific, strongly immunogenic and shared among different patients. The identification of new TAA to be used as targets of antitumour vaccination for this form of cancer represents an important focus for future studies.

19.4

Risk factors and prevention of colon cancer

Despite advances in surgical technique and adjuvant therapy, there has been only a modest improvement in survival for patients who present with advanced neoplasms. Most colorectal cancers arise from precursor adenomatous polyps that develop into invasive carcinoma, typically over a five to ten year or longer period. Hence, effective primary and secondary preventive approaches must be developed to reduce the morbidity and mortality from colorectal cancer. Central to this is the identification of at risk subjects, who can be targeted for chemoprevention coupled with modification of lifestyle.

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19.4.1 Diet and excess weight Diet has long been regarded as the most important environmental influence on colorectal cancer and this is reflected in the volume of studies testing the dietary hypothesis. However, diet is related to other aspects of lifestyle that may influence risk and people eat food rather than nutrients. There have been almost 20 studies on excess weight and colon cancer covering more than 3,000 cases, and there is consistent evidence of a positive association with obesity (Bergstrom et al., 2001). Colon cancer rates are high in populations with high total fat intakes and are lower in those consuming less fat (Rose et al., 1986). The evidence from case control studies consistently supports an inverse association between vegetable intake and colon cancer, although the evidence from cohort studies, particularly the most recent ones, is less convincing (Michels et al., 2000; Potter, 1997). The role of dietary fibre has also been widely studied, but the cohort studies suggest no clear association between fibre intake and colorectal cancer, and the results of case control studies have been inconsistent (Kim, 2000; Potter, 1997). Total meat consumption has been studied in two meta-analyses that showed no statistically significant overall association (Sandhu et al., 2001, Norat et al., 2002). Data on red meat and processed meat suggest positive associations with the risk of colorectal cancer. 19.4.2 Physical activity Studies indicate a consistent inverse relationship, with a 50% reduction in risk among those with the highest level of exercise (IARC 2002; Kiningham, 1998; Colditz et al., 1997; Friedenreich, 2001). 19.4.3 Smoking Although earlier studies of smoking and colorectal cancer showed no association, in more recent studies long-term smokers are found to be at elevated risk, with relative risks typically in the range of 1.5 to 3.0 (Giovannucci, 2001). 19.4.4 Alcohol The association between colorectal cancer and alcohol is not clear. Although the majority of studies suggest a positive association between alcohol consumption and colorectal cancer, a substantial portion of studies show no association (Potter, 1997). 19.4.5 Hormone therapy Protective effects of both hormone replacement therapy (HRT) and oral contraceptives (OC) have been postulated. The majority of evidence shows an

Synbiotics and colon cancer 543 inverse relationship between postmenopausal HRT and colorectal cancer (Crandall, 1999; Rossouw et al., 2002). 19.4.6 Chemoprevention The sequential process of tumorigenesis provides opportunities for the development and testing of both primary and secondary prevention strategies. Chemoprevention, is defined as the use of natural or synthetic agents to reverse the process of carcinogenesis. Epidemiological studies have consistently shown that chronic intake of nonsteroidal anti-inflammatory drugs (NSAIDs), principally aspirin, can reduce the incidence of colorectal adenomas and carcinomas. The conversion of arachadonic acid to prostaglandins is catalysed by the cyclooxygenase family of enzymes, COX-1 and COX-2. Aspirin and sulindac inhibit both enzymes, whereas celecoxib inhibits only COX-2. These agents induce apoptosis by both Cox-dependent and COX-independent mechanisms. The inhibition of COX-2 leads to an increase in arachadonic acid, which, in turn stimulates the conversion of sphingomyelin to ceramide, a mediator of apoptosis (Sheng et al., 1998). Inhibition of COX-2 may also lead to apoptosis by altering prostaglandin production and by decreasing angiogenic factors (Chan et al., 1998; Tsujii et al., 1998). Aspirin, sulindac, and selective COX-2 inhibitors also exert their effects by COX-independent mechanisms (Elder et al., 1997). Some of these effects may be mediated by the ability of aspirin, sulindac, and sulindac metabolites to inhibit the activation of nuclear factor-kB (NF-kB) (Yamamoto et al., 1999) or to interfere with the binding of peroxisomeproliferator-activated receptor delta (PPARd) to DNA (He, 1999). Sulindac sulfone, a metabolite of sulindac that inhibits neither COX-1 nor COX-2, causes apoptosis of colon-carcinoma cell lines and is chemopreventive in animal models (Piazza et al., 1997). Other COX-independent mechanisms are currently not well characterised. Evaluation of NSAIDs, including newer selective cyclo-oxygenase-2 inhibitors, in carcinogen-induced and genetically manipulated animal models of colorectal cancer demonstrates that these drugs are effective chemopreventive agents. In 1983 Waddell and Loughry reported that sulindac, an indene acetic analogue of the NSAID indomethacin, caused the regression of adenomatous polyps in a patient with familial adenomatous polyposis. This serendipitous discovery was confirmed in uncontrolled studies, case reports, and controlled trials. In a placebo-controlled, randomised study of 22 patients with familial adenomatous polyposis and established adenomatous polyps (Waddell and Loughry, 1983), the effect of 150 mg of sulindac orally twice a day was greater after six months of treatment than after nine months, suggesting the emergence of resistance to sulindac. This effect may have been mediated by a decrease in the expression of COX-2 or other molecular events such as a mutation in the Kras gene (Keller et al., 2001). More recently, the selective cox-2 inhibitor Celebrex was also shown to be effective in familial adenomatous polyposis

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(Steinbach et al., 2000) and was approved by the Food and Drug Administration as an adjunct to usual care in these patients. In addition to demonstrating efficacy, chemopreventive agents must be safe and well tolerated for chronic administration and should be relatively cost-effective. Other chemopreventive agents that have been assessed, include folate, calcium, and oestrogens. In the Nurses' Health Study, supplementation with folate was protective against colorectal cancer, with the greatest risk reduction among women taking high daily doses of folate; this reduction became significant only after 15 years of use (Giovannucci et al., 1998). The long time needed for clinical benefit suggests that folate acts early in colon carcinogenesis. Some, but not all, studies of humans consuming high calcium diets or receiving calcium supplements have shown decreased proliferation of colorectal epithelial cells, changes in bile acid composition, or decreased cytotoxicity of faecal water (Baron et al., 1995; Lipkin and Newmark, 1985; Bostick et al., 1995; Holt et al., 1998; Lapre et al., 1993). During the past 20 years, mortality from colorectal cancer has decreased slightly in men but much more in women (Greenlee et al., 2000); this may be due to the use of HRT (Wysowski et al., 1995). Oestrogens may prevent colorectal cancer by decreasing the production of secondary bile acids, by decreasing the production of insulin-like growth factor 1, by exerting direct effects on the colorectal epithelium, or by a combination of these mechanisms (McMichael and Potter, 1980; Ma et al., 1999; Campagnoli et al., 1993; Thomas et al., 1993).

19.5

Screening of colorectal cancers

Screening is the term used to describe the investigation of asymptomatic individuals in order to detect disease at an early stage when it is more amenable to treatment. Fewer than 30% of eligible persons in the US have had a screening test for colorectal cancer (MMWR, 1999). 19.5.1 Population screening The American Gastroenterological Association guidelines for screening recommends, for average risk people, offering to screen men and women aged 50 years and older, to stratify patients by risk, to offer various options for screening, to follow up a positive screening test with a colonoscopy, and appropriate and timely surgery for detected cancers. Follow-up surveillance is required after polypectomy and surgery (Winawer et al., 2003). The screening strategy is as follows: offer yearly screening with faecal occult blood test (FOBT) using a guaiac-based test with dietary restriction or an immunochemical test without dietary restriction. Two samples from each of three consecutive stools should be examined without rehydration. Patients with a positive test on any specimens should be followed up with colonoscopy (Winawer, 2003). The

Synbiotics and colon cancer 545 most widely investigated screening modality has been faecal occult blood testing (FOBT), a meta-analysis of around 443,000 people aged 40 or over in five countries concluded that FOBT screening resulted in a 16% reduction in colorectal cancer mortality, and when adjusted for attendance for screening, this improved to a 23% reduction (Towler et al., 1998). The AGA also recommends offering flexible sigmoidoscopy every five years, or offering screening with FOBT every year combined with flexible sigmoidoscopy every five years, or colonoscopy every ten years, or double contrast barium enema every five years (Winawer et al., 2003). Unlike the situation ten years ago, there is now no uncertainty about whether screening can reduce the rates of death from colorectal cancer. What remains uncertain are issues regarding the relative cost effectiveness of the various tests, and specifically whether routine colonoscopic screening should be the test of choice. 19.5.2 Screening patients with IBD In patients with long-standing, extensive inflammatory bowel disease, surveillance colonoscopy with systematic biopsies should be considered. This applies to both ulcerative colitis and Crohn's colitis because the cancer risk is similar in both diseases (Winawer et al., 2003). 19.5.3 Screening patients after removal of adenomatous polyps Patients who have had numerous adenomas, a malignant adenoma (with invasive cancer), a large sessile adenoma, or an incomplete colonoscopy should have a short interval follow up based on clinical judgement. Patients who have advanced or multiple adenomas (3) should have their first follow-up colonoscopy in three years. Patients who have one or two small (<1cm) tubular adenomas should have their first follow-up colonoscopy at five years (Winawer et al., 2003). 19.5.4 Family history of colorectal cancer People with a first degree relative (parent, sibling, or child) with colon cancer or adenomatous polyps diagnosed at age < 60 years or two first degree relatives diagnosed with colorectal cancer at any age should be advised to have screening colonoscopy starting at age 40 or ten years younger than the earliest diagnosis in their family, whichever comes first and repeated every five years. People with a first degree relative with colon cancer or adenomatous polyp diagnosed at age >60 years or two second degree relatives with colorectal cancer should be advised to be screened as average risk persons, but beginning at age 40 years. People with one second degree relative (grandparent, aunt or uncle) or third degree relative (great-grandparent or cousin) with colorectal cancer should be advised to be screened as average risk persons (Winawer et al., 2003).

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19.5.5 Hereditary nonpolyposis colorectal cancer People with the genetic and clinical diagnosis of HNCC or who are at risk of HNCC should have a colonoscopy every one to two years beginning at age 20± 25 years, or ten years earlier than the youngest age of colon cancer diagnosis in the family (Winawer et al., 2003). 19.5.6 Familial adenomatous polyposis (FAP) People with a diagnosis of FAP should have annual sigmoidoscopy, beginning at age 10±12 years, to determine if they are expressing the genetic abnormality (Winawer et al., 2003).

19.6

Diagnosis and treatment of colorectal cancers

Early diagnosis of colorectal cancer is often difficult clinically, as symptoms and signs present only late in the disease. 19.6.1 Symptoms Presenting features of colon cancer include rectal bleeding, changes in bowel habit, abdominal pain, tenesmus, iron deficiency anaemia, emergency presentation with large bowel obstruction or perforation and symptoms of malignancy such as weight loss and anorexia. 19.6.2 Endoscopy Colonoscopy is an extremely sensitive diagnostic test for colorectal cancer and has the major advantages of allowing both biopsy and polypectomy and does not involve exposure to ionising radiation. It also has some disadvantages. In a variable proportion of cases (5±30%) the caecum is not reached (Theoni and Laufer, 1994), intravenous sedation is nearly always required, the localisation of the tumour can be inaccurate, and there is a small but significant risk of complications (Atkin, 1999; Waye et al., 1996). The procedure-related mortality is approximately 1 in 5,000 for colonoscopy and 1 in 50,000 for sigmoidoscopy and for double contrast barium enema. Wireless capsule endoscopy is an emerging technique for visualising the gastrointestinal tract (GIT). In 1981, an Israeli physician, Dr Gavriel Iddan, began development of a video camera that would fit inside a pill. With the progress of technology, the pill has been put into clinical use in recent years. About the size of a large vitamin, the capsule is specially sealed with biocompatible material, resistant to stomach acid and digestive enzymes. It relays video pictures of the GIT, via a wireless radiofrequency transmitter, as it moves naturally via peristalsis. Its main role is in the diagnosis of small bowel disorders and it is not useful in large bowel lesions as the folds of the lower intestine make visualisation of the entire colon difficult.

Synbiotics and colon cancer 547 19.6.3 Double contrast barium enema Double contrast barium enema is a radiological procedure using air and barium as contrast to highlight the lining of the large bowel. It may be employed as a sensitive, safe alternative to colonoscopy. Where the sigmoid is not well visualised, e.g., in the presence of diverticular disease, double contrast barium enema should be combined with flexible sigmoidoscopy. In all cases the rectum should be visualised by rigid or flexible sigmoidoscopy (Jensen et al., 1990). 19.6.4 Computed tomography (CT) colonography Thin-section, helical, CT followed by off line processing (`virtual colonoscopy') can yield high-resolution, three-dimensional images of the colon. CT colonography is a sensitive method for detection of colorectal cancer, but not for polyps less than 10 mm (Miao et al., 2000). 19.6.5 Tumour markers Biochemical markers for colorectal cancer are potentially useful in screening for early disease, aiding diagnosis, predicting likely responses to specific therapies, and surveillance of patients undergoing curative resection and monitoring the treatment of advanced disease. Although multiple markers have been described, confusion remains about how best to use these factors. Carcinoembyronic antigen (CEA) is a high molecular weight glycoprotein belonging to the immunoglobulin superfamily of molecules (Hammarstrom, 1999; Duffy, 2001). It has been postulated to play a role in a number of biological processes including cell adhesion, immunity and apoptosis (Hammarstrom, 1999; Duffy, 2001). As with screening, inadequate sensitivity severely limits the value of CEA for the diagnosis of early or low stage colorectal cancer. In addition, as CEA can be elevated in the absence of malignancy (e.g. patients with benign liver disease and in cigarette smokers) specificity is also impaired (Duffy, 2001; Fletcher, 1986). Serum CEA levels may identify a subgroup of node-negative or Dukes' B colon cancer patients who have a poor prognosis and therefore define a subset of patients that could benefit from adjuvant chemotherapy (Harrison et al., 1997). It is important to state that there are no studies at present showing a benefit from adjuvant chemotherapy where patients have been selected for treatment based on a high CEA level. Preoperative CEA levels may also provide a baseline level that may be of value in the interpretation of subsequent serial results. An elevated preoperative concentration should return to normal within approximately six weeks following complete surgical resection of a colorectal cancer (Filella et al., 1994). Failure to do so suggests residual or metastatic disease. Serial monitoring with CEA has been shown to detect recurrent or metastatic disease with a sensitivity of approximately 80%, specificity of approximately 70% (Duffy, 2001; Fletcher, 1986). CEA is also useful in monitoring chemotherapy in patients with advanced disease. Although the oldest, CEA is

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still the best available serum marker for colorectal cancer. One of its major limitations in monitoring, however, is that 20±30% of patients fail to produce elevated serum levels, despite the presence of advanced disease. For follow up of these patients, other markers are therefore necessary. After CEA, CA 19.9 is the most widely used tumour marker. It is the best available marker for pancreatic cancer but it is less sensitive than CEA for detecting colorectal carcinoma (Duffy, 1998). Elevated preoperative levels of CA 19.9 have been found to correlate with adverse patient outcome (Lindmark et al., 1995; Nakayama et al., 1997; Reiter et al., 1997; Reiter et al., 2000), despite being of little value in early diagnosis of the disease (Moertel et al., 1993). Furthermore, serial determinations of CA 19.9 provide little extra information to that of CEA in monitoring patients. Newer markers are under scrutiny, but there is insufficient data available to recommend their use in the early detection of colorectal cancer. Studies suggest that they may provide independent prognostic information. These markers include the following: · · · ·

CA 242 TPA TPS TIMP-1

CA 242 is not sensitive for detection of early stage colorectal cancer. There are preliminary studies suggesting that it may complement CEA in the surveillance of patients diagnosed with cancer (Hall et al., 1994). TPA (tissue polypeptide antigen) measures fragments of cytokeratin 8, 18, and 19 and TPS (tissue polypeptide-specific antigen) detects fragments of cytokeratin 18. These have been subjected to limited evaluation in colon cancer. Tissue inhibitor of metalloproteinase 1(TIMP-1) is a multifunctional glycoprotein that inhibits metalloproteinase activity, stimulates cell growth and inhibits apoptosis (Chambers and Matrisian, 1997). TIMP-1 has been shown to have a specificity of 95% with a sensitivity of 65% for colon cancer and 42% for rectal cancer (Holten-Andersen et al., 2002). A combination of these tumour markers may provide stronger prognostic information compared with existing staging systems. 19.6.6 Cell and tissue markers It is well established that multiple molecular alterations occur at the genome level, during the transition from normal mucosa to invasive carcinoma (Fearon and Vogelstein, 1990). Genes undergoing mutation during this process might be expected to provide markers that are relatively specific to malignant and premalignant disease and also aid early diagnosis. Cells containing these abnormal DNA sequences are shed into the lumen by exfoliation and can be detected in the stools (Duffy, 1995). These markers will be discussed further below.

Synbiotics and colon cancer 549 19.6.7 Surgery Surgery remains the definitive treatment for apparently localised colorectal cancer, offering the only chance of cure. The colon and the rectum are anatomically distinct and present different difficulties to the surgeon. The principles of radical surgical resection for colonic tumours have generally remained unchanged in the past few decades. The procedure involves ligation of the major vascular pedicle, obtaining tumour free margins, and resection of any contiguous involved organ. Ligation of the vascular pedicle enables wide excision of lymph nodes draining the tumour, which is helpful in histopathological staging. In patients with colon cancer there is no evidence that radicality of excision has an effect on outcomes. Total mesorectal excision (TME) is recommended for most rectal cancers where the patient is fit for radical surgery (Heald et al., 1998; Wiig et al., 1998). The mesorectal excision should be total for tumours of the middle and lower thirds of the rectum, and care should be taken to preserve the pelvic autonomic nerves wherever possible without compromising tumour clearance (Havenga et al., 2000). Anastomotic leakage is an important and potentially fatal complication of colorectal cancer surgery, and measures to minimise it should be taken. With a low rectal anastomosis, should be considered a defunctioning stoma (Dehni et al., 1998) and with low rectal anastomosis after TME a colopouch may be necessary (Huber et al., 1999). Certain rectal cancers are amenable to local excision, and there is evidence from a randomised trial that this is associated with less morbidity than radical surgery (Winde et al., 1996). Further surgery for pedunculated polyp cancers is indicated if there is histological evidence of tumour at, or within 1mm of the resection margin, there is lymphovascular invasion, or the invasive tumour is poorly differentiated (Wolff et al., 1990; Chapman et al., 2000). Evidence from several randomised controlled trials, case control studies, and cohorts indicate that laparoscopic surgery for colorectal cancer is feasible, and can reduce postoperative pain, analgesia use, hospital stay and blood loss in the short term (Maxwell-Armstrong et al., 2000). But these have the potential for bias as blinding is impractical. Reliable outcomes are also lacking. Patients with liver and lung metastases should be considered for resection or, in the case of liver disease, in situ ablation (Scheele et al., 1995; Shirouzu et al., 1995). In patients with advanced local or recurrent disease, careful consideration should be given to surgical excision or palliative intraluminal procedures, e.g., stenting of laser ablation (Seifert et al., 1998). 19.6.8 Pathology and staging Pathological examination of the resected tumour is vital in assessing prognosis and the need for adjuvant therapy. Staging identifies patients who might benefit from adjuvant chemotherapy, and circumferential resection margin (CRM) helps select patients with rectal cancer that might benefit from postoperative radiotherapy (Adam et al., 1994). Pathological reporting of

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colorectal cancer resection specimens should include information on tumour differentiation, staging (Dukes' and TNM systems), margins (peritoneal and CRM) and extramural invasion (Shepard, 1997; Adam et al., 1994; Talbot et al., 1980). The American Joint Committee on Cancer (AJCC) has designated staging by the TNM classification (AJCC, 1997). T refers to the primary tumour, where TX is where the primary tumour cannot be assessed, T0 is no evidence or primary tumour, Tis is carcinoma in situ: intraepithelial or invasion of the lamina propria, T1 the tumour invades the submucosa, T2 tumour invades muscularis propria, T3 tumour invades through the muscularis propria into the subserosa, or into nonperitonealised pericolic or perirectal tissues, T4 tumour directly invades other organs or structures, and/or perforates visceral peritoneum. N refers to lymph nodes, where NX is regional lymph nodes cannot be assessed, N0 no regional lymph node metastasis, N1 is metastasis in one to three regional lymph nodes, N2 in four or more regional lymph nodes. M refers to distant metastasis, MX is distant metastasis cannot be assessed, M0 is no distant metastasis, M1 is distant metastasis. Staging is as follows; Stage 0 is Tis, N0, M0; Stage 1 is T1, N0, M0, T2, N0, M0; Stage 2 is T3, N0, M0, T4, N0, M0; Stage 3 is any T, N1, M0 or any T, N2, M0; Stage 4 is any T, any N, M1. Dukes' staging was originally described for rectal cancer but now applied to all colorectal adenocarcinomas. It was created in 1929 by Sir Cuthbert E. Dukes, a pathologist in St Mark's Hospital in London. Dukes' A is where tumour is confined to the bowel wall, B is tumour invasion through the bowel wall into the surrounding tissue, C is lymph node involvement, with C1 where tumour has not reached the apical (highest) regional lymph node, and C2 where the apical node is involved. Dukes himself did not describe a D but this was later added to refer to distant metastases. Five-year-treated survival according to Dukes' staging is 90% for A, 65% for B, 30% for C, with only 5% five-year survival if distant metastases are present (Dukes' D). 19.6.9 Chemotherapy and radiotherapy Both radiotherapy and chemotherapy can improve survival rates after potentially curative surgery, and chemotherapy prolongs overall survival of patients with advanced disease. The anatomical differences between colon and rectal cancer and the ease of surgical intervention determine the pattern of recurrence, which defines the most appropriate adjuvant and relapse treatments. About 50% of recurrences of rectal cancer occur in the pelvis and are, therefore, amenable to local radiotherapy. By contrast, relapse of colon cancer is generally at distant sites ± liver, lungs and bone ± and systemic chemotherapy seems more appropriate. Adjuvant chemotherapy Fluorouracil has remained the cornerstone chemotherapy for colorectal cancer for over 40 years. It is a prodrug that is converted intracellularly to various

Synbiotics and colon cancer 551 metabolites that bind to the enzyme thymidylate synthase, inhibiting synthesis of thymidine, DNA, and RNA. There is evidence showing absolute survival benefit (of 4±13% at five years) from adjuvant chemotherapy for patients with Dukes' C colon cancer (Figueredo et al., 1997a; O'Connell et al., 1997; Marsoni, 1995). The evidence for Dukes' B tumours shows no overall benefit from adjuvant chemotherapy (Marsoni, 1995; Figueredo et al., 1997b). The recommended adjuvant regimen in patients with Dukes' C tumours is bolus fluorouracil and low dose folinic acid (FUFA), administered over five days every four weeks for six months (Figueredo et al., 1997b; O'Connell et al., 1997). Efforts to improve the efficacy of 5-fluorouracil have confirmed the benefit of including folinic acid without the need for levamisole. Co-administration with folinic acid increases the degree of inhibition of thymidylate synthase, depletes cellular thymidine, and induces apoptosis. In addition, protracted venous infused 5-fluorouracil is reported to achieve higher response rates and has lower toxicity in patients with metastatic disease. Preliminary results of the SAFFA study (short course adjuvant 5-fluorouracil, 5-fluorouracil plus folinic acid) appear to at least support the reduced toxicity of this regimen in the adjuvant setting (Saini et al., 2000). Adjuvant radiotherapy Reports of the use of preoperative radiotherapy in rectal cancer have indicated a reduction in the local recurrence rate from more than 25% to less than 10%, and this was subsequently translated into a survival advantage (Swedish Rectal Cancer Trial, 1997). However, the surgical approach to rectal cancer has also changed. Postoperative radiotherapy is generally less efficacious than preoperative treatment. Chemotherapy for metastatic disease There is evidence from two systematic reviews that chemotherapy for metastatic colorectal cancer can improve survival, and should be considered in all cases (Simmonds, 2000; Jonker et al., 2000). This form of treatment is given with palliative intent, and a major aim should be to alleviate symptoms or delay their onset. Up to 20% of patients who relapse after apparently curative resection of colorectal cancer present with disease macroscopically confined to the liver. Two meta-analyses (Harmantas et al., 1996) and two randomised trials (Lorenz, 2000; Kemeny et al., 1999) have found that response rates are higher with hepatic artery infusion (HAI) of fluorouracil but improvements in survival are meagre. Any advantages achieved through the use of HAI are offset by technical difficulties associated with the technique. Radiotherapy for advanced disease The potential roles here are to improve operability of unresectable disease (Habor-Gamma et al., 1998), in the curative treatment of inoperable disease, and in palliative management of symptoms in patients with persistent or locally recurrent disease (Spanos et al., 1993).

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19.6.10 Follow-up Patients who have undergone curative resection for colorectal cancer should undergo formal follow-up in order to facilitate the early detection of metastatic disease. Interval CT scanning and CEA estimation may be of value in follow-up of patients who have undergone curative resection for colorectal cancer (Rosen et al., 1998; Renehan et al., 2002; Jeffery et al., 2002) but further studies are required to define an optimum approach. Colonoscopy after curative resection for colorectal cancer should be carried out as for adenomatous polyps. Clinicians should investigate promptly any symptoms or signs of recurrence.

19.7 Pre-, pro- and synbiotic influences on colon carcinogenesis Many studies have confirmed that the normal microflora plays a role in the onset of colon cancer. It therefore is practical to propose that altering the intestinal microflora may influence tumour development. A protective role for pre-, proand synbiotics has been shown in animal studies, (Gallaher and Khil, 1999). The exact mechanisms by which pre-, pro- and synbiotics may inhibit colon carcinogenisis are as yet uncertain. However, based on results from the vast amount of in vitro and in vivo studies performed to date, it is possible to speculate on some possible mechanisms. Rafter (2002) suggested the following mechanisms by which lactic acid bacteria may inhibit colon cancer: · · · · · · ·

altering the physicochemical environment in the colon enhancement of the host's immune response inactivating potential carcinogens and mutagens altering the composition of the intestinal flora to a more beneficial flora anti-mutagenic or anti-tumourigenic activities altering the metabolic activity of the flora increase activities of protective enzymes and reduce activities of enzymes involved in carcinogenesis.

19.7.1 Altering the physicochemical environment in the colon Modler et al. (2001) suggested that large bowel cancer may be influenced directly by reducing the intestinal pH causing the growth of putrefactive bacteria to be reduced. However pH has not been consistently reduced in studies of probiotic consumption. 19.7.2 Immune response to probiotics Probiotics have been shown to exert various immune enhancing effects following oral consumption. These include enhanced macrophage phagocytosis

Synbiotics and colon cancer 553 (Perdigon et al., 1988), promotion of IgA responses (Isolauri et al., 1993; Malin et al., 1996; Link-Amster et al., 1994), and altered cytokine production (Miettinen et al., 1996), leading to a shift in the balance between Th1 and Th2 immune responses. As part of the innate immune system, when an antigen is ingested, the host immune system has to discriminate rapidly between potential pathogens, commensal bacteria, and food antigens. This is done with the use of a restricted number of preformed receptors. A new class of cell surface receptors, the Toll-like receptors, specifically interact with the bacterial walls or antigens (Krutzik et al., 2001). Dendritic cells are crucial in determining the local development of an adaptive immune response. Two subsets of monocyte-derived dendritic cells (MDC) exist; MDC1, which responds to lipopolysaccharide, lipotechoic acid or CpG DNA by means of the Toll mannose receptor which leads to NF-jB activation and potentiates Th1 responses, whereas MDC2 responds to immune complexes and cellular factors and leads to a Th2 response (Hemmi et al., 2000). Hence, according to the nature of the antigen a Th1/Th2 polarisation will occur. Th1 type responses are characterised by production of proinflammatory cytokines; interferon-gamma, tumour necrosis factor-alpha (TNF-), Interleukin-12 (IL-12), and IL-2. Th2 cells mediate humoral immunity through the actions of IL-4, IL-5, and IL-6. It is important to maintain a balance between Th1 and Th2 responses in relation to oral tolerance to ingested antigens. Inappropriate polarisation can eventually lead to disease, for example, allergy (Th2) or autoimmunity (Th1). The involvement of regulatory cell types which produce anti-inflammatory cytokines like TGF- and IL-10 is an area of increased research interest. The Th3 cell subset is generated in the gut-associated lymphoid tissue (GALT) and suppresses the function of effector cells by releasing the inhibitory cytokine TGF- . Th3 cells are also implicated in IgA production and in the regulation of oral tolerance by blocking the Th1 or Th2 response Another T cell suppressor cell population is T regulator 1 which secretes IL-10 (Newberry et al., 1999). Probiotics have been shown to influence the balance between the arms of the T helper cell response. McCarthy et al. (2003) showed that when fed probiotics, Interleukin-10 knockout mice, had reduced levels of IL-12 and TNF- both systemically and mucosally, and TGF- levels were maintained. Interleukin-10 knockout mice develop colitis, which is similar to Crohn's disease in humans (Th1 type disease), and this study illustrates that probiotic consumption reduced production of Th1 type cytokines and there was an associated attenuation in colitis. It is postulated that the immune response to probiotics depends on the host's immune status and for example, in atopic individuals, consumption may lead to an improved Th1 response. The immune response may also vary with different strains of probiotic. Specific strains with immunoregulatory and/or anticancer effects need to be clearly identified and their mechanism of action characterised.

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19.7.3 Inactivating potential carcinogens and mutagens Mutagens and potential carcinogens are ingested every day as part of the normal diet. One possible mechanism by which pre-, pro- and synbiotics may protect against colon cancer is by binding and degrading potential mutagens and carcinogens. High meat consumption is considered a risk factor for colon cancer due to the production of mutagenic heterocyclic amines (HCA), which are formed during cooking (Sinha and Rothman, 1999). LAB have been shown to bind HCA in vitro dependent on the pH of the culture medium (Mortomi and Mutai, 1986; Zhang and Ohta, 1991; Orrhage et al., 1994). Moritomi and Mutai (1996) demonstrated in vitro that L. casei YIT 9018 bound the mutagenic pyrolysate Trp-P-2 formed in cooked meat, and prevented the mutagenic effect of Trp-P-2 in the Salmonella mutagenicity assay. Consumption of lactobacilli in healthy human feeding studies has been shown to reduce the mutagenicity of faeces and urine associated with the ingestion of carcinogens in cooked meat, (Lidbeck et al., 1992; Hayatsu and Hayatsu, 1993). A lyophilised culture of Bif. longum has been shown to have an inhibitory effect on 2-amino-3-methylimidazol [4,5-f] quinoline (IQ) induced colon carcinogenesis (100% inhibition) in male colon cancer and to a lesser extent (50% inhibition) in mammary carcinogenesis in female rats (Reddy and Rivenson, 1993). A possible explanation for the complete inhibition in colon carcinogenesis in this model may be that the Bif. longum may bind the IQ in the lumen preventing interaction of the mutagen with the mucosa. Rowland and Grasso (1975) demonstrated that lactobacilli have the ability to degrade nitrosamines, mutagens associated with cooked meat. Wollowski et al. (1999) showed that Lactobacillus bulgaris 191R fed to rats reduced DMH induced DNA damage in colon cells as measured in the comet assay. 19.7.4 Altering the composition of the intestinal flora Consumption of pre-, pro- and synbiotics is proposed to increase the levels of beneficial bacteria such as lactobacilli and bifidobacteria and suppress the growth of putrefactive bacteria involved in the production of carcinogens and tumour promoters, such as bacteroides, clostridia and coliforms. Gibson et al. (1995a) demonstrated that consumption of oligofructose and inulin significantly increased the numbers of faecal bifidobacteria while decreasing the numbers of bacteroides, clostridia, fusobacteria and Gram-positive cocci. 19.7.5 Antimutagenic or antitumorigenic activities The anti-tumorigenic activities of LAB have been demonstrated in animal studies. The ability of pre-, pro- and synbiotics to inhibit chemically induced carcinogenesis has been investigated in numerous studies. Aberrant crypt foci (ACF) are early preneoplastic lesions in the colon from which adenomas and carcinomas may develop. ACF have consistently been observed in experimentally induced colon carcinogenesis in laboratory animals and have also been

Synbiotics and colon cancer 555 shown in the colonic mucosa of patients with colon cancer (McLellan et al., 1991; Wargovich et al., 1996; Pretlow et al., 1992). ACF express mutations in the apc and ras oncogenes, which are involved in cancer development (Vivona et al., 1993). ACF formation is often used in animal studies as end points for investigating potential anti-tumorigenic agents as several inhibitors of ACF formation have been shown to reduce the incidence of colonic tumours in laboratory animals (Wargovich et al., 1996). The multiplicity of aberrant crypts is also measured in many studies and ACF with four or more crypts is considered predictive of tumour incidence (Pretlow et al., 1992). A study by Reddy et al. (1997) investigated the effect of prebiotics specifically oligofructose and inulin on ACF formation in azoxymethane (AOM) induced carcinogenesis in rats. It was found that consumption of inulin and to a lesser extent oligofructose significantly decreased the total number of ACF per colon compared to controls. Multiplicity for crypts with two to three aberrant crypts per focus was also significantly reduced compared to control. Taper et al. (1997) demonstrated that the consumption of oligofructose or inulin significantly inhibited the growth of transplantable mouse tumours, EMT6 and TLT in mice. Probiotics have also been shown to inhibit colon cancer. Singh et al. (1997b) demonstrated that consumption of lyophilised Bifidobacterium longum significantly inhibited tumour incidence and multiplicity in AOM treated animals compared to controls. Similar anti-tumorigenic effects have been seen with synbiotics. Rowland et al. (1998) investigated the effect of a derivative of inulin (Raftiline HP) alone and in combination with Bifidobacterium longum on ACF induction by AOM. Bifidobacterium longum consumption resulted in a 26% reduction in small ACF (one to three aberrant crypts per focus) compared to control with inulin inhibiting ACF formation by 41%. However when administered together as a synbiotic small ACF formation was inhibited by 80%. The synergistic effect was particularly evident as the synbiotic inhibited large ACF formation (>4 aberrant crypts/focus) by 59% but the inulin and Bifidobacterium longum alone were ineffective. The experiment described above was a short-term experiment for 12 weeks and very little data is available for pre-, pro- or synbiotics in long-term carcinogenesis experiments. Femia et al. (2002) investigated the effect of consumption of a prebiotic Synergy 1, the probiotics Lactobacillus rhamnosus GG (GG) and Bifidobacterium lactis Bb12 (Bb12), and a synbiotic containing all three for 31 weeks on AOM-induced carcinogenesis in rats. The groups consuming the prebiotic had a significantly lower number of colorectal adenomas, compared with rats which did not receive the prebiotic, while consumption of the two strains of probiotics were ineffective. Similarly, the number of colorectal cancers was significantly lower in the prebiotic consuming group and the number of cancers in the group treated with probiotics alone was lower than untreated rats but did not reach significance. The authors suggest that in the case of AOM induced carcinogenesis the protective effect of probiotics alone may be

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restricted to malignant tumours, and the effect of the addition of prebiotic to the fibre content of the diet should also be considered. Consumption of Lactobacillus salivarius ssp. salivarius UCC118 in interleukin-10 knockout mice (colitis model) was associated with a trend towards reduced tumour development and attenuation of gastrointestinal inflammation. Gastrointestinal neoplastic transformation was evident in 50% of mice from the placebo group while only 10% of the probiotic group demonstrated any evidence of neoplastic change (O'Mahony et al., 2001). Prebiotics have been shown to stimulate apoptosis in the colon of rats, consuming a high fat Western type diet and treated with the carcinogen DMH (Hughes and Rowland, 2001). Apoptosis was significantly higher in the colon of rats fed diets supplemented with either oligofructose or HP inulin compared to rats fed the basal diet. 19.7.6 Altering the metabolic activity of the flora The normal colonic microflora produces enzymes such as -glucuronidase, glucosidase, nitrate reductase, nitroreductase and azoreductase, the activities of which have been implicated in colon carcinogenesis. Bifidobacteria and lactobacilli have lower activities of these xenobiotic-metabolising enzymes than do bacteroides, clostridia and enterobacteriae. Therefore, decreasing the levels of bacteria with high activities of theses enzymes while increasing the levels of bacteria with lower activities is desirable. Many toxic and carcinogenic compounds are metabolised in the liver and conjugated with glucuronic acid and are then excreted via the bile into the small intestine. Within the colon bacterial -glucuronidases hydrolyse the glucuronide conjugates releasing potential carcinogens or mutagens into the lumen and increasing the risk of tumour formation. Plant glycosides consist of low-molecular weight substances linked to sugar moieties and are found in fruit and vegetables and also in drinks produced from plants, e.g., wine. Plant glycosides are poorly digested in the upper GIT and reach the colon in their safe glycosidic form. -glucosidases cleave the sugar moieties releasing toxic, mutagenic or carcinogenic aglycones (Hughes and Rowland, 2000). Nitroreductases reduce aromatic and heterocyclic nitrocompounds, which are found in cigarette smoke and diesel exhaust, to potentially toxic N-nitroso and N-hydroxy compounds. Colonic nitrate reductases convert nitrate ingested in food and water to nitrite. Oxides of nitrogen derived from nitrite may further react with amines and amides to produce N-nitroso compounds many of which are highly carcinogenic. Reddy et al. (1974) showed that treatment with the carcinogen 1,2-dimethylhydrazine (DMH) or Methylazoxymethanol (MAM)glucuronic acid, which is formed following bacterial deconjugation of DMH, produced less colonic tumours in germ-free rats than in rats with a normal flora. This finding clearly demonstrates the role of bacterial derived -glucuronidases in the promotion of carcinogenesis. Diet has been shown to influence the activities of these enzymes in the intestinal tract. High-risk diets are consistently

Synbiotics and colon cancer 557 shown to increase the activity of -glucuronidase compared to low risk diets (Eiryamremu and Adamson, 1995). Hambly et al. (1995) showed a 2.5 fold increase in activity on a high-risk diet. Consumption of pre- and probiotics has been shown to decrease the activity of -glucuronidase. Ling et al. (1994) demonstrated that consumption of Lactobacillus GG alone or Lactobacillus GG with fibre for two or four weeks significantly reduced faecal -glucuronidase and nitroreductase activities as well as glycocholic acid hydrolase activities in healthy volunteers. Activities of these enzymes returned to baseline levels following cessation of feeding. -glucosidase activities were not significantly altered by intervention. Azoreductase promotes the reduction of azo food dyes to substituted phenyl and naphthyl amines, which are potent carcinogens. Laboratory rats, which have been experimentally fed beef and Lactobacillus have reduced faecal amounts of beta-glucuronidase, azoreductase, and nitroreductase (Simon and Gorbach, 1986). As suggested by Rafter (2002), `it is important to note that the studies to date do not always find reductions in the same enzymes, although findings with -glucuronidase and nitroreductase are the most consistent'. 19.7.7 Modulating the activity of host enzymes The activity of detoxifying enzymes plays an important role in the protection of the colonic mucosa from carcinogens and mutagens. The Glutathione-Stransferase (GST) family (alpha [], pi [] and mu []) of proteins are important phase II detoxification enzymes and a reduction in their activity is associated with an increased risk of colon cancer (Szarka et al., 1995). Treptow et al. (1999) demonstrated that resistant starch (a prebiotic), increased levels of GST in the rat colon. Consumption of lactulose, alone or in conjunction with Bif. longum, was shown to significantly increase GST levels in the rat colon (Challa et al., 1997). SCFA, the main fermentation products of the microflora have been shown to induce GST in a Caco-2 cancer cell line. It is also interesting to note an increase in GST in rectal biopsies following consumption of Brussels sprouts was mirrored by an increase in GST activity in blood lymphocytes. GST levels in lymphocytes may be used as a marker for cancer risk or to assess the effect of intervention on cancer progression. Lactic acid bacteria have been shown to increase the activity of NADPHcytochrome P-450 reductase (CYC) activity in the colon (Pool-Zobel et al., 1996). CYC is a phase I xenobiotic metabolising enzyme involved in the biotransformation of noxious xenobiotics before excretion. Increased activities of some enzymes are associated with an increase in cancer risk. Ornithine decarboxylase (ODC) is the first and rate-limiting enzyme in the synthesis of polyamines. Polyamines are involved in cell proliferation and differentiation and in macromolecular synthesis. In humans ODC levels are elevated in neoplastic colonic mucosa compared to normal appearing mucosa, in dysplastic polyps compared to nondysplastic polyps and in colonic adenocarcinomas compared to adjacent non-involved mucosa (Reddy, 1999). Studies have shown that probiotic

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administration in animal models of chemically induced colon carcinogenesis reduces the levels of ODC (Singh et al., 1997b). Ras activation is one of the earliest and most frequently occurring genetic alterations associated with human cancers, especially colon cancer (Barbacid, 1990). Bif longum consumption has been shown to reduce ras-p21 expression and cell proliferation in AOM induced carcinogenesis in rats (Singh et al., 1997a,b; Reddy, 1998). The evidence outlined above suggests possible mechanisms by which pre-, pro- or synbiotics may protect against colon cancer. However, it must be noted that as yet there is no direct experimental evidence that pre-, pro- or synbiotics play a role in cancer suppression in man.

19.8

Predicting tumour formation: biomarkers

The long time period between exposure to a carcinogen and tumour formation in humans poses a difficult problem when studying the relationship between suspected aetiological agents of colon cancer and the end point of tumour formation. As mentioned in section 19.6.5, CEA is the most widely used cancer marker, however, it is not a marker of cancer risk and therefore is not particularly useful in cancer risk lowering studies. Because of these problems, there is much interest in developing non-invasive reliable markers predictive of tumour formation, which are affected by changing risk. Such biomarkers have been proposed and developed from an understanding of the chain of events leading to colon cancer and an understanding of the changes, which occur in the colon before the manifestation of cancer. With regard to colon cancer there are two main types of biomarkers (i) tissue biomarkers and (ii) biochemical biomarkers. 19.8.1 Tissue biomarkers Tissue biomarkers are analysed in tissue biopsies, which may be obtained by a number of techniques of varying levels of invasiveness. In the normal colonic mucosa proliferation occurs in the lower third of the crypt, and as cells mature they move to the top of the crypt where they are eliminated by apoptosis and eventually sloughed off. However in the abnormal situation, e.g., polyp or tumour formation, proliferation occurs throughout the entire crypt often with the zone of maximum activity occurring at the top of the crypt. In animal models of carcinogenesis, the carcinogen often increases cell proliferation and causes a shift in the zone of proliferation. Mucosal cell proliferation is easily measured in biopsy samples. Immunostaining for proliferating cell nucleus antigen (PCNA), is a commonly used method to investigate changes in proliferative activity. Incorporation of tritiated-thymidine followed by autoradiography allows determination of the rate as well as the zone of proliferation. To date, studies assessing the association of proliferation and cancer risk have produced variable results and the usefulness of proliferation as a biomarker is still under consideration (Mills et al., 2001; Sandler et al., 2000). However,

Synbiotics and colon cancer 559 inclusion of apoptosis and differentiation markers in studies assessing proliferative activity may strengthen this parameter as a biomarker. The presence of DNA adducts in the colonic mucosa is associated with increased risk of colon cancer (Pfohl-Leszkowicz et al., 1995). DNA adducts are formed when carcinogenic or mutagenic substances (from the diet or environment) e.g., polycyclic aromatic hydrocarbons (PCA) covalently bind to DNA. Oxidative DNA adducts have been shown to be increased in all stages of colorectal carcinogenesis, (Schmid et al., 2000; Hendrickse et al., 1994). Mucosal cells, which have been sloughed off in the colon, are excreted in the faeces. DNA adducts can be measured in faecal samples and as such seem a promising choice as a non-invasive biomarker. 19.8.2 Genotoxic damage The Ames test has traditionally been used to investigate the mutagenic potential of compounds associated with cancer. Mutagenicity is determined based on the ability of the test substance to induce mutations in Salmonella typhimurium. However, the ability of this test to identify human carcinogens is limited as the mutagenic potential of substances is evaluated using prokaryotic cells. In recent years, the use of methods, which measure genotoxic damage in eukaryotic cells, have proved to be more promising as an indicator of cancer risk. Genotoxic agents cause cellular DNA damage but are not cytotoxic to the cell. One of the most commonly used methods to assess genotoxic damage is the microgelelectrophoresis assay or comet assay, which was developed by È stling and Johansson in 1984, and modified by Singh et al., in 1988 as the O alkaline comet assay. Briefly, a single cell suspension is prepared either from a cell line or cells isolated from the target organ and treated with the agent under investigation. Cell numbers and viabilities are assessed. The cells are then immobilised in agarose on a glass slide and treated to lyse the cell membrane. This is followed by a period of alkaline treatment to allow unwinding of DNA. An electric current is then applied to the DNA and broken DNA fragments (damaged DNA) migrate away from the nucleus producing tail lengths, which vary based on the amount of DNA damage. This method has been used to study the genotoxic potential of substances associated with cancer risk in freshly derived colon cells from healthy subjects (Pool-Zobel and Leucht, 1997). Studies have shown that some lactic acid bacteria inhibit MNNG and DMH induced genotoxicity in colon cells isolated from rats (Wollowski et al., 1999; Pool-Zobel et al., 1996). Currently this technique is being employed in the Syncan project (described below) to investigate if synbiotic intervention in subjects at risk of colon cancer has an effect on genotoxic damage in the colon. 19.8.3 Gene expression Investigations of gene expression in tumours, polyps and in normal tissue will help to identify the genes involved in carcinogenesis and to assess their

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importance. As discussed in sections 19.3.1 to 19.3.12, the expression of oncogenes, tumour suppressor genes and DNA repair genes are often altered in cancer. Using molecular techniques, such as gene array analysis, the expression of these genes can be investigated and over- and under-expressed genes can be identified. As discussed earlier, cancer arises from an accumulation of mutations in a single cell. Genotyping of neoplasms can be used to detect mutated genes whose loss of function may contribute to carcinogenesis. 19.8.4 Biochemical markers Biochemical markers can be measured in blood, urine and faeces and samples are obtained by less invasive methods than for tissue biomarkers. There are two types of biochemical markers (i) gut flora associated biomarkers which include bacterial enzymes and faecal metabolites and (ii) mammalian enzymes for example GSTs and the Cytochrome P450 family. The influence of pre-, pro- and synbiotics on these markers is discussed in sections 19.7.6 and 19.7.7 above. 19.8.5 Faecal water activity Colonic tumours arise from damage to the colonic mucosa, which is a result of the interactions with gut luminal contents. Faecal water (FW), which is the aqueous phase of faeces, contains most of the free reactive and soluble factors, which likely interact with the mucosa in vivo (Rafter et al., 1987). The cytotoxic and genotoxic potential of FW is considered a measure of the potential of the dissolved factors to induce damage in the colonic mucosa. Venturi et al. (1997) first used FW genotoxicity testing in the comet assay to assess the ability of FW from healthy individuals to induce genotoxic damage in Caco-2 cells. Studies have shown that there is an inter- and intra-individual variation of faecal water genotoxicity even in subjects consuming the same diet (Oûwald et al., 2000; Woods, 2002). However, Oûwald et al. (2000) suggested that FW genotoxicity is suitable as a biomarker for application in intervention studies when each subject is analysed as his or her own control. Rieger et al. (1999) demonstrated that a diet high in fat and meat but low in dietary fibre (considered high risk for colon cancer) increased the genotoxic potential in healthy volunteers (Rieger et al., 1999). The Syncan study, discussed later, is currently investigating if genotoxic damage in colon cells as measured by the comet assay is associated with the genotoxic potential of FW. Stimulation of proliferation in the colon is, in part, to compensate for the loss of cells, which may have been damaged through cytotoxic mechanisms, causing an increase in mitotic activity, increasing the risk of colon cancer. In vitro faecal water cytotoxicity towards colon cells is often employed as a biomarker in intervention studies. Glinghammer et al. (1997) demonstrated that a shift from a dairy-product rich diet (low cancer risk) to a dairy product-free diet (high risk) increased cytotoxicity of FW from healthy volunteers. The exact mechanism by which dairy products lower cancer risk is unknown however it may be

Synbiotics and colon cancer 561 speculated that, in this study, the increase in cytotoxicity may have been due to the reduction of calcium in the diet.

19.9

Future trends

An EU-funded research project (the SYNCAN project QLK1-1999-00346) is currently under way which aims to evaluate whether synbiotics can reduce the risk of colorectal cancer. The project brings together eight institutions in seven countries in an attempt to identify and develop novel biomarkers for colon cancer risk and to assess the effect of synbiotic intervention on these biomarkers (biomarker network). In vitro studies were performed to screen potential synbiotic combinations in order to determine the most suitable combination of prebiotic and probiotic for the study. Candidate pre-, pro- and synbiotics were evaluated in batch fermentations and also in human gut models. Fermentation supernatants were distributed to the biomarker network and their influence on biomarkers evaluated. Raftilose Synergy 1 (prebiotic) and Lactobacillus rhamnosus GG and Bifidobacterium lactis Bb12 (probiotics) were chosen as the best synbiotic combination. The ability of this synbiotic to reduce the risk of colon cancer was investigated in a rat study and is currently under investigation in a human intervention study, (www.syncan.be). To the best of our knowledge this is the only study investigating the effect of synbiotics on colon cancer risk in humans. The Crownalife project began in 2001 and aims to investigate the composition of the GIT flora in the elderly (>65) and in younger adults (20± 45). The project will also investigate the effect of synbiotic intervention on the composition of the flora in the elderly. As part of the study, some of the biomarkers outlined earlier used to assess cancer risk will also be investigated, however, this is not the main aim of the study (www.crownalife.be). As discussed previously, animal studies have shown that synbiotics play a role in the prevention of colon cancer. However, most of these studies did not screen candidate pre- and probiotics in order to determine the optimal synbiotic combination. In general there is a lack of studies that concentrate on the development of synbiotics from properly selected prebiotics and probiotics and the confirmation of their effectiveness in vivo. Synbiotic selection should be targeted towards the disease, as a synbiotic combination effective, for example, in ameliorating antibiotic associated diarrhoea may be ineffective in lowering cancer risk. The design of effective synbiotics may further be improved by targeting prebiotics for a specific probiotic. Rabiu et al. (2001) isolated -galactosidases from five bifidobacterium strains and used these enzymes in glycosyl transfer reactions to synthesise oligosaccharides from lactose. Fermentation tests showed an increased growth rate compared to oligomate 55, a commercial prebiotic galacto-oligosaccharide, for four out of five of the bifidobacterium strains on the product derived using their own enzyme. One of the novel prebiotics was further tested in the presence of mixed populations of intestinal bacteria and was seen to

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have an equivalent prebiotic activity to that of Oligomate 55. However, this novel prebiotic exhibited a lower stimulatory effect on bacteroides and lactobacilli than did Oligomate 55 suggesting that it is more selective. The potential of plant cell wall polysaccharides as sources of novel prebiotics has recently been investigated. Specific glycanases can be used to produce novel oligosaccharides from plant cell wall polysaccharides (Van Laere et al., 2000). In recent years, there has been much interest in the use of Lactobacilli as vectors for vaccine delivery. Attenuated pathogens are effective vaccines against the pathogen itself, however these organisms are often strongly immunogenic and are therefore not suitable for use in immunocompromised individuals for whom they may be potentially pathogenic. As lactic acid bacteria have been consumed by a great many people for many years without causing health problems, they are an attractive vehicle for the delivery of vaccines. Isolauri et al. (1995) administered LGG concomitantly with the D RRV reassortment rotavirus vaccine to young infants and evaluated the immune response following vaccination. Results suggested that the immunogenicity of an oral live rotavirus vaccine may be improved by concomitant and subsequent administration of Lactobacillus GG. Kruisselbrink et al. (2001) used a recombinant lactobacillus to induce tolerance in mice against a component of the house dust mite, demonstrating the usefulness of probiotic bacteria in the treatment of allergic and autoimmune diseases. Steidler et al. (2000) engineered a Lactococcus lactis strain to secrete an anti-inflammatory cytokine (IL-10). The therapeutic effect of the engineered strain was comparable to conventional steroid treatment in murine models of inflammatory bowel disease. The potential of probiotic bacteria to be used as delivery vehicles seems very promising. Symptoms of colorectal cancer present at a late stage in the disease process and therefore early prevention and detection of preneoplastic changes should be the main area of focus in the coming years. Non-invasive cancer risk biomarkers, similar to those discussed above, which herald early events in the cancer process, can be used to identify individuals at risk of colon cancer and also to assess efficacy of preventive measures. Synbiotics offer great potential as a preventive strategy. Synbiotic intervention in animal models of colon carcinogenesis has yielded very promising results. Current studies in humans should further elucidate the mechanisms involved in their preventive action and their possible role as an early intervention strategy. In this area there is a need for well-designed and executed intervention studies. The synbiotic combination should be selected from well characterised pre- and probiotics and based on its ability to affect the disease process. The study design should be double-blinded and placebo controlled and should investigate a well-defined population. The use of proved biomarkers is essential in assessing the efficacy of the intervention. Progress in the area of detection of cancer and the development of early preventive measures, such as synbiotics, will lead to a dramatic change in the morbidity and mortality pattern of colorectal cancer in the developed world.

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19.10

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20 Identifying antimutagenic constituents of food S. KnasmuÈller, B. J. Majer and C. Buchmann, University of Vienna, Austria

20.1

Introduction

In 1973 Bruce Ames and his co-workers postulated that `mutagens are carcinogens' (Ames et al., 1973). In the following years, this hypothesis was strengthened by numerous experimental data and in the 1980s it was shown that proto-oncogenes are converted to oncogenes by alterations of the genetic material. This observation provided an explanation for the causal link between DNA-damage and cancer induction. According to the multistep hypothesis of cancer formation which is widely accepted at present, mutations lead to the formation of `initiated' cells which develop into tumours in the presence of promoting factors acting mainly via epigenetic mechanisms (Pitot, 2002). DNA-damage in somatic cells is also considered to accelerate ageing processes (Beckman and Ames, 1998) and to be causally involved in arthritis and other degenerative disorders (Sun and Cheung, 2002). Furthermore, damage of the genetic material in germ cells leads to heritable diseases and affects fertility. In order to protect humans against DNA-damage and its consequences, a broad variety of genotoxicity test procedures has been developed (OECD, 1997; European Chemicals Bureau, 2003) which are used for routine screening of chemicals and to study the molecular mechanisms of DNA-damage and repair. Numerous DNA-reactive carcinogens have been identified in the human diet and also in the environment with these test systems. Typical examples for dietary carcinogens are nitrosamines, aflatoxins, heterocyclic aromatic amines (HCAs), polycyclic aromatic hydrocarbons (PAHs), certain mycotoxins and heavy metals to name just a few. Apart from external DNA-damage caused by chemicals and by radiation, the integrity of the genetic material may also be affected by

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endogenous damage caused by oxidative stress and errors arising during the replication process. The search for dietary anti-mutagenicity is based on the assumption that prevention of exo- and endogenous DNA-damage will have beneficial health effects such as protection against cancer and increase of the life span. The first dietary constituents which were found to elicit DNA-protective effects, were certain vitamins (A, C, E) and it was shown half a century ago that they protect against radiation induced DNA-damage and cancer (for review see Gebhart, 1974). In the following years, it became apparent that vitamins also prevent the induction of mutations by chemicals. In the 1970s, large screening trials were conducted, mainly by Japanese groups who tested a large number of plant and spice extracts for putative protective properties in bacterial mutagenicity tests (Kada et al., 1982). In these early years, mainly directly acting mutagens (e.g. alkylating agents and radiomimetic compounds, such as 4-nitroquinoline 1oxide were used to invoke damage. Since humans are not exposed to these compounds under normal conditions, it is problematic to assess the human relevance of these experiments. Important insights into the cancer protective effects of dietary constituents came also from the pioneering work of Wattenberg and his co-workers who demonstrated that several plant constituents (e.g. metabolites of glucosinolates, allyl sulfides, diterpenoids and many more) prevent the induction of neoplasia caused by PAHs (Wattenberg, 1985, 1992). Since the anti-carcinogenic effects could in most cases be attributed to the induction of glutathione-S-transferase (GST), which protects against DNAreactive metabolites, it became apparent that the prevention of tumour formation by these compounds is related to DNA-protection. The efforts to identify protective food constituents were also strongly stimulated by epidemiological studies, which indicated that the diet plays a major role in the etiology of human cancer. According to the estimates of Doll and Peto (1981) and Doll (1992) approximately 30±35% of all human cancers are related to nutritional factors and might be prevented by dietary strategies. Steinmetz and Potter compiled and evaluated the results of a large number of epidemiological studies which showed that consumption of certain types of vegetables is inversely related with cancer incidence in humans (Steinmetz and Potter, 1991). Many of the plant groups, which reduce cancer incidence in humans, such as Brassicas and Allium species, contain compounds that possess strong antimutagenic effects. In the following chapter we will give a brief overview of the most relevant modes of action of antimutagens and describe the most commonly used in vitro and in vivo approaches as well as human biomonitoring models. The subsequent sections concern extrapolation problems encountered with different methodologies and describe new research strategies and the development of new tools for future research.

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20.2 Methods for identifying antimutagenic constituents in foods 20.2.1 Mechanisms of DNA-protection In the 1980s attempts were made to classify antimutagens in regard to their mode of action: Kada et al. (1986) designated compounds acting outside the cell as `desmutagens' and compounds which act intracellularly as `bioantimutagens'. According to the time schedule used in anticarcinogenicity studies `suppressing agents' are effective when they are administered before the carcinogen whereas `blocking agents' cause protection after exposure of test animals to a cancer inducing agent (Wattenberg, 1992). Since the formation of initiated cells is considered to be causally related to induction of DNA-damage, antimutagens fall into the first category while blocking agents counteract tumour promotion and progression and may act via mechanisms other than DNA-protection. De Flora and Ramel (1988, 1990) published a schematic overview on the different mechanisms of antimutagens and anticarcinogens (see Fig. 20.1). Table 20.1 is based on this scheme, but is confined to the most relevant DNA-protective mechanisms elicited by dietary constituents. Antioxidants are typical examples for compounds, which inactivate DNA-reactive molecules, and it is notable in this context that reactive oxygen species (ROS) also play an important role in tumour

Fig. 20.1 Overview of different modes of action, by which dietary compounds prevent DNA damage.

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Table 20.1 Mechanisms of antimutagens and anticarcinogens Mode of action

Examples

Inhibition of formation of mutagens

Inhibition of endogenous nitrosamine formation by vitamins and plant phenolics Binding of polycyclic aromatic hydrocarbons and heterocyclic aromatic amines by fibres and Lactobacilli Short-chain fatty acids interfere with the cellular uptake of dimethylnitrosamine Putrescin prevents the cellular uptake of paraquat Inhibition of -C hydroxylation of nitrosamines by glucosinolate metabolites Induction of PAH-metabolites by phenolic acids Scavenging of reactive oxygen species. Induction of glucuronosyl-transferase by isothiocyanates and cruciferous vegetables. Induction of glutathion-S-transferase by coffee specific diterpenoids. Spice ingredients such as cinnamaldehyde, vanillin, etc.

Direct binding of mutagens Inhibition of cellular uptake

Inhibition of activation of promutagens Inactivation of DNA-reactive molecules Induction of detoxifying enzymes

Modification of DNA-repair and replication

promotion. Many of the currently known antimutagens act simultaneously on different levels and this feature enhardens the assessment of the relevance of a specific mode of action in humans. For example, the coffee-specific diterpenoids kahweol and cafestol have a strong impact on a variety of xenobiotic drug metabolising enzymes and also induce a specific form of DNA-repair (Huber et al., 2002a, 2002b, 2003). Also the degradation products of glucosinolates, carotenoids and allyl sulfides are typical examples of compounds which cause protection on different levels (Dragsted et al., 1993; Watzl and Leitzmann, 1999). Most of the DNA-protective compounds, on which research has focused during the last years, are of plant origin. These bioactive compounds have no direct nutritional value such as micro- or macronutrients. Many of them protect plants against bacteria, fungal infections and insects, therefore they can be regarded as `natural pesticides' (Ames et al., 1990). Typical examples are pungent spice ingredients, allyl sulfides in Allium species, glucosinolates in cruciferous vegetables and benzoxazinones in Gramineae. The latter groups are present in intact plants in biologically inactive form as glucosides; upon destruction of the cell wall, highly bioactive and toxic molecules are formed enzymatically. For mammals and man, all these plant constituents are xenobiotics and as such eliminated via reactions mediated by phase I and phase II enzymes. Phase I reactions are catalysed mainly by cytochrome P-450 enzymes and lead to formation of electrophiles which undergo conjugation reactions triggered by phase II enzymes such as UDP-glucuronosyltransferase (UGT), GST, N-acetyltransferase (NAT) and sulfotransferase (SULT) (Oesch

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and Arand, 1999). The detoxification products are excreted via the bile or the urine. Many genotoxic carcinogens such as aflatoxins, PAHs and HCAs are biologically inactive per se and require conversion to DNA-reactive intermediates by phase I reactions, in some cases (e.g. HCAs) also phase II enzymes (e.g. SULT and NAT) are involved in the activation reactions (King et al., 2000). Many plant constituents inhibit activation reactions or act via induction of detoxifying (phase II) enzymes (Fig. 20.1). The molecular mechanisms upregulating the activity of xenobiotic drug-metabolising enzymes have been investigated in a number of studies (see for example Kensler, 1997, Prochaska and Talalay, 1988). As initially proposed by Wattenberg (1985), two groups of enzyme inducers exist, namely bi- and monofunctional agents. Bifunctional inducers (e.g. flavones) elevate the activity of phase II and of phase I enzymes. They are planar polycyclic aromatics, which are ligands for the Ah-receptor and increase enzyme activity directly through the xenobiotic response element (XRE). Monofunctional inducers (e.g. isothiocyanates) transcriptionally activate inducible phase II enzymes via the antioxidant response element (ARE) but have no impact on phase I enzyme activities. No specific structural characteristics of these agents have been identified so far. Since Ah-receptor-dependent phase I enzymes can activate pro-carcinogens, monofunctional inducers are more desirable candidates for chemoprevention in man. Another promising mechanism of DNA-protection is the interaction of dietary constituents with DNA-repair and -recombination processes. Kada and his coworkers showed that flavourings (e.g. coumarin, vanillin, cinnamaldehyde and tannins) protect bacteria against UV and chemically induced DNA-damage (Ohta, 1993, Shimoi et al., 1985). Since this phenomenon was seen only in repair proficient strains and not in derivatives lacking specific repair functions, it was concluded that the protective effects are due to interaction of these compounds with DNA-replication and/or repair processes. Also caffeine elicited protective effects under specific experimental conditions, which were attributed to its interaction with DNA-repair processes. In subsequent experiments it was found that the aforementioned compounds were also protective in cultured mammalian cells, when they were added to cultured cells after induction of DNA-damage (Ohta, 1993; Sanyal et al., 1997) and some mechanistic data are available which suggest that vanillin promotes DNA-rejoining catalysed by polymerase (Sasaki et al., 1990). It was suggested that the effects of caffeine could be due to its interaction with mismatch repair (Beland and Kadlubar, 1990), but newer findings did not report on such a relation (Kumar et al., 2001; Vaidya and Pasupathy, 2001). However, data on the protective effects of different spice ingredients and tannins in laboratory rodents are scarce and depend strongly on the experimental design. Therefore it is unclear at present if protective effects can be expected in humans. Recently, Huber et al. (2003) reported on results obtained with the coffee specific diterpenoids kahweol and cafestol which increased the activity of O6-methylguanine-DNA-methyltransferase (MGMT) in rat liver. MGMT protects against DNA-alkylating

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agents such as dietary nitrosamines and mice overexpressing this enzyme are less susceptible to xenobiotic tumourigenesis (Becker et al., 2003). 20.2.2 Methodological aspects Overview on endpoints used in antigenotoxicity experiments The formation of mutations is a complex process. It begins with primary lesions of the DNA (e.g. DNA-adducts, breaks or base dimers), which are either repaired or lead to cell death or replication of the damaged cells. The latter process finally results in the phenotypical manifestation of mutations. Mutations can occur either in individual genes (point mutation or frameshifts) or take place on the chromosomal level (structural and numerical chromosomal aberrations). The induction of aneuploidy is not caused by alterations of the DNA but by disturbances of the spindle apparatus. As mentioned above, numerous experimental procedures have been developed which enable the detection of DNA-alterations. According to the scheme described above, these approaches can be grouped into three major categories, namely (i) trials which detect primary damage (ii) indirect methods which detect phenomena associated with DNA-alterations in particular processes associated with DNA-repair and (iii) experiments which detect mutations in a strict sense (see Fig. 20.2). Additionally, chemical methods have been developed which provide indirect evidence for potential DNA-damaging or protective activities, e.g., the effects of ROS and antioxidative activities can be monitored in lipid peroxidation measurements and by use of spectrophotometric methods, the DNA-reactivity of compounds can be monitored in vitro in reactions with individual DNA-bases and enzyme measurements provide information on processes which lead to DNA-protection. The major advantage of indicator assays, which detect primary DNA-damage or `epiphenomena' causally related to DNA-alterations, is that no cell divisions are required. Therefore such measurements can be carried out with non- or slowly dividing cells in culture or with primary cells isolated from various inner organs. The formation of oxidised DNA-bases can be monitored either in cells from various organs or in the urine. In most experiments the formation of 8-OHdG is measured by HPLC. For the measurement of DNA-adducts the 32P-postlabelling method is most frequently used. DNA is extracted from a variety of organs and split enzymatically into nucleotides, which are labelled with phosphor, subsequently the unchanged bases are removed and the bases with adducts are analysed by thin layer chromatography (for a detailed description see Randerath and Randerath, 1991). Specific DNA-bases can also be detected and quantified immunologically by use of antibodies. The UDS test measures DNA-synthesis caused by repair processes and is based on the autoradiographic determination of incorporation of 3H-labelled thymidine into DNA (Williams, 1977). The Single Cell Gel Electrophoresis Assay (SCGE or comet assay) is increasingly used in in vitro and in vivo studies with rodents and in human intervention trials. In this approach, the formation of `comets' caused by the

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Fig. 20.2 Classification of different endpoints used in genotoxicity/antigenotoxicity studies.

migration of DNA in an electric field is used to quantify DNA-damage (see Fig. 20.3). The comet assay allows the detection of single- and double-strand breaks, apurinic sites and conformational alterations of the DNA. In order to assess which lesions account for comet formation, DNA-restriction enzymes can be used which discriminate between oxidised purines, pyrimidines and bulky adducts (Collins et al., 1996). At present, attempts are made to validate and standardise this relatively new technique which is also increasingly used in antimutagenicity studies (for a description of the guidelines see Tice et al., 2000, Hartmann et al., 2003). In gene mutation assays with bacteria (e.g. in the Salmonella/microsome assay) amino acid auxotrophy (e.g. hisÿÿ!his+) or resistance towards antibiotics are used as endpoints. In experiments with mammalian cells, resistance towards antimetabolites, which interfere with the synthesis of DNAbases, is employed for the detection of mutations (e.g. TGr, HPRT). In higher organisms (plants, insects, mammals) gene mutations can be detected by scoring for morphological alterations in somatic cells (e.g. eye colour or wing spots in Drosophila, spot test in mice, stamen hair mutations in Tradescantia). These

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Fig. 20.3 COMETS, which are formed as a consequence of DNA migration in an electric field. The SCGE assay is increasingly used in DNA protection studies in humans.

changes can be monitored either in the exposed parental generations or by analysis of the progeny. Typical examples for such assays are the specific locus test with mice, the sex linked recessive lethal assays with Drosophila melanogaster and gene mutation experiments with Arabidopsis thaliana. The detection of gene mutations in the inner organs of rodents became possible with the construction of transgenic animals, which carry bacterial genes encoding for lactose synthesis, such as lacI or lacZ (Gossen et al., 1989; Kohler et al., 1990a, 1990b). Following the treatment of the animals, the inner organs are removed. After the extraction of the DNA the target sequences are excised and transferred via vectors into bacteria for further analysis. However, these procedures are quite costly and time consuming and currently not suitable for screening trials with putative protective dietary components. Gene mutations can also be detected in HPRT assays which are conducted with rodent cells or with human T-lymphocytes (Chu and Malling, 1968; Cole and Skopek, 1994). Chromosome mutations can be detected microscopically in metaphase cells. By use of fluorescent in situ hybridisation (FISH), individual chromosomes or chromosome regions can be distinguished, and alterations such as translocations can be detected easily. Micronucleus (MN) formation results either from chromosome breakage (clastogenicity) or aneuploidy and the scoring of these extranuclear bodies is by far less time consuming than conventional karyotype analysis. By use of FISH-probes, which bind to the centromer region, it is possible to discriminate between MN caused by chromosome breakage (clastogenicity) and aneuploidy (Pinkel et al., 1986). Inter- and intra-chromosomal recombination processes can also alter the expression of genes and it has been postulated that this mode of action is involved in the induction of cancer (Schiestl, 1989; Schiestl et al., 1994). Several test systems have been developed which enable the detection of such events, for example Somatic Mutation And Recombination Tests (SMART) with Drosophila or the spot test with mice (Fahrig, 1975; Fahrig and NeuhauserKlaus, 1985) as well as specific assays with Saccharomyces cerevisiae (Fahrig, 1984). Also the formation of sister chromatid exchanges apparently arises from recombinogenic effects (Perry and Thomson, 1984). As mentioned above, it is

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well known that gene and chromosome mutations are causally related to cancer induction and other diseases. The role of aneuploidy in the induction of cancer is still not fully understood, but evidence is accumulating that certain forms of cancer, for example leukaemia, are causally related to this phenomenon (Ohshima and Bartsch, 1994; Sen, 2000). Other endpoints, such as DNA-adduct formation, formation of oxidised bases or induction of repair processes and DNA-migration (which is monitored by the SCGE assay) are not necessarily related to persisting mutations. For example `comets' disappear as a function of time due to repair processes (Collins et al., 1997a) and it has been shown for HCAs that the organ specific formation of DNA-adducts does not correlate with mutation induction in the inner organs of mice (Ochiai et al., 1998). It should be kept in mind that indication for the prevention of `true mutations' has a higher predictive value in regard to protective effects in humans, than the detection of indirect phenomena associated with DNA-damage; in other words the different endpoints used in antigenotoxicity studies are not equal in regard to their reliability as predictors for cancer prevention. In vitro approaches for the detection of DNA-protective effects The most commonly used in vitro test systems for the identification of antimutagens are summarised in Table 20.2. Since the structure of the genetic material is similar in all organisms, a broad variety of indicator cells including prokaryotes, plants and insects can be used in genotoxicity tests (see Fig. 20.4). The most commonly employed method for the detection of antimutagens are bacterial test systems, in particular the Salmonella/microsome assay which was developed by Bruce Ames (for a description of the method see Maron and Ames, 1983). These experiments are easy to perform and standardised protocols are available. The major advantage of bacterial based assays is that high cell densities, which are required for the detection of mutations, can be achieved within a short cultivation period. Recently we evaluated the database on dietary compounds which protect against HCAs (Schwab et al., 2000). More than 600 individual compounds and complex mixtures were tested and approximately 95% of the results come from bacterial in vitro tests. Also other dietary compounds, which protect against other classes of genotoxic dietary carcinogens (aflatoxins, polycyclic aromatic amines and nitrosamines), were mainly tested in bacterial systems. One of the major disadvantages of antimutagenicity tests with bacterial cells and also of trials with conventional mammalian cells, which are used in routine genotoxicity testing, such as V-79 and CHO cells, is that these indicator cells do not possess certain important drug metabolising enzymes, i.e., they lack certain phase I enzymes that catalyse the activation of promutagens. Also phase II enzymes are not or only moderately expressed (Wiebel et al., 1984; Wiebel, 1993), therefore exogenous enzyme homogenates from rodents and co-factors (e.g. S-9 homogenate) are added to mimic the activation of procarcinogens in rodents. As a consequence, these test systems do not reflect the induction of detoxifying

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Fig. 20.4 Formation of his+ revertants as a consequence of gene mutations in Salmonella typhimurium strains (Salmonella/microsome assay). The right part shows an untreated plate, the left part the increase of revertant colonies as a consequence of chemical induction of mutations.

phase II enzymes and it is also unclear whether or not inhibition of phase I enzymes takes place intracellularly (KnasmuÈller et al., 2002). We recently showed that changes in the molarity and of the pH of the enzyme homogenates decrease their activation capacity, therefore any compound which causes such effects will be classified as an antimutagen (Schwab et al., 2000). It has also been postulated by Rutten and Gocke (1988) on the basis of experimental data obtained with cinnamaldehyde that compounds that cause division delays, which are not usually taken into consideration in screening trials, mimic false positive antimutagenic effects. Fig. 20.5 shows the differences between the representation of metabolic pathways in conventional cells (lacking drug metabolising enzymes) and in primary cells. When we compared the results of bacterial antimutagenicity tests with data from in vivo experiments with rodents it became apparent that many compounds which act as antimutagens in vitro are ineffective or even cause synergistic effects in the living animal (Schwab et al., 2000). On the basis of this observation we concluded that only simple mechanisms such as direct binding effects but not protective mechanisms, which involve alterations of the metabolism of genotoxic carcinogens, can be adequately detected in conventional in vitro assays. Promising alternatives are the use of stable cell lines which have retained the activities of enzymes that are usually lost during cultivation, such as the Reuber rat hepatoma line (Roscher and Wiebel, 1988), or the human derived liver cell lines Hep3B and HepG2 (Knowles et al., 1980). In experiments with these cell

Table 20.2 In vitro test systems used in antigenotoxicity studies Indicator system

Endpoint

Advantages/disadvantages

Examples

Induction of amino-acid auxotrophy (Salmonella/ microsome test or E.coli WP/2 strains) or comparison of strains with different repair capacity

Lack of adequate representation of drug metabolising enzymes false positive results due to inactivation of S9-mix and division delays. Fast and cost effective; can be used for screening trials

Investigation of protective effects of fibres and pigments against heterocyclic amines (HCAs), for review see Schwab et al. (2000)

Tests with conventional mammalian cell lines (V-79, CHO)

Micronucleus induction, chromosomal aberrations, unscheduled DNA-synthesis, comet formation and induction of gene mutations (e.g. HPRT)

Drug metabolising enzymes are not represented. Chromosomal aberration analysis is very time consuming.

CA-experiments were used in earlier studies of protective effects of vitamins towards PAHs.

Primary cells: hepatocytes, colon cells and primary lymphocytes

Comet formation, micronuclei, sister chromatid exchange and chromosomal aberrations

For MN/CA/SCE-experiments cultivation procedures are complicated and not standardised; comet assay is standardised and more often performed. Peripheral lymphocytes have only a low expression of xenobiotic drug metabolising enzymes.

Genotoxicity tests Bacterial mutagenicity tests

Experiments with metabolically competent hepatoma cell lines (HepG2 etc.)

Enzyme induction assays Experiments with hepatoma cells (HepG2; target enzymes, quinone reductase measurements with the murine hepatoma line Hepa 1c1c7)

Comet formation and micronuclei Cells are slowly dividing and induction effects vary in different subclones. Induction of drug metabolising enzymes can be detected

Increasingly used in anti-mutagenicity tests: Chrysin Ô B(a)P (Uhl et al., 2003a), effects of cruciferous vegetables and isothiocyanates (Kassie et al., 2003c)

In HepG2-cells `target-enzymes' involved in the detoxification of mutagens (UGT, GST, etc.) are monitored. In the murine hepatoma line quinone reductase is used.

Flavonoid effects in HepG2-cells (Uhl et al., 2003a) Induction of QR by isothiocyanates (Zhang and Talalay, 1998)

Antioxidative effects Different endpoints which can be Formation of TBARS, TOSC, monitored chemically X/XO, DPPH, TROLOX, etc. (see Aruoma, 2003)

Changes in QR activity do not necessarily reflect the upregulation of other relevant enzymes QR assays can be automatised for screening trials.

Easy to perform, useful for Numerous screening trials have been screening assays, do not provide published (Aruoma, 2003) information on absorption in vivo, often carried out at nonphysiological pH-values

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Fig. 20.5 Schematic representation of antimutagenic effects reflected in in vitro models which require addition of exogenous activation mix (a) and in cells possessing phase I and phase II enzymes (b). In experimental models with enzyme homogenates, DNAor protective compounds may act outside the cells either directly with the mutagens or bind to reactive intermediates . It remains inhibit activating enzymes unclear whether these reactions also take place inside the cells. Detoxifying enzymes are not represented in most models. In primary cells and/or lines which have maintained the activity of drug metabolising enzymes (e.g. HepG2), inhibition of activating enzymes and of metabolites takes place inside the cells and additionally another important mechanism can be detected of chemoprotection, namely the induction of detoxifying enzymes (b). R stands for repair of damaged DNA.

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lines no addition of liver enzymes is required for the activation of promutagens. The largest database is currently available on HepG2 cells. Commonly used endpoints are MN formation (Natarajan and Darroudi, 1991) and DNAmigration (Uhl et al., 1999, 2000). These tests detect the genotoxic effects of a broad variety of dietary carcinogens, including compounds which give negative results in conventional in vitro tests, but are mutagenic and carcinogenic in animal experiments, such as safrol, the pesticide hexamethylphosphoramide (HMPA) and the mycotoxins ochratoxin A and fumonisin B1 (Natarajan and Darroudi, 1991; Ehrlich et al., 2002a, 2002b). Furthermore it was shown that the cells can discriminate between structurally related carcinogens and noncarcinogens, e.g., benzo[a]pyrene (B(a)P) and pyrene. We showed recently in a comparative investigation that Hep3B cells are in general less sensitive towards genotoxins than HepG2 cells, due to their lower enzyme activities (Majer, 2003). The major advantage of the use of these cell lines for the detection of DNA-protective compounds is that phase I and phase II enzymes (for example UGT, GST, NAT, CYP1A) are represented in an inducible form and results from a number of antimutagenicity studies are available (KnasmuÈller et al., 1998; Laky et al., 2002; Uhl et al., 2003a; Kassie et al., 2003a, 2003b, 2003c). Over the past few years, genetically engineered cell lines were constructed that express individual human drug metabolising enzymes and combinations thereof (Crespi, 1995). These cell lines are useful tools for mechanistic studies, but since the enzymes are not inducible, the use of such cell lines does not enable the detection of protective compounds that act via enzyme induction. Edenharder et al. (2002) reported recently on the successful use of such cell lines in antimutagenicity studies with HCAs. Several attempts have been made to use primary cells in genotoxicity and antimutagenicity studies. For example, Anderson and co-workers used peripheral human lymphocytes and sperm cells in comet assays to study the protective effects of flavonoids and other compounds (Anderson et al., 1997). However, the metabolic capacity of these cells is very low, and many genotoxins do not give positive results unless exogenous enzyme homogenate is added. A number of efforts have also been made to use primary liver cells. Eckl and Raffelsberger (1997) developed a cultivation protocol for rat liver cells that enables the detection of chromosomal aberrations (CA) and micronuclei. However, this procedure is not yet standardised and validated, and a major problem is that the activities of the drug metabolising enzymes decline rapidly after the isolation of the cells (Wiebel et al., 1984, Glatt et al., 1987). A promising alternative was developed by Fahrig and co-workers (1998) who used three-dimensional sandwich cultures with human hepatocytes and showed that the cells have high levels of enzyme activities up to two weeks after isolation. However, none of these models has ever been used in antimutagenicity studies to our knowledge. Prohaska and Talalay (1988) developed a test with a murine hepatoma cell line (Hepa 1c1c7), which does not focus on the detection of DNA-protection but on the induction of protective enzymes. The test system is based on the

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spectrophotometric measurement of NAD(P)H:quinone reductase (QR) in microtitre plates and suitable for screening trials. The rationale behind this approach is the assumption that QR is induced via the same regulatory pathway as other drug metabolising enzymes and can be used for the identification of inducers. A large number of compounds, in particular isothiocyanates, have been tested with this system (Zhang and Talalay, 1998). One of the most potent compounds was sulforaphane, which is contained in broccoli and other cruciferous plants. A further development of this model is a derivative of the cell line that is less susceptible to bifunctional inducers and can be used to distinguish between mono- and bifunctional compounds (GerhaÈuser et al., 1997, Prochaska and Talalay, 1988). However, a comparative study with HepG2 cells showed that strong quantitative differences exist between GST-induction (one of the most important detoxifying enzyme systems) and QR-induction and it has been postulated that these two enzymes are independently regulated in human cells (Williamson et al., 1997). Several in vitro methods have been developed enabling the detection of antioxidative effects, which are not based on the measurement of oxidation of DNA, but on chemical-physical approaches. Examples are the determination of oxygen radical absorbance capacity (ORAC), ferric reducing antioxidant power (FRAP), total oxidant scavenging capacity (TOSC), trolox equivalent antioxidant capacity (TEAC), the 1,1-diphenyl-2-picrylhydrazil (DPPH-assay) and the xanthine/xanthine oxidase (X/XO). Since some of these assays are performed at non-physiological pH-values, it is problematic to extrapolate the results to the physiological environment (for review see Aruoma, 2003). Furthermore it is important to note that many compounds, which are highly effective in these in vitro models, are poorly absorbed from the intestinal tract and therefore are inactive in inner organs. Typical examples are anthocyans, chlorophylls and curcumin (and its metabolites) from turmeric, which is one of the most active radical scavengers in vitro (Srinivas and Shalini, 1991). In vivo tests In the `old days', i.e., in the 1970s and 1980s, the most frequently used endpoints in antimutagenicity studies with rodents were bone marrow micronucleus assays with polychromatic erythrocytes and chromosomal aberration experiments with peripheral blood cells. These endpoints are sensitive towards radiation and directly acting compounds, such as alkylating agents and cytostatic drugs, but they are quite insensitive towards important classes of dietary carcinogens, such as nitrosamines and HCAs. Another disadvantage of these models is that they do not provide information on target organs of tumour formation. The alkaline elution method, which was developed in the 1980s, is based on the determination of DNA-fragmentation by use of filters and radiolabelled thymidine and enables to carry out measurements in a variety of inner organs of rodents. However, the machinery required is expensive and complicated and this method was hardly ever used in antimutagenicity trials. Another approach,

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which was already developed in the 1960s, is the host-mediated assay, in which indicator organisms (mainly bacteria) were injected intrasanguineously or intraperitoneally into chemically treated rodents. The indicator cells were recovered after short exposure periods (one to two hours) and plated on selective media for analyses of DNA-damage. By development of isogenic E. coli-strains differing in their DNA-repair capacity, it was possible to carry out viability counts with cells recovered from a variety of inner organs and this procedure was used successfully in a number of experiments (KnasmuÈller and Mohn, 1986; KnasmuÈller et al., 1986, 1989). However, with certain compounds, e.g., HCAs, the bacterial metabolism influenced the results of the experiments and comparisons with data from DNA-adduct measurements indicated that the findings of host mediated assays do not reflect the situation in inner organs (KnasmuÈller et al., 1996). The detection of gene mutations in transgenic animals is briefly described in section 20.2.2. This approach enables measurements in multiple organs, but the method is expensive and has been used only in a few protection studies with HCAs (Dashwood, 2002; Yang et al., 2003). Another example are the experiments of Manjanatha et al. (2003) who investigated the effects of isoflavones and -estradiol on DMBA induced gene mutations in different organs of transgenic animals; Heddle et al. (2003) reported on the effects of a Western-style diet on the mutation spectra in the lacI gene in mice and found no impact on the mutation frequencies. At present, the most widely used technique for antigenotoxicity studies with rodents is the SCGE-assay. Methods have been developed which enable measurements in a broad variety of organs. Initially, intact cells were isolated with specific techniques after chemical exposure of the animals (genotoxin putative Ô antigenotoxin). After viability determination, which is a critical parameter to obtain reliable results, the cells are transferred to agarose coated slides, lysed, submitted to electrophoresis and scored for induction of comets (McKelvey-Martin et al., 1993). Sasaki et al. (2000) developed a faster protocol, where the organs are homogenised and only the nuclei isolated for comet analysis. Potential acute toxic effects have to be assessed with this approach by histopathology. As endpoints, different parameters, such as tail length, percentage of DNA in tail and tail moment are used in SCGE-experiments and it is still a matter of debate which of them is the most suitable. Guidelines for the comet assay, which are also relevant for protection studies, have been published by Tice et al. (2000) and Hartmann et al. (2003). Information on the statistical analysis of the data is given in the articles of Duez et al. (2003) and Wiklund and Agurell (2003). In most antimutagenicity studies, liver and colon were used as target organs. Typical examples for protection studies with the SCGE technique are experiments with Lactobacilli, fibres, Brassica vegetables and prebiotics (Wollowski et al., 2001; Kassie et al., 2002; Humblot et al., 2002). DNA-adduct measurements can be used for analysing protective effects towards HCAs, PAHs, aflatoxin, and other adduct forming carcinogens. They enable the measurement of organ specific effects and have been used in a

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number of protection studies, for example Huber et al. (1997) studied the inhibition of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-DNAadduct formation in the liver and colon of rats with a variety of different dietary constituents. The SMART wing spot test with Drosophila melanogaster was also used in a few antimutagenicity studies. Protective effects were seen, for example, with coffee and chlorophyllin and its constituents towards HCAs (Negishi et al., 1994) and with turmeric against urethane (el Hamss et al., 1999). Human biomonitoring studies For many years, the most widely used techniques to monitor human exposure to genotoxic carcinogens were cytogenetic experiments with peripheral lymphocytes. These methods were widely used in occupational exposure studies and in experiments concerning the effects of lifestyle factors such as alcohol consumption and smoking. A number of studies were conducted in which the potential protective effects of vitamin supplementation were investigated (IARC, 1986; Wang, 1999; Nyberg et al., 2003). Also radioprotective effects were studied in humans with these techniques. Karyotype analysis is a very time-consuming procedure and a possible alternative is the MN assay with peripheral lymphocytes. Since MN-formation requires cell proliferation, the use of cytochalasin B, which leads to the formation of binucleated cells, ensures that cells used for MN-scoring, have undergone nuclear division (see Fig. 20.6). Typical examples for protection studies are described in the articles of Fenech and co-workers (1994, 1997b, 1998) who showed that folate in combination with vitamin B6 and B12 supplementation led to a decrease of the MN-frequency in the Australian population; in another study they showed a reduction of MN in peripheral blood cells by wine consumption (Fenech et al., 1997a). Micronuclei can also be monitored in exfoliated cells from the oral and nasal cavity, bladder, cervix and oesophagus. It has been stressed that the majority of human cancers (>90%) are of epithelial origin (Cairns, 1975) and that this simple method might have an equally or higher predictive value than studies with blood cells. A number of chemoprevention trials have been conducted with exfoliated cells, most of them with buccal mucosa cells. Experiments with vitamin E, -carotene and vitamin A showed that increased MN-frequencies, and premalignant lesions caused by smoking, snuff or betel chewing declined after the interventions. In other studies it was shown that the incidence of MN caused by Schistosoma infections in bladder cells decreased as a consequence of medical treatment. A review on the results of intervention trials with exfoliated cells is given in the article of Majer et al. (2001). At present, the most widely used approach for the detection of dietary DNAprotective compounds are SCGE experiments with peripheral blood cells. Since strong inter-individual variations exist, the most adequate study design is the use of intervention (cross-over) trials. The currently available database is summarised in the reviews of Moller and Loft (Moller et al., 2000; Moller and Loft, 2002). Mainly

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Fig. 20.6 Formation of micronuclei in binucleated human hepatoma (HepG2) cells. MN are a commonly used endpoint in vitro and in rodent studies, and can also be used in human intervention trials.

vitamin supplements were investigated, additionally also results from experiments with fruits, vegetables and beverages (e.g. green tea) are available. It is important that the results of intervention studies depend strongly on the use of controlled diets and that the number of subjects involved as well as seasonal variations, gender and age have an impact on comet formation. Furthermore, it was stressed that the inclusion of placebo groups and washout periods would substantially strengthen the scientific value of such studies. It is also possible to use lymphocytes collected before and after the intervention to investigate the sensitivity of the blood cells towards oxidative damage caused by exposure to either hydrogen peroxide (Moller and Loft, 2002) or carcinogenic chemicals (HoÈlzl, 2003). The use of restriction enzymes (endonuclease III and formamidopyrimidine DNA glycosylase) enables discrimination between the extent of induction of endogenously formed oxidised purines and pyrimidines (Collins et al., 1996). The article of Nyberg et al. (2003) is the first study in which the impact of dietary factors on somatic mutation frequencies was monitored in humans by use of the HPRT-assay with T-lymphocytes. They found that the mutation frequencies were significantly reduced in relation to consumption of citrus fruits, vegetables and berries. For carotenoid consumption a significant U-shaped association was observed. Another important parameter for DNA-protection, which can be used in intervention trials, are DNA-adduct measurements. Currently available methods allow the quantification of adducts caused by environmental or occupational exposure to PAHs and aflatoxins. For other adduct-forming carcinogens it is difficult to carry out measurements in humans. Since humans are exposed only to very low levels of HCAs via the diet, the detection of DNA-adducts formed

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by these compounds is extremely difficult, although model studies with accelerated mass spectrometry (AMS) with 2-amino-3,8-dimethylimidazo[4,5f]quinoxaline (MeIQx) were successful (Turteltaub et al., 1997). The latter method, however, is by far too expensive to be used in protection studies. Bacterial mutagenicity tests have been used to prove that dietary components alter the excretion of genotoxins; for example Malaveille et al. (1996, 1998) showed that plant phenolics are probably responsible for the reduction of urinary mutagenicity in smokers. They also showed that this effect is paralleled by a decrease of DNA-adducts in the bladder. With HCAs, a number of studies has been published showing that urinary mutagenicity, which can be monitored after consumption of HCA-containing fried meat in an amine-sensitive Salmonella typhimurium tester strain (YG1024), is altered by consumption of Brassica vegetables or lactobacilli (Tavan et al., 2002; DeMarini et al., 1997; Steinkellner et al., 2001; Ohyama et al., 1987; Murray et al., 2001; Reddy and Rivenson, 1993). The interpretation of the biological consequences of alterations of urinary mutagenicity is problematic. For example, a decrease of HCA-induced effects is not necessarily indicative of a protective effect as it cannot be excluded that the decrease is due to enhanced formation of electrophilic metabolites that bind to DNA. A more promising approach are measurements of specific metabolites whose biological relevance is known. Frandsen et al. (2002) developed a highly sensitive method that enables the monitoring of the urinary excretion of 5-OHPhIP, a derivative of the DNA-reactive precursor, a nitrenium-ion formed from the N-hydroxylamine, which is considered to be the ultimate carcinogenic metabolite. Knize and co-workers developed a protocol for the determination of N2-OH-PhIP-N2 glucuronoide, the main detoxification product of this abundant HCA (Kulp et al., 2000). They also showed in preliminary studies that the consumption of broccoli leads to an increase of the urinary excretion of this detoxification product. Also the inhibition of endogenous nitrosamine formation can be monitored in humans in the urine using a non-toxic derivative as a model for other nitrosamines. Uptake of the amino acid proline and simultaneous consumption of nitrite containing red beet juice results in urinary excretion of the non-carcinogenic nitrosamine nitrosoproline (NPRO) and it was demonstrated that additional intervention with dietary constituents which prevent endogenous nitrosation reactions, e.g. with vitamin C, leads to a substantial decrease of NPRO excretion (Bartsch et al., 1990). The prevention of oxidative DNA-damage can be monitored by analysis of 7hydroxy-8-oxo-20 -deoxyguanosine (8-OHdG) in the urine and this methodology has been used in a number of intervention trials (for review see Moller and Loft, 2002). It is also possible to monitor 8-OHdG levels in peripheral lymphocytes and sperm cells. Inter-laboratory comparisons showed that the results obtained with this method depend strongly on the experimental protocol used, and that oxidation processes in the samples may have a strong impact on the outcome. At present efforts are being made to develop a reliable and standardised protocol for this approach.

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Etheno()-modified DNA-bases (dA, dC, dG) are generated by reactions of DNA with a major lipid peroxidation product, trans-4±hydroxynonenal, and reflect oxidative stress. A highly sensitive immunoaffinity-HPLC-fluorescence method for dA measurements has been developed by Nair (1999), and recently results from an intervention study were published (Hanaoka et al., 2002), in which a trend towards decreased dA could be found in subjects on a low salt ± high antioxidant diet (Hanaoka et al., 2002). Enzyme measurements, e.g., GST and UGT-measurements, can be carried out with samples of individuals before and after dietary prevention and provide valuable information on protective effects towards specifical chemical carcinogens which are detoxified via these enzymes. It was shown that coffee and Brassica vegetables increase the activity of GST in humans and in another study it was demonstrated that the activity of UGT which plays a crucial role in the detoxification of HCAs, can be induced (Pantuck et al., 1984; Steinkellner et al., 2001). Effects of nutrients on the antioxidant's defence system can be monitored by measurement of specific enzymes (glutathione-peroxidase, superoxide dismutase) and indirectly by determination of the ratios of reduced/oxidised glutathione in blood.

20.3 Limitations of methods for identifying antimutagenic compounds Over the last 50 years, a huge bulk of information (several thousands of articles) has accumulated which concerns the identification of DNA-protective compounds. In most cases, the main aim was to identify dietary components, which should facilitate the development of nutritional strategies that protect humans against DNA-damage and its consequences. However, the extrapolation of the results of antigenotoxicity experiments in vitro and with laboratory animals to protective effects in humans is a critical issue that depends strongly on the experimental model used (type of indicator organism, endpoint) and also on the conditions (effective dose of the putative compound, choice of the genotoxin, etc.). We discussed some of the limitations of the different methods in the preceding section; in the following we shall briefly address additional important points. 20.3.1 Limitations of the predictive value of different endpoints and test systems As mentioned above, in vitro models with indicator cells lacking activating/ detoxifying enzymes reflect only `simple' protective mechanisms (direct binding of genotoxins and their DNA-reactive metabolites), but not protective modes of actions, which are due to alterations of the metabolism of DNAreactive carcinogens. A promising alternative is the use of primary cells or cell lines that retain the activity of xenobiotic drug metabolising enzymes in an

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inducible form. However, a further limiting factor for all in vitro approaches is that they do not take into account the uptake of dietary anti-genotoxins in the intestinal tract. Also potential effects of the intestinal microflora are not represented in the currently used in vitro approaches. Many dietary supplements which are highly effective in in vitro tests and which are at present marketed are only poorly absorbed in the GI-tract and it is unlikely that they lead to beneficial effects in internal organs. In general, more relevant information can be expected from the results of experiments with laboratory rodents. The development of experimental models with human flora associated animals enables to mimic the impact of the intestinal human microflora (Mallett et al., 1987). As mentioned above, the SCGE-assay is at present one of the most widely used methods to assess prevention of DNA-damage in rodents and also in humans. We recently showed in model experiments that the prevention of HCAinduced DNA-migration in rats by a cruciferous vegetable (garden cress) is paralleled by a decrease of the formation of preneoplastic lesions in liver and colon (Kassie et al., 2002). This indicates that the comet assay indeed enables conclusions to be drawn on cancer-protective effects. However, this may not necessarily be true for studies, in which the impact of dietary factors on endogenous or ROS-induced damage is monitored. It is known that the comet assay is extremely sensitive to oxidative damage, but such lesions may be repaired and have no significant consequences in terms of health risks (Collins et al., 1997b). Collins et al. (1998) investigated the 8-oxo-dG levels in different European populations and found no significant association between specific forms of cancer and oxidised DNA base levels in the lymphocytes, whereas a significant association was seen with the incidence of coronary heart disease. On the other hand, a large number of data indicates that ROS are causally involved in the induction of initiated cells and also in tumour promotion (Athar, 2002). Taken together, it is still difficult to establish direct links between certain endpoints and human health risks, therefore the results of antigenotoxicity studies cannot be taken as firm proofs of beneficial health effects in humans, but only as an indication which requires further verification in studies with humans or in laboratory experiments, in which prevention of cancer and other diseases is used as a firm endpoint. 20.3.2 Specificity of protection In many older studies, genotoxins were used to which humans are not exposed under normal conditions, and no attempts were made to specify the molecular mechanisms that account for the protective effects, therefore the results of these experiments are mainly of academic interest. In past years, the number of DNAprotection studies increased, in which representatives of compounds were used that are considered to be involved in the etiology of human cancer, such as nitrosamines, PAHs, HCAs, aflatoxins, heavy metals, and ROS and strong efforts were made to clarify the underlying mechanisms. In this context it is important to mention the risk assessment of Lutz and Schlatter who attempted to

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rank the importance of different dietary factors in regard to human cancer risks (Lutz and Schlatter, 1992, 2000). It should be kept in mind that the protection spectrum of dietary constituents depends strongly on their mode of action. For example, chlorophylls which act via direct binding inactivate only molecules with planar structures, such as HCAs and PAHs. Compounds which induce GSTs are likely to have a broader protection spectrum, since these enzymes can inactivate a broad variety of DNA-reactive electrophiles (Mantle et al., 1987). Inducers of specific DNArepair enzymes (e.g. involved in excision repair) might be protective against a broad variety of DNA-damaging molecules, but as mentioned above, it is unclear if, and to what extent, results obtained in bacterial tests are relevant for humans. It is possible that the intrinsic formation of mutations is more important in the formation of exogenous (chemical and physical) factors in regard to cancer induction and other health effects, but only a few studies have been conducted in which the impact of dietary factors on endogenous (spontaneous) mutation frequencies were studied. Examples of recent experiments are the in vitro experiment of Klein et al. (2003), who studied the effect of antioxidants on the HPRT-mutation spectrum in cell lines; Heddle (2003) and Zhang et al. (1996) reported on the effect of dietary factors on spontaneous mutation frequencies in transgenic animals. Nyberg et al. (2003) conducted a study in which they investigated the impact of fruit and vegetable consumption on HPRT mutations in humans. 20.3.3 Time-kinetics of protection The time dependency of protective effects by dietary factors is of crucial importance for the assessment of human relevance, but has been neglected in many studies. For example inhibition of DNA-damage and cancer caused by tobacco-specific nitrosamines with isothiocyanates has been attributed to inhibition of -hydroxylation, the first step in the activation of nitrosamines (Hecht et al., 1994). We found that this effect lasts only for a few hours, which would mean that protection could be achieved only when smokers consume Brassica vegetables continuously. On the other hand, the protective effects seen with Lactobacilli towards HCAs last over a longer time period, i.e. up to twelve hours (Zsivkovits et al., 2003; KnasmuÈller et al., 2001; Hayatsu and Hayatsu, 1993). In the case of ethanol, the effect of nitrosamine-induced genotoxicity in rats is biphasic. Simultaneous administration of the alcohol with nitrosamines causes a protective effect (due to competitive inhibition of the activating enzyme CYP2E1), whereas chronic administration over several days leads to an increase of the activity of this enzyme and as a consequence to a strong enhancement of nitrosamine-induced genotoxicity (KnasmuÈller et al., 1994). The induction of detoxifying enzymes (GST, UGT, etc.) takes, in contrast to the inhibition of activating enzymes, much longer (6 hours) and therefore can be detected only

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when the exposure periods are sufficiently long (Lamb and Franklin, 2000; Cho and Kim, 1998). 20.3.4 Dose-effect relationships In many laboratory experiments, high doses of genotoxic carcinogens were used which exceeded by far realistic exposure conditions in humans. The use of such experimental conditions is often necessary, since otherwise no measurable effects can be induced. This is true, for example, for CA, MN and comet-experiments with nitrosamines, PAHs and HCAs. Only for DNA-adduct measurements low exposure levels are sufficient. Also the concentration of putative antimutagens, which were used in protection studies, was unrealistically high in many experiments. For example the effects of isothiocyanates or lactobacilli seen in animal experiments would be achieved in humans only after consumption of 10± 20 litres of yoghurt/p/d or extremely large amounts of Brassica vegetables (Uhl et al., 2003b; Zsivkovits et al., 2003). In order to claim protective effects in humans, it has to be shown that they take place under realistic exposure conditions. The promising results obtained with certain dietary compounds have led to the production of dietary supplements, which contain active ingredients in much lower amounts than those in natural foods. A number of studies indicate that many mutagens act biphasic, i.e., they are protective only at low dose levels but elicit DNA-damage by themselves at higher concentrations. Typical examples are spice ingredients, caffeine, tannic acid, and flavonoids (Uhl et al., 2003a; Sanyal et al., 1997) which caused U-shaped dose response curves in in vitro experiments with human cells. With -carotene similar effects were seen in human studies (Nyberg et al., 2003) and with selenium (Se) in dogs (Waters et al., 2003). A possible explanation for this phenomenon might be that these antimutagens are DNA-reactive by themselves and induce a sort of adaptive response, similar to that seen with low radiation doses, such as induction of DNA-repair enzymes and detoxifying enzymes. For a more detailed discussion of this topic, see KnasmuÈller et al. (2002). The dose-effect relations of DNA-protective compounds are largely unknown; in most experiments only a few dose levels were tested. It may be tentatively assumed that linear dose-effect relations exist for compounds, which directly bind to DNA-reactive molecules, whereas for protective mechanisms caused by enzyme-inhibition or induction, threshold doses are likely to exist.

20.4

Future trends

A retrospective look shows that the quality of anti-mutagenicity studies has improved over the years. For the major part of the scientific community, it has become clear that the initial assumption that any method that can be used for the detection of mutagens can be used also for the detection of anti-mutagenicity, is a false paradigm. As a consequence, attempts have been made to develop in

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vitro tests that reflect a broad variety of protective mechanisms. Another important task concerns the development of human biomonitoring methods. These provide information of specific observations from prevention studies with animals and can also be expected in humans. Improvements of biochemical and chemical analytical methods will enable the measurement of important enzymes related to DNA-damage and of metabolites of carcinogens with known biological effects in humans. The use of genomics technologies (principally DNA-microarrays) in genetic toxicology is still at its early stages and the working group of the ILSI/HESI is currently concerned with the development and validation of this powerful new technology (Newton, 2003; Pennie, 2002, http://hesi.ilsi.org). Some data are already available. In short, attempts were made to characterise the genotoxic responses of selected model compounds by use of microarrays (e.g. induction of DNA-repair enzymes, etc.) and the sensitivity of gene expression changes was compared with that of traditional genetic endpoints. Available data show that the number of genes altered by a particular compound was not as sensitive as more traditional genetic endpoints. These results are probably also relevant for the potential use of DNA-microarrays for the identification of anti-mutagens. Only a few reports on the use of microarrays from chemoprevention studies are available, for example, Izzotti et al. (2003) conducted experiments with Nacetylcysteine (NAC) and other chemoprotective agents with rats and analysed the expression profiles of 4800 genes in different organs. Werle-Schneider et al. (2003) used a customised cDNA-array to study EGCG-induced changes in the expression of 140 different DNA-repair and -related genes and found alterations in 15 different genes. On the basis of these findings, they postulated that the mode of protection of EGCG involves alterations of DNA-repair processes. Another approach is the use of targeted arrays, which enable the monitoring of the expression of oncogenes, tumour-suppressor genes and biotransformation genes in human intervention studies. Such studies are currently in progress at the University of Maastricht (van Breda et al., 2003).

20.5

Sources of further information and advice

A number of books have been published which describe the methodology of mutagenicity-tests. The Handbook of Mutagenicity Test Procedures by Kilbey et al. (1984), has for many years been the best source of information. However, newer techniques, such as FISH and comet-assays, are not described. Other books, describing methodological aspects are the UKEMS-Guidelines (Kirkland, 1990) and Environmental Mutagenesis (Phillips and Venitt, 1995). Articles on antimutagenic effects are published frequently in journals, such as Carcinogenesis, Mutation Research, Mutagenesis, Food and Chemical Toxicology, Environmental Molecular Mutagenesis, Nutrition and Cancer and Cancer Detection and Prevention. The Journal of Epidemiology, Biomarkers and Cancer Prevention focuses mainly on the results of human studies.

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The Conferences on Antimutagenesis and Anticancerogenesis are biannual events, which are supported by the Environmental Mutagen Society, and Mutation Research regularly publishes presentations of these conferences in special issues. Data on antigenotoxicity studies are also frequently presented at the meetings of the Environmental Mutagen Societies. The Institute of Food Research (IFR, Norwich, U.K.) has organised several conferences on nutrition, DNA-damage and cancer and the contributions have been published by the UK Royal Society of Chemistry (http://www.rsc.org). Contributions of a large conference held in Vienna were published in a special issue of Food and Chemical Toxicology (2002, vol. 40/8). Waters et al. (US-EPA) have developed antimutagenicity profiles for a number of compounds. The results appeared in a number of articles (Waters et al., 1990, Brockman et al., 1992, Waters et al., 1998). The International Agency of Cancer (IARC, Lyon, France) has started to publish a series of books on cancer prevention, which include data on DNAprotection.

20.6

References

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21 Glucosinolates and the prevention of cancer F. Kassie, University of Giessen, Germany and S. KnasmuÈller, University of Vienna, Austria

21.1

Introduction

The inverse association between high consumption of fruits and vegetables and the risk of cancer was known as early as 1933.1 However, it is only during the last three decades that active research in this area has been carried out. Among vegetables, the cancer protective effect of cruciferous vegetables is quite remarkable and their consumption is associated with a reduced risk of cancer of the lung, stomach, colon and rectum with a possible reduction in endometrial and ovarian cancer, and a decrease in total cancer incidence.2 The potent anticarcinogenic effect of cruciferous vegetables is ascribed to their characteristic constituents, glucosinolates, which upon hydrolysis give rise to bioactive products, isothiocyanates (ITCs) and indoles. At present, attempts are being made to grow cruciferous vegetables that yield a higher amount of glucosinolates and possibly stronger chemopreventive effects. In this overview the occurrence, chemistry and cancer protective effects of glucosinolates are presented. Also, comments on the future trends of use and consumption of glucosinolates and sources of further information on glucosinolates are included. 21.1.1 Occurrence of glucosinolates Glucosinolates are plant secondary metabolites contained in Cruciferae as well as in at least 500 species of non-cruciferous dicotyledonous angiosperms.3 Among the cruciferous plants, members of the Brassica olearcea species such as cabbage, kale, Brussels sprouts, cauliflower, broccoli and kohlrabi are the most important dietary sources of glucosinolates, the amount and type of

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Table 21.1 Commonly consumed dietary glucosinolates and their sources Glucosinolate

Chemical name

Glucoiberverin

3-(methylthio)propyl cabbage, broccoli, Brussels sprouts, Sauerkraut, turnip 3-(methylBrussels sprouts, cauliflower, broccoli, sulfinyl)propyl turnip, Sauerkraut 4-methylsulfinyl-3- broccoli, horseradish, cabbage butenyl 2-propenyl Brussels sprouts, cabbage, cauliflower, mustard 3-butenyl cabbage, Brussels sprouts, turnip 2-hydroxy-3-butenyl Brussels sprouts, cabbage, turnip benzyl garden cress, horseradish, mustard phenylethyl watercress, turnip, horseradish 2-hydroxy-3-butenyl white mustard indol-3-ylmethyl Brussels sprouts, broccoli, cabbage, mustard, cauliflower, kohlrabi, horseradish 1-methoxyindol-3Brussels sprouts, broccoli, cabbage, ylmethyl horseradish, kohlrabi, cauliflower

Glucoiberin Glucoraphanin Sinigrin Gluconapin Progoitrin Glucotropaeolin Gluconasturtiin [Glu]sinalbin Glucobrassicin Neoglucobraassicin

Occurrence

glucosinolate differing not only among different vegetables but even among different plant organs. For instance, whereas the glucosinoolates gluconastrutiin and glucotropaeolin are contained predominantly in less commonly consumed cruciferous plants, water cress and garden cress, respectively, glucorabrassicin and sinigrin are the main glucosinolates of Brussels sprouts, cauliflower and cabbage. Generally, seeds contain the highest amount of glucosinolates and young plants are richer in glucosinolate content than late vegetative or reproductive stage plants.4 Table 21.1 shows the most studied glucosinolates and their primary dietary sources. 21.1.2 Chemistry and hydrolysis of glucosinolates Chemically, glucosinolates are -thioglucoside N-hydroxysulfates with a sulfur linked -D-glucopyranose moiety and side chain R, the latter being variable and may be alkyl, alkenyl, arylalkyl, alkylthioalkyl, -hydroxyalkyl or indolylmethyl (Fig. 21.1).5 In nature, glucosinolates are stored in vacuoles being separated from the plant enzyme myrosinase. However, when the plant tissue is damaged during cutting, chewing, freeze-thawing or any other form of plant processing, myrosinase becomes free and hydrolysis of glucosinolates takes place. Ingested glucosinolate can be hydrolysed in the gut by myrosinase of the microflora.6 Hydrolysis of glucosinolates leads to the cleavage of the thioglucoside linkage giving rise to D-glucose and unstable thiohydroximate-Osulfonate, which may undergo further reactions through intermolecular rearrangement, yielding nitriles, thiocyanates and ITCs (Fig. 21.1). The glucosinolate hydrolysis products are highly influenced by the side chain of

Glucosinolates and the prevention of cancer 617

Fig. 21.1 Hydrolysis of glucosinolates by myrosinase and formation of isothiocyanates and other products (adapted from Pessina et al.5).

the glucosinolate, the pH of the medium, and the presence of cofactors. For instance, whereas glucosinolates with a side chain of indoles give rise to indoles, glucosinolates with -hydroxy-alkyl side chain oxazolidine-2-thiones generate oxazolidine-2-thiones. Hydrolysis at neutral pH favours the formation of ITCs; at low pH and in the presence of Fe++ ions other compounds such as nitriles, epithionitriles, thiocyanates or oxazolidine-2-thiones may be produced.7 Among the different glucosinolate hydrolysis products, ITCs and indoles are the most important ones with regard to effects on human health.

21.2

The role of glucosinolates in the prevention of cancer

Although glucosinolates are not as such biologically active by themselves, their hydrolysis products, ITCs and indoles, are remarkably potent cancer chemopreventive agents. The evidence on the anticarcinogenic effects of ITCs and indoles stems from epidemiological and experimental studies. 21.2.1 Evidence from epidemiological studies The first epidemiological study on the inverse relationship between consumption of high quantities of cruciferous vegetables and the risk of cancer was described more than nine decades ago by Stocks and Carn,1 who reported reduction of cancer of different organs upon consumption of cauliflower, cabbage, watercress and other non-cruciferous vegetables. During the last three decades numerous studies have been carried out on the cancer protective activities of cruciferous vegetables and a summary of the results of these studies has been published recently.2 After analysing data from six prospective cohort studies and 74 casecontrol studies, the authors concluded that high consumption of one or more Brassica vegetables (cabbage, broccoli, cauliflower, Brussels sprouts, kale,

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kohlrabi, etc.) is associated with a reduced risk of cancer of the lung, stomach, colon and rectum with a possible reduction in endometrial and ovarian cancer, and a decrease in total cancer incidence. Also, studies carried out a few years ago to investigate the effect of cruciferous vegetables in cancer target organs previously not studied, or for which no protection was found, showed a statistically significant reduction of cancer of the urinary bladder,8 prostate,9 breast10 and nonHodgkin's lymphoma11 with increasing cruciferous vegetable intake. The most recent epidemiological studies examined the relationship between the level of urinary ITCs or dithiocarbamates, glucosinolate and ITC metabolites, and the risk of developing cancer in individuals regularly consuming cruciferous vegetables. 12±14 Since cruciferous vegetables contain, in addition to glucosinolates, a number of potentially cancer protective phytochemicals such as flavonoids, phenolics, vitamins, dithiolthiones and fibres, the results of such studies unveil the particular role of glucosinolates and their breakdown products in cancer protection. In the above-mentioned studies, individuals who excreted a higher level of ITCs or dithiocarbamates had a lower risk of cancer, the inverse relationship being more prominent in individuals with homozygous deletion of glutathione S-transferase (M1 and T1). These enzymes play a role in the metabolic conversion of ITCs to dithiocarbamates, thereby decreasing the half-life of isothiocyanates in the body and by extension their cancer protective effects. 21.2.2 Evidence from experimental studies with laboratory animals The groundwork for the study of chemopreventive activities of constituents of cruciferous vegetables arose from the report of Sasaki15 in the 1960s, in which the author showed inhibition by a synthetic ITC, -napthyl isothiocyanate, of 3methyl-4-dimethyl-aminoazobenzene-induced liver tumourigenesis in rats. A decade latter, Wattenberg16 demonstrated that benzyl ITC (BITC), a naturally occurring ITC contained at higher concentrations in garden cress, and indole-3carbinol (I3C), a breakdown product of glucobrassicin, inhibited the tumorigenic effect of dimethylbenzanthracene and benzo(a)pyrene-induced mammary and forestomach tumorigenesis, respectively. These results inspired other investigators to study the chemopreventive effects of cruciferous vegetable preparations or different naturally occurring ITCs and indoles towards diverse groups of carcinogens.7,17,18 A few examples of these results are presented in Table 21.2. Several experimental studies demonstrated that freeze-dried, cooked or raw cruciferous vegetables protect against chemically induced preneoplastic lesions or frank neoplasia when fed to the animals before, during or after the carcinogen.7,22,23 The carcinogens against which the vegetables showed protection belong to different chemical classes and include polycyclic aromatic hydrocarbons, aflatoxins, heterocyclic aromatic amines and hydrazines (Table 21.2). The protective activity of ITCs and indoles, unlike that of whole vegetables, depended on the time of administration and dose level, target tissue, type of anticarcinogenic compound and class of carcinogen. For instance, phenethyl

Table 21.2 Some examples on inhibition of chemically induced preneoplastic lesions or frank tumours by cruciferous vegetables, isothiocyanates and indoles Carcinogen

Vegetable/glucosinolate hyrdrolysis product

Experimental animal

Target organ

Reference

Dimethylbenzanthracene Aflatoxin B1 Methylnitrosourea 2-amino-3-methylimidazo [4,5-f]quinoline (IQ) Dimethylbenzanthracene

Brussels sprouts ground beet/cabbage cabbage garden cress, Brussels sprouts, cabbage

rat rat rat rat

mammary gland liver mammary gland liver, colon

19 20 21 22,23

BITC, PEITC, sulforaphane BITC, PEITC

rat

mammary gland

16,24

mouse

lung, forestomach

16

BITC PEITC PEITC PEITC I3C, DIM I3C, DIM I3C I3C I3C I3C I3C

rat mouse rat hamster rat mouse rat trout mouse mouse rat

colon, intestine lung oesophagus lung, pancreas mammary gland forestomach tongue liver liver lung colon

25 26 27 28 29 29 30 31 32 33 34

Dimethylbenzanthracene and benzo(a)pyrene Methylazoxymethanol NNK N-nitrosobenzylmethylamine N-nitrosobis(2-oxopropyl)amine Dimethylbenzanthracene Benzo(a)pyrene 4-nitroquinoline Aflatoxin B1 Diethylnitrosamine NNK IQ

620

Functional foods, ageing and degenerative disease

ITC (PEITC) reduced the incidence and multiplicity of 4-(Methylnitrosoamino)1-(3-pyridyl)-1-butanone (NNK)-induced lung tumours35 or N-nitrosobis(2oxopropyl)amine (BOP)-induced lung and pancreas tumours28 when given prior to the carcinogens. Administration of PEITC during the post-initiation period did not have any effect on either incidence or multiplicity of tumours.36 However, recently, ITCs were found to be protective against azoxymethaneinduced colonic preneoplastic lesions,37 NNK-induced lung adenomas and benzo(a)pyrene-induced mouse lung tumours38 when administered subsequent to the carcinogen. In the latter study, the dose of PEITC administered was higher (15 mol/g diet) than that usually needed at pre-initiation or initiation stages (1 or 3 mol/g diet). With indoles the time of administration in relation to the carcinogen is even more critical than it is with ITCs, since administration of the compounds subsequent to the carcinogen may lead to enhancement of tumorigenesis. For example, whereas feeding of I3C to trout during the preinitiation period reduced aflatoxin B1-induced liver tumours and diethylnitrosamine induced-rat glutathione S-transferase-P (GST-P) positive liver foci, administration of I3C after the carcinogens increased the incidence of liver tumour and the size and number of GST-P foci.39,40 The protective activity of ITCs is also tissue and carcinogen-specific. Administration of PEITC to mice before benzo(a)pyrene reduced the incidence and multiplicity of forestomach tumours induced by the carcinogen but not multiplicity or incidence of lung or skin tumours.41 Also, when PEITC was fed to rats prior to or during treatment with NNK, the incidence of lung tumours was reduced but not the incidence of liver or nasal tumours.35 BITC is an effective chemopreventive agent at pre-initiation or initiation stage against dimethylbenzanthracene-induced mouse forestomach and lung tumours16 but not towards NNK and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-induced lung and mammary gland tumourigenesis, respectively.26,42

21.3

Mechanisms of action

21.3.1 Modulation of carcinogen activating phase I enzymes Generally, most chemical carcinogens act indirectly in that they need activation primarily by means of cytochrome P450 enzymes before they bind to DNA or other critical molecules of the cell and cause damage. Therefore, cytochrome P450 enzymes are important targets to inhibit tumorigenesis. One of the mechanisms by which ITCs exert chemopreventive effects is through inactivation of P450 enzymes through a direct interaction of the ITC with nucleophilic residues in the enzyme or by interaction of the isocyanate metabolite (ITCs are metabolised by P450 enzymes to isocyanates) with available nucleophiles in the active site or as a result of a covalent modification of the enzyme by atomic sulfur produced through oxidative desulfuration.43 Inactivated P450 enzymes are not able to metabolise procarcinogens to ultimate carcinogens and damage to macromolecules of the cell is prevented.

Glucosinolates and the prevention of cancer 621 Alteration of the activity of P450 enzymes and the resulting decreased activation of carcinogens by ITCs has been extensively investigated and much of the work has been done with PEITC and BITC.7,18 Administration of PEITC and BITC inhibited the metabolic alpha hydroxylation in vivo of the nitrosamines NNK, Nnitrosomethylbenzylamine, N-nitrosonornicotine, N-nitrosopyrrlidine and Nnitrosomethylamine,18 which is critical for DNA adduct formation. This is brought about through inactivation of different P450 enzymes, among which CYP2B1, CYP1A and CYP2E1 are the most important ones. As CYP1A and CYP2E1 are known to be involved in the metabolic activation of a wide spectrum of carcinogenic compounds, their inactivation by ITCs may give a blanket chemopreventive effect. Despite their proved chemopreventive effect, whole cruciferous vegetables and indoles, unlike ITCs, induce the activity of phase I enzymes. The correlation between increased activity of phase I enzymes and decreased tumorigenicity of chemical carcinogens is unclear. However, since cruciferous vegetables or their constituents, indoles, induce both phase I and phase II enzymes, increased activation by phase I enzymes of procarcinogens may render them more susceptible to detoxification by phase II enzymes. For instance, although I3C is a very potent inducer of the cytochrome P4501A gene family, which is involved in the activation of polycyclic aromatic hydrocarbon group of carcinogens, I3C was also reported to be a potent inhibitor of benzo(a)pyrene and 7,12dimethylbenzanthracene-induced forestomach and rat mammary gland tumorigenesis, respectively, in mice.29 21.3.2 Increased carcinogen detoxification through induction of phase II enzymes Induction of activities of phase II enzymes is in general considered to be a detoxifying mechanism. The most important phase II enzymes are glutathione Stransferase (GST), UDP-glucuronosyl transferase (UDPGT) and quinone reductase. They catalyse conjugation of phase I reaction products with endogenous ligands such as glutathione, glucuronic acid and sulfate giving rise to more polar metabolites that are readily excreted. Cruciferous vegetables and their constituents, ITCs and indoles, are very potent inducers of GST enzymes. A large amount of data is available on this subject and some of this data has been discussed in reviews.7,24 ITCs are not only inducers of GSTs but they are also metabolised by these enzymes to dithiocarbamate products, thereby their biological half-life and chemopreventive effects are greatly reduced. These findings indicate that the cancer protective property of ITCs is mediated probably through a mechanism other than induction of GST enzymes such as induction of other phase II enzymes or mechanisms different from induction of phase II enzymes. Indeed, we recently reported induction by garden cress, Brussels sprouts and red cabbage of UDPGT activity, which was paralleled by the lower incidence of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ)-induced colonic and hepatic preneoplastic lesions in F344 rats fed with these vegetables.22,23

622

Functional foods, ageing and degenerative disease

21.3.3 Induction of apoptosis Until a very recent time, reports on chemoprotective effect of ITCs or indoles were limited to experimental protocols in which the compounds were administered before and/or during treatment with a carcinogen. The only exception was the report two decades ago by Wattenberg,44 in which BITC was found to inhibit DMBA-induced mammary carcinogenesis in rats when given subsequent to the carcinogen. However, the mechanism of protection was unclear. Lately, many studies in cell culture have demonstrated induction by ITCs of apoptosis, a process through which genetically damaged or improperly dividing cells are eliminated. The mechanism of induction of apoptosis is, however, different and appears to depend on the specific cell line employed in the study or the individual ITC. Sulforaphane-induced apoptosis in HT-29 human colon cancer cells occurred in the absence of any change in p53 protein levels.45 On the contrary, in Jurkat T-lymphocytes, sulforaphane increased p53.46 In studies with human colon adenocarcinoma cells differing in p53 gene status (wild-type or mutant) the same degree of apoptosis was induced by PEITC, indicating the absence of a role for p53. However, in normal cells, p53 played an essential role in PEITC-induced apoptosis since normal mouse fibroblasts (p53 /) but not p53 deficient embryo fibroblasts (p53 ÿ/ÿ) underwent apoptosis.47 Also, a number of studies showed that caspases are involved in ITC-induced apoptosis. In the aforementioned report by Fimognari and colleagues,46 in addition to p53, proapoptotic gene, Bax was expressed together with cytochrome c release and cleavage of poly(ADP-ribose)polymerase, suggesting that caspases were activated to execute the cells. Consistent with this report, PEITC-induced apoptosis in HeLa cervical cancer cells was associated with caspase 3 activation since a caspase-3 activation inhibitor prevented apoptosis.48 Very recent reports indicate that ITCs induce apoptosis of tumour cells in vivo when given subsequent to the carcinogen.37 Administration of Nacetylcysteine conjugates of BITC and PEITC after a single dose of benzo(a)pyrene reduced lung tumour multiplicity. The mechanism of protection was found to be increased rate of apoptosis in lung tissues mediated via activation of mitogen activated protein kinase pathway. Although not investigated as intensively as ITCs, there are some reports that indicate induction by indoles of apoptosis. In one study comparing induction of apoptosis by I3C in tumorigenic and non-tumorigenic breast epithelial cells, the compound selectively induced apoptosis in the former cells but not in the latter.49 The mechanism behind this effect was found to be up regulation of Bax/ Bcl2 ratio and down regulation of Bcl-x1 in tumour cells. 21.3.4 Antioxidant effect The other but less studied mechanism of the cancer protective effects of ITCs and indoles is their antioxidant effect. Indoles are directly acting antioxidants since they scavenge reactive oxygen species50 whereas ITCs are indirect

Glucosinolates and the prevention of cancer 623 antioxidants involving enhancement of the antioxidant capacity of cells through increase in either the activity of phase II enzymes such as quinone reductase and GST or the synthesis of glutathione.51

21.4

Future trends

Despite regional differences in the total amount and particular type of cruciferous vegetable consumed, these vegetables have always been part of the human diet in many parts of the world. In general, consumption is the highest in affluent, better-educated urbanised populations. At the moment the individual average daily intake of cruciferous vegetable or glucosinolates is not known. A study carried out by Fenwick and Heaney two decades ago indicated an intake of 12±16 mg glucosinolate per person per day in the UK.52 The growing evidence of the cancer protective effects of cruciferous vegetables and the widely popular advocacy by the World Cancer Research Fund of five portions of vegetables and fruits might lead to a higher per capita consumption of cruciferous vegetables and glucosinolates in the future. However, the means to increase dietary intake of glucosinolates should be a higher intake of naturally grown cruciferous vegetables rather than products bought from the pharmacy or consumption of those cruciferous vegetable lines that produce enhanced levels of glucosinolates after being genetically manipulated. This is because of the possible detrimental effect on human health of extremely high amounts of glucosinolates. Data from laboratory studies with very high doses of ITCs and indoles showed that the compounds induce carcinogenic effects in their own right.53,54

21.5

Sources of further information and advice

World Cancer Research Fund and American Institute of Cancer Research, Food, Nutrition and the Prevention of Cancer: A Global Perspective. American Institute of Cancer Research, Washington, DC, 1997. International Agency for Research on Cancer (IARC), World Health Organisation, Handbook on Fruits and Vegetables, Lyon, France, in press. Fahey J W, Zalcmann A T and Talalay P, `The chemical diversity and distribution of glucosinolates and isothiocyanates among plants', Phytochemistry 2001 56 5±51. Hecht SS, `Inhibition of carcinogenesis by isothiocyanates', Drug Metabol Rev 2000 32 95±341.

21.6 1.

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22 Dietary fiber and the prevention of cancer J. Slavin, University of Minnesota, USA

22.1

Introduction: defining dietary fiber

Large international differences in rates of cancer as well as results from migratory studies that find individuals take on the cancer demographics of the population to which they migrate. This suggests a strong role for environmental factors including diet on cancer incidence. Most published studies on dietary fiber and cancer relate to colon cancer, although other studies support a role for dietary fiber and decreased risk of other types of cancers. Dietary fiber is known to decrease serum estrogen concentrations which suggests that dietary fiber also may protect against hormone-dependent cancers such as breast, endometrial, ovarian, and prostate. The term `dietary fiber' was not coined until 1953, but the anti-constipating effects of high-fiber foods have been long appreciated. In 430 BC, Hippocrates compared the superior laxative effects of coarse wheat and refined wheat (McCance and Widdowson, 1955). Graham (of graham cracker fame) denounced the harmful effects of refined carbohydrate foods during the 19th century, and the first Kellogg's and Post cereals were formulated in response to increasing interest in dietary fiber. In the 1920s, J. H. Kellogg published extensively on the attributes of bran, claiming it increased stool weight, promoted laxation, and prevented disease. Dietary fiber was studied throughout the 1930s and then forgotten. Denis Burkitt is usually credited with popularizing the contemporary idea that dietary fiber may protect against the development of Western diseases, including diabetes, hypercholesterolemia, heart disease, diverticular disease, and cancer (Burkitt, 1971). Whether isolated dietary fiber has the same physiologic properties as the dietary fiber found naturally in grains, fruits, and vegetables with associated substances is not known and is difficult to study.

Dietary fibre and the prevention of cancer 629 Interest in dietary fiber has been limited by our inability to agree on definitions for dietary fiber and recommended intake levels. New definitions for dietary fiber and recommendations for fiber intake were published in the Dietary Reference Intakes (DRIs) (DRI, 2002). Dietary Fiber consists of nondigestible carbohydrates and lignin that are intrinsic and intact in plants. Added Fiber consists of isolated, nondigestible carbohydrates that have beneficial physiological effects in humans. Total Fiber is the sum of Dietary Fiber and Added Fiber. Two categories of fiber are described, dietary fiber; fiber that is in its natural state, and functional fiber, fiber that is isolated, manufactured, synthetic, or enzyme-produced. Functional fiber does not have to be plant based. Other important recommendations of the committee are that Functional Fiber must show a beneficial physiological effect in order to be classified as Functional Fiber. Additionally, the committee recommended phasing out the terms soluble and insoluble dietary fiber. Two properties, viscosity and fermentability, were recommended as meaningful alternative characteristics for the terms soluble and insoluble fiber. The Panel on Dietary Reference Intakes for Macronutrients (DRI, 2002) was responsible for reviewing the research on dietary fiber and disease prevention and deciding whether to set a recommended intake level for dietary fiber. Prior to this report, there was no RDA for dietary fiber. The panel also found in its deliberations that there was no official definition of dietary fiber. Thus, a Panel on the Definition of Dietary Fiber was formed to review existing literature on dietary fiber and determine the best scientific definition of dietary fiber (DRI, 2001). We know all fiber is not created equal. Dividing dietary fiber into soluble and insoluble fiber was an attempt to assign physiological effects to chemical types of fiber. Scientific support for the claims that soluble fibers lower serum cholesterol while insoluble fibers increase stool size is inconsistent at best. A meta-analysis testing the effects of pectin, oat bran, guar gum, and psyllium on blood lipid concentrations found that 2 to 10 g/day of viscous fiber was associated with small but significant decreases in total and LDL cholesterol concentrations (Brown et al., 1999). Oat bran lowers serum lipids while wheat bran does not (Anderson et al., 1991). Resistant starch, generally a soluble fiber, does not affect serum lipids (Jenkins et al., 1998). Thus, not all soluble fibers are hypocholesterolemic agents and other traits such as viscosity of fiber play a role and must be considered. The insoluble fiber association with laxation also is inconsistent. Fecal weight increases 5.4 g/g wheat bran fiber (mostly insoluble), 4.9 g/g fruits and vegetables (soluble and insoluble), 3 g/g isolated cellulose (insoluble), and 1.3 g/g isolated pectin (soluble) (Cummings, 1993). Many fiber sources are mostly soluble but still enlarge stool weight, such as oat bran and psyllium. Not all insoluble fibers are particularly good at relieving constipation, for example isolated cellulose. The disparities between the amounts of soluble and insoluble fiber that are measured chemically and the physiological effects led a National

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Academy of Sciences Panel to recommend that the terms soluble and insoluble fibers gradually be eliminated and be replaced by specific beneficial physiological effects of a fiber, perhaps viscosity and fermentability. The DRI committee used the new definitions for Dietary, Functional, and Total Fiber in their report. Additionally, they set an Adequate Intake (AI) for Total Fiber in foods of 38 g and 25 g/day for young men and women, respectively, based on intake level observed to protect against coronary heart disease. Adequate Intake (AI) is the recommended average daily intake level based on observed or experimentally determined approximations or estimates of nutrient intake by a group (or groups) of apparently healthy people that are assumed to be adequate ± used when an RDA cannot be determined. There was insufficient evidence to set a Tolerable Upper Intake Level (UL) for Dietary Fiber or Functional Fibers. They conclude that the recommended intake of dietary fiber should also provide protection against cancer, but there is not enough research to set a recommended fiber intake based on cancer prevention. Median intake of Dietary Fiber ranged from 16.5 to 17.9 g/day for men and 12.1 to 13.8 g/day for women. Thus, there is a large fiber gap to fill between usual intake of dietary fiber and recommended intakes. 22.1.1 Methods of measuring dietary fiber When we accept a physiologic definition of dietary fiber, measuring dietary fiber becomes problematic. The difficulty in devising a method for assessing total dietary fiber can be appreciated if one considers the diverse nature of dietary fiber. A simple, reproducible method for removing protein, fat and soluble sugars and starch from food while retaining both water-soluble and water-insoluble components of dietary fiber is difficult from an analytic standpoint. In the United States, dietary fiber has been defined by default by the AOAC method for total dietary fiber (TDF). If any food or fiber supplement could be analyzed as dietary fiber by this official method, it could be labeled as dietary fiber on the Nutrition Facts panel. This worked well for most fibers, but isolated compounds, such as fructo-oligosaccharides, resistant starch, and polydextrose were not captured as dietary fiber by this official fiber method. Thus, these compounds were not allowed to make fiber claims, despite published data that they delivered many of the physiological effects of accepted fibers.

22.2 The relationship between dietary fiber intake and cancers of the gastrointestinal tract 22.2.1 Cancer background Cancers of the gastrointestinal tract represent the second most common cancer in the United States and are second only to cancer of the respiratory tract as a cause of cancer-related mortality the United States. The development of colon cancer involves a complex interplay between environmental and genetic factors. As the

Dietary fibre and the prevention of cancer 631 colorectal epithelium progresses from normal histology to one that is hyperproliferative, adenomatous, and finally malignant, multiple molecular alterations, including the activation of proto-oncogenes, the inactivation of tumor suppressor genes, and mutations in mismatch repair genes, occur (Fearton and Jones, 1992). Environmental factors are also intimately involved in the process and many represent risk factors that can be altered, unlike genetic risk factors. Dietary factors are particularly relevant in the process, with estimates that 35% of all cancers are attributable to diet and that up to 90% of colorectal cancer in the United States could be avoidable with alterations in diet (Doll and Peto, 1981). Dietary treatments have been tested as chemopreventive measures in the process of colorectal cancer. However, establishing a cause-and-effect relationship between diet and colorectal cancer is a difficult task. Studies in diet and cancer include ecologic studies where dietary variables are compared across populations. Case-control studies can examine differences in diet between people who have colon cancer compared to those who do not. A serious limitation of retrospective studies is the accuracy with which intakes of dietary factors can be established. It is difficult to recall dietary intakes in former years and disease tends to alter diet. A stronger epidemiologic design is the cohort study in which subjects exposed to a particular agent are followed over time and their cancer incidence is compared with those who have not been exposed. These studies are costly and require large numbers of subjects. Intervention studies allow investigators to make a dietary change and then follow the course of disease. Intervention studies are limited by the slow progressive nature of the cancer process and the large number of subjects needed for statistical power. Strategies to avoid these problems include studying individuals who are at high risk of developing cancer to determine whether a chemopreventive agent can prevent the development of cancer. Secondly, studies use intermediate biomarkers of the cancer process as the end point rather than waiting for the cancer to occur. Chemoprevention trials in high-risk individuals with measurement of intermediate biomarkers is commonly used to determine the role of dietary ingredients on colon cancer causation. Many studies exist using animal models to study the relationship between dietary fiber and colon cancer. Animal studies offer greater control of variables, allow for a broader range of interventions, and are generally less expensive than human studies. They suffer from species differences, which for dietary fiber are particularly problematic since the gastrointestinal tract of the rat varies significantly from the human. Other research in dietary fiber and cancer is conducted in cell culture cancer lines. Mechanistic studies of dietary fiber and its potential role in colon cancer prevention are also conducted in healthy human subjects. Studies on dietary fiber and cancer are limited because of problems in defining, measuring, and quantitating dietary fiber. Because fiber has a physiological definition, it is difficult to agree on a chemical method to measure dietary fiber. Epidemiologic studies generally rely on food frequency

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instruments that estimate dietary fiber. These estimates are woefully inadequate if we are trying to estimate fermentable carbohydrate that reaches the colon (Hill, 1998). He suggests that we return to foods for epidemiologic studies rather than attempting to measure dietary fiber, which obviously is problematic. Whole grain intake also is associated with lower risk of colon cancer (Jacobs et al., 1998). Data summarized from Italian case-control studies of whole grain intake and cancer risk support that whole grain intake protects against colon cancer (Chatenoud et al., 1998). Despite these limitations and frustrations, we need to move ahead in the area of dietary fiber and cancer. This chapter will describe the mechanistic support for a relationship between dietary fiber and cancer prevention. Additionally, it will highlight epidemiological evidence and human intervention studies that have been conducted on this topic. Finally, the public health message for dietary fiber and cancer prevention will be offered. 22.2.2 Why would dietary fiber protect against cancer? Dietary fiber has physiological effects throughout the gastrointestinal tract that may explain its protectiveness against cancer. Although we generally think of dietary fiber as most active in the large intestine, it is known that fiber affects hormones in the upper digestive tract. These changes may alter satiety, slow digestion, and aid in weight maintenance. Additionally, insulin response has been considered most relevant in diabetes prevention, although it has been linked to risk of colon cancer and breast cancer. Potential mechanisms for the protective nature of dietary fiber against colon cancer are listed in Table 22.1. The fermentation of dietary fiber in the gut is considered its most important physiological effect. Fermentation of carbohydrate in the colon produces short chain fatty acids that help maintain the integrity of the gut (Topping and Clifton, 2001). More than 75% of dietary fiber Table 22.1 Mechanisms by which fiber can protect against the development of cancer Increased stool bulk decreased transit time dilution of carcinogens Binds with bile acids or other potential carcinogens Lower fecal pH inhibit bacterial degradation of normal food constituents to potential carcinogens Changes in microflora Fermentation by fecal flora to short chain fatty acids Decrease in colonic pH Inhibition of carcinogens Increase in lumenal antioxidants Peptide growth factors Alteration of sex hormone status Change in satiety resulting in lowered body weight Alterations in insulin sensitivity and/or glucose metabolism

Dietary fibre and the prevention of cancer 633 in an average diet is broken down in the large intestine resulting in the production of carbon dioxide, hydrogen, methane and SCFAs including butyrate, propionate and acetate. Propionate and acetate are metabolized in colonic epithelial cells or peripheral tissue. Butyrate may regulate colonic cell proliferation and serve as an energy source for colonic cells. Propionate acid is transported to the liver, and may suppress cholesterol synthesis, a potential explanation for how soluble dietary fiber lowers serum cholesterol. According to calculations by Cummings and McFarlane (1997), if approximately 20 grams of fiber is fermented in the colon each day, approximately 200 mM of SCFA will be produced, of which 62% will be acetate, 25% propionate, and 16% butyrate. Colonic absorption of SCFA is concentration dependent with no evidence of a saturable process. The mechanism by which SCFA cross the colonic mucosa is thought to be passive diffusion of the unionized acid into the mucosa cell. SCFAs are respiratory fuels for the colonic mucosa. In isolated human colonocytes, butyrate is actively metabolized to both CO2 and ketone bodies, which accounts for about 80% of the oxygen consumption of colonocytes. Butyrate is almost completely consumed by the colonic mucosa, while acetate and propionate enter the portal circulation extending the effects of dietary fiber beyond the intestinal tract. Butyrate may be an important protective agent in colonic carcinogenesis (Valazquez et al., 1996). Trophic effects on normal colonocytes in vitro and in vivo are induced by butyrate. In contrast, butyrate arrests the growth of neoplastic colonocytes and inhibits the preneoplastic hyperproliferation induced by some tumor promoters in vitro. Butyrate induces differentiation of colon cancer cell lines and regulates the expression of molecules involved in colonocyte growth and adhesion. The effects of butyrate on colonic tumor cell lines in vitro seem to contradict what has been shown in vivo (Hague et al., 1997). Butyrate appears to have two contrasting effects. It serves as the primary energy source for normal colonic epithelium and stimulates growth of colonic mucosa, yet in colonic tumor cell lines it inhibits growth and induces differentiation and apoptosis. Since SCFAs are volatile, they are quickly absorbed from the lumen. SCFAs acidify the gut, which may affect development of colon cancer because changes in gut pH will affect solubility of metabolites and activities of bacterial enzymes (Thornton, 1981). Other mechanisms on how dietary fiber affects colon cancer have been examined in animal models. Reddy et al. (1998) reported that the concentration of fecal secondary bile acids and fecal mutagenic activity were significantly lower during wheat bran supplementation compared to control, whereas an oat bran diet supplemented at a level to achieve the same level of fiber had no impact on these measures. Other studies have examined fiber's ability to increase fecal bulk and speed intestinal transit. Dietary fibers differ in their ability to hold water and their resistance to bacterial degradation in the gut. Pectin is effective in holding water, but is quickly fermented in the gut and cannot be found in feces. Wheat bran consistently has been found to have the

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most effect on stool bulk, probably because it is slowly fermented and survives transit through the gut. Milling of wheat bran may affect the laxative properties of the bran, with larger particle sizes causing larger increases in fecal weight. Dietary fiber sources such as wheat bran are complex matrices and attempts have been made to isolate the effects of chemical components of wheat bran. In an animal study, rats were fed wheat bran, dephytinized wheat bran, and phytic acid alone and aberrant crypt foci were measured after treatment with azoxymethane (Jenab and Thompson, 1998). Wheat bran without phytic acid was less protective than intact wheat bran suggesting that the protective effects of wheat bran include fiber and phytic acid. Certain components of dietary fiber are more protective against colorectal cancer. Insoluble fibers have consistently been found to decrease cell proliferation while soluble fibers may even increase cell proliferation. Lu et al. (1998) found that lignin, a component of insoluble dietary fiber is a free radical scavenger. They suggest that the ability of dietary fiber to protect against colorectal cancer may be determined by the amount of lignin in dietary fiber as well as the free radical-scavenging ability of the lignin. A usual criticism of animal studies in this area is the large amount of dietary fiber that is fed. Dietary fibers have been fed at levels of 30% of the diet and more. These levels of intake have no bearing on typical or recommended intakes in humans. Yet animal studies allow investigators to screen a wide range of different dietary fibers at many doses.

22.3 Epidemiological evidence on the protective role of dietary fiber The relationship between dietary fiber and colon cancer has been studied extensively by epidemiologists since the hypothesis linking fiber to colon cancer was popularized by Denis Burkitt. Besides looking for relationships between dietary fiber and colon cancer, researchers have looked for relationships between colon cancer and foods high in dietary fiber, including vegetables, fruits, grains, and legumes. The majority of descriptive, case-control, and cohort epidemiologic studies support an inverse relationship between consumption of vegetables and fruits and colorectal cancer risk (Steinmetz and Potter, 1991). Besides dietary fiber, fruits, vegetables and grains contain a variety of anticarcinogenic compounds (Table 22.2). Few well-designed cohort studies have examined the relationship between intake of high-fiber foods and colorectal cancer. The relationship between fruit and vegetable intake and colorectal cancer was examined in the Iowa Women's Health Study (Steinmetz et al., 1994). This study is a prospective cohort study of 98,030 postmenopausal women who were asked to complete a questionnaire on health and diet. The cohort was followed for four years. Dietary intake was assessed with the Willett semiquantitative food frequency questionnaire. The occurrence of colorectal cancer was documented and a total of 212 cases and

Dietary fibre and the prevention of cancer 635 Table 22.2 Dietary fiber Lignans Isoflavones Coumarins Phytates Dithiothiones Carotenoids Tocopherols Ascorbate

Potential anticarcinogens in fruits, vegetables, legumes and grains Folate Isothiocyanates Indoles Glucosinolate Plant sterols Protease inhibitors Allium compounds Flavonoids Other phenolic compounds

35,004 noncases remained for analysis. Total intake of both vegetables and fruits did not reduce the relative risk of colorectal cancer. Garlic consumption was linked to reduced risk of colorectal cancer, perhaps because garlic is a good source of glutatione-S-transferase which has been shown to inhibit experimentally induced carcinogenesis in animals. Epidemiologic evidence that whole grains protect against colorectal cancer is also strong. In an expanded meta-analysis, odds ratios were < 1 in 9 of 10 mentions of studies of colorectal cancers and polyps and the relationship to whole grain intake (Jacobs et al., 1998). Whole grain intake was estimated by intake of whole grain bread, brown rice, and intake of whole grain cereals. Intervention studies to test this hypothesis are lacking. Two analyses, conducted as meta-analysis, have summarized the observational and case-control epidemiologic studies on dietary fiber and colorectal cancer. Trock et al. (1990) analyzed 37 epidemiologic studies that examined the relationship between colorectal cancer and fiber, vegetables, grains and fruits, either alone or in combination. Overall, 80% of the studies reported up to that time supported the protective role of dietary fiber in colorectal cancer. Howe et al. (1992) conducted a combined analysis of data from 13 case-control studies in populations with different colorectal cancer rates and dietary practices. The risk of colorectal cancer decreased incrementally as dietary fiber intake increased. Consumption of more than 31 g of fiber/day was associated with a 50% reduction in risk of colorectal cancer compared to diet incorporating < 11 g/day. The authors estimate that the risk of colorectal cancer in the U.S. population could be reduced by about 31% from an average increase in fiber intake from food sources of about 13 grams/day. In contrast, Giovannucci (2003) recently concluded that more recent epidemiologic studies have not supported a strong influence of dietary fiber or fruits and vegetables on colorectal cancer. Marchand et al. (1997) report a protective role of fiber from vegetables against colorectal cancer which appears independent of its water solubility property and of the effects of other phytochemicals. High intake of vegetables, fruits, and grains was associated with decreased risk of polyps in a case-control study (Witte et al., 1997). Lubin et al. (1997) found no significant protection against adenomatous polyps in a case-control study. They did find a significant interaction between water and fiber intake. They suggest that fiber and water

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increase the volume of colonic contents which dilutes and adsorbs exogenous and endogenous toxic compounds present in the colonic contents. Also, the increased volume of bowel contents promotes peristalsis, reducing the duration of the contact of colonic contents with the mucosa. Slattery et al. (1998) examined eating patterns and risk of colon cancer in a population-based case-control study. The prudent patterns which included vigorous exercise, smaller body size, and higher intakes of dietary fiber and folate were associated with lower risk of colon cancer. In contrast, the western style was associated with increased risk of colorectal cancer. Mai et al. (2003) reported that within a cohort of older women characterized by a relatively low fiber intake, there was little evidence that dietary fiber intake lowered the risk of colorectal cancer. Peters et al. (2003) assessed the relation of fiber intake and frequency of colorectal adenoma within the prostate, lung, colorectal and ovarian (PLCO) cancer screening trial. High intakes of dietary fiber were associated with lower risk of colorectal adenoma. In this case-control study of over 38,000 subjects, subjects with the highest amounts of fiber in their diets (36 g/day) had the lowest incidence of colon adenomas. Their risk of having an adenoma detected by sigmoidoscopy was 27% less than that of the people who ate the least amount of fiber (12 g/d). Fiber from fruits and from grain/cereals was significantly associated with lower adenoma risk, while fiber from vegetables and legumes was not. The data from large cohort studies are not consistent. In the Health Professional Follow-up Study (Giovannucci et al., 1992), dietary fiber was inversely associated with risk of colorectal adenoma in men. All sources of fiber (vegetables, fruits, and grain) were associated with decreased risk of adenoma. The Nurses' Health Study found no protective effect of dietary fiber on the development of colorectal cancer in women (Willett et al., 1990). In the Iowa Women's Health Study, a weak and statistically non-significant inverse association was found between dietary fiber intake and risk of colon cancer (Steinmetz et al., 1994). The European Prospective Investigation into Cancer and Nutrition (EPIC) is a prospective cohort study comparing the dietary habits of more than a halfmillion people in ten countries with colorectal cancer incidence (Bingham et al., 2003). They found that people who ate the most fiber (those with total fiber from food sources averaging 33 grams a day) had a 25% lower incidence of colorectal cancer than those who ate the least fiber (12 grams per day). The investigators estimated that populations with low average fiber consumption could reduce colorectal cancer incidence by 40% by doubling their fiber intake. Few epidemiologic studies have collected biomarkers of dietary fiber intake. Cummings et al. (1992) collected data from 20 populations in 12 countries and found that average stool weight varied from 72 to 470 grams/day and was inversely related to colon cancer risk. Dukas et al. (2003) reported that in the Nurses' Health Study, women in the highest quintile of dietary fiber intake (median intake 20g/d) were less likely to experience constipation than women in the lowest quintile (median intake 7 g/d).

Dietary fibre and the prevention of cancer 637 It is known that different dietary fibers have different effects on stool weight. As summarized by Cummings (1993), wheat bran is most effective is increasing stool weight with each gram of fiber fed as wheat bran increasing stool weight by 5.4 grams. In contrast, soluble fibers like pectin only increase stool weight by 1.2 grams per gram of fiber fed as pectin. Yet psyllium, a fiber that is at least 70% soluble increases stool weight by 4.0 grams per gram fiber fed as psyllium. Thus the laxation properties of a fiber source cannot be predicted based on the solubility of the fiber. The relationship between stool weight and protection from colorectal cancer could be strong while the relationship between dietary fiber intake and colorectal cancer weak because of all these inconsistencies in fiber measurement and physiological effect.

22.4

Dietary fiber and hormonally related cancers

The hypothesis of a protective influence of reproductive factors and exogenous hormones on colorectal cancer was first proposed by McMichael and Potter (1980). This followed observations of ecological correlation between breast and colon cancer, higher than expected incidence of colorectal tumors among nuns, and descriptive sex and site specific data showing a cross-over of colorectal cancer rates around age 50 (Potter, 1995). Women have a similar or higher incidence of colorectal cancer than men before age 50 and a lower incidence thereafter. Potter et al. (1993) concluded that both parity and age at first pregnancy were likely not associated with colorectal cancer risk, whereas the available evidence suggests risk reduction associated with hormone replacement. Epidemiologic findings in this area vary greatly, with a meta-analysis of hormone replacement therapy and colon cancer in women concluding a 0±25% risk reduction among users of hormone replacement therapy (Hebert-Croteau, 1998). Few studies have examined the effects of dietary fiber on hormone metabolism, although colon cancer is affected by diet and hormones. Rose et al. (1991) reported that when wheat bran was added to the diet of premenopausal women, it significantly reduced serum estrogen concentrations, while neither corn bran nor oat bran had an effect on estrogen levels. Dietary fiber intake was increased from about 15 grams/day to 30 grams/day in this study, an increase similar to that recommended by the National Cancer Institute. Goldin et al. (1994) reported that a high-fiber, low-fat diet significantly decreased serum concentrations of estrone, estrone sulfate, testosterone, and sex hormone binding globulin in pre-menopausal women. Dietary fiber also caused a lengthening of the menstrual cycle by 0.72 day and a lengthening of the follicular phase by 0.85 day, changes thought to lower overall risk of developing hormonally dependent cancer. Bagga et al. (1995) also found that increased intake of dietary fiber significantly decreased serum estradiol and estrone, which should decrease risk of hormonally dependent cancers. Gender differences in bowel function were more profound than differences caused by increased fiber intake in a study comparing physiological effects of

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cereal and vegetable fiber (Lampe et al., 1993). Despite consuming identical diets to men, women had significantly smaller stools and slower transit times. Women digested more fiber and the concentration of secondary bile acids was greater in men than women. Other data support that menstrual cycle affects transit time, and that transit slows during pregnancy, which support the relationship of hormonal status and bowel function. 22.4.1 Insulin and colon cancer Giovannucci (1995) has proposed that the etiology of insulin resistance and colorectal cancer are related. He suggests that diets high in fat and energy and low in complex carbohydrates and a sedentary lifestyle lead to insulin resistance and that the associated hyperinsulinemia, hypertriglyceridemia, and glycemia lead to increased colon cancer risk through the growth-promoting effect of insulin on the increased availability of energy. La Vecchia et al. (1997) examined the relationship between diabetes mellitus and the risk of colorectal cancer in an Italian case-control study. They found that subjects with non-insulin-dependent diabetes have a slightly increased risk of colorectal cancer. Allowance for potential confounding factors, including body mass index, diet, and physical activity could not explain the excess colorectal cancer risk among subjects with diabetes.

22.5

Clinical studies of the protective role of dietary fiber

Several randomized intervention studies with high-fiber diets as a component of chemoprevention have been published. Alberts et al. (1990) studied the effects of wheat bran fiber (an additional 13.5 grams/day as wheat bran cereal) on rectal epithelial cell proliferation in patients with resection for colorectal cancers. They found that the wheat bran fiber cereal inhibited DNA synthesis and rectal mucosal cell proliferation in this high-risk group, which they argued should be associated with reduced cancer risk. They suggested that such a fiber regimen might be used as a chemopreventive agent for colorectal cancers. The study is weakened by poor compliance to the intervention over the four years of the study. A double-blind, placebo-controlled randomized trial with supplements of fiber and calcium and measurement of labeling index in rectal biopsies was conducted (Alberts et al., 1997). They concluded that nine months of high-dose wheat bran fiber and calcium carbonate supplementation in study participants with a history of recently resected colorectal adenomas did not have a significant effect on cellular proliferation rates in rectal mucosal biopsies, comparing threeand nine-month results to baseline results. In a randomized trial of intake of fat, fiber and beta carotene to prevent colorectal adenomas, patients on the combined intervention of low fat and added wheat bran had no large adenomas at both 24 and 48 months, a statistically significant finding (MacLennan et al., 1995). The Toronto Polyp Prevention trial

Dietary fibre and the prevention of cancer 639 demonstrated no significant difference between low-fat/high-fiber and high-fat/ low-fiber dietary groups with regard to the recurrence of adenomatous polyps (McKeown-Eyssen et al., 1994). Two widely publicized intervention studies do not support the protective properties of dietary fiber against colon cancer (Schatzkin et al., 2000; Alberts et al., 2000). The studies found no significant effect of high-fiber intakes on the recurrence of colorectal adenomas. Both papers described well-planned dietary interventions to determine whether high-fiber food consumption could lower colorectal cancer risk, as measured by a change in colorectal adenomas, a precursor of most large-bowel cancers. Perhaps the fiber interventions were not long enough, the fiber dose was not high enough, and recurrence of adenoma is not an appropriate measure of the effectiveness of fiber in preventing colon cancer. Yet the results from the studies are clear. Increasing dietary fiber consumption over three years did not alter recurrence of adenomas. BonitonKopp et al. (2000) found that the addition of 3.5 g/day psyllium (ispaghula husk) decreased polyp recurrence, with an adjusted odds ratio of 1.67 (p 0.042) for the psyllium fiber intervention on polyp recurrence Despite the inconsistency in the results of fiber and colon cancer studies, the scientific consensus is that there is enough evidence on the protectiveness of dietary fiber against colon cancer that health professionals should be promoting increased consumption of dietary fiber (Kim, 2000).

22.6 The relationship between dietary fiber intake and different cancers 22.6.1 Breast cancer Limited epidemiologic evidence has been published on fiber intake and human breast cancer risk. Since the fat and fiber content of the diet are generally inversely related, it is difficult to separate the independent effects of these nutrients, and most research has focused on the fat and breast cancer hypothesis. International comparisons show an inverse correlation between breast cancer death rates and consumption of fiber-rich foods. An interesting exception to the high-fat diet hypothesis in breast cancer was observed in Finland, where intake of both fat and fiber is high and the breast cancer mortality rate is considerably lower than in the United States and other Western countries where the typical diet is high in fat. The large amount of fiber in the rural Finnish diet may modify the breast cancer risk associated with a high-fat diet. A meta-analysis of 12 case-control studies of dietary factors and risk of breast cancer found that high dietary fiber intake was associated with reduced risk of breast cancer (Howe et al., 1990). Dietary fiber intake has also been linked to lower risk of benign proliferative epithelial disorders of the breast (Baghurst and Rohan, 1995). Not all studies find a relationship between dietary fiber intake and breast cancer incidence, including a prospective cohort study reported by Willett and colleagues (1992). Jain et al. (2002) also found no association among total

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dietary fiber, fiber fractions, and risk of breast cancer. Still, nutrition differences, including dietary fiber intake, appear to be important variables that contribute to the higher rate of breast cancer experienced by younger African American women (Forshee et al., 2003). Considerable evidence suggests that both breast and colon cancers are hormone-mediated diseases (Rose, 1993). Few studies have examined the effects of dietary fiber on hormone metabolism while fat content of the diet was held constant. Rose and colleagues (1991) reported that when wheat bran was added to the usual diet of premenopausal women it significantly reduced serum estrogen concentrations, whereas neither corn bran nor oat bran had an effect. Dietary fiber intake was increased from about 15 g per day to 30 g per day in this study, an increase similar to that recommended by the National Cancer Institute. Goldin et al. (1994) reported that a high-fiber, low-fat diet significantly decreased serum concentrations of estrone, estrone sulfate, testosterone, and sex hormone binding globulin in premenopausal women. Dietary fiber also caused prolongation of the menstrual cycle by 0.72 day and of the follicular phase by 0.85 day, changes thought to reduce overall risk of developing breast cancer. 22.6.2 Other cancers Few studies have been reported in this area. Studies of dietary fiber and endometrial cancer have reported both increases and decreases in risk (Barbone et al., 1993; Goodman et al., 1997; McCann et al., 2000). Ovarian cancer risk is decreased with higher intakes of dietary fiber (McCann et al., 2001; Risch et al., 1994). No significant relationships have been reported between dietary fiber intake risk of prostate cancer (Andersson et al., 1996; Rohan et al., 1995).

22.7

Conclusions

The relationship between cancer and dietary fiber remains complex. Although not all data support the relationship, the difficulty in measuring dietary fiber and the poor databases for dietary fiber content of foods make it a difficult relationship to study. Stronger protective support has been found for whole foods high in dietary fiber, such as cereal fiber, fruits, and whole grains. Dietary fiber may be just part of the protective puzzle with other components including antioxidants, phenolic compounds, and associated substances also providing protection against colorectal cancer. Other cancer sites are equally elusive as to their connection with dietary fiber intake. Since fiber intake is linked to lower body mass index, it will be protective against breast and prostate cancer. Also, breast cancer may be prevented by high fiber intakes, especially if the fibers consumed are high in phytoestrogens that alter sex hormone metabolism. Despite many years of research and nutrition education, dietary fiber intakes are not increasing. We must continue to promote consumption of foods high in

Dietary fibre and the prevention of cancer 641 complex carbohydrates, including resistant starch, oligosaccharides, and dietary fiber (Marlett et al., 2002). As many consumers depend on processed foods as the mainstay of their diets, efforts should be made to increase the fiber content of popular foods to assist consumers in obtaining recommended levels of unavailable carbohydrate. Differences in fiber composition must be considered since recent studies find that cereal fiber and not vegetable fiber is protective against cancer.

22.8

References

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ALBERTS DS, EINSPAHR J, RITENBAUGH C, AICKIN M, REES-MCGEE S, ATWOOD J, EMERSON S, MASON-LIDDIL N, BETTINGER L, PATEL J, BELLAPRAVALU S, RAMANUJAM PS, PHELPS J, CLARK L, (1997) The effect of wheat bran fiber and calcium supplementation on recal mucosal proliferation rates in patients with resected adenomatous colorectal polyps. Cancer Epid Biomarkers Prev; 6: 161±169. ALBERTS DS, MARINEZ ME, KOR DL, GUILLEN-RODRIGUEZ, MARSHALL JR, VAN LEEUWEN JB, REID ME, RITENBAUGH C, VARGAS PA, BHATTACHARYYA AB, EARNEST DL, SAMPLINER RE and THE PHOENIX COLON CANCER PREVENTION New Eng J Med; 324(16): 1156±1162.

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(2003) Dietary fibre and colorectal adenoma in a colorectal cancer early detection programme. Lancet; 361: 1491± 1495. POTTER JD, (1995) Hormones and colon cancer. J Natl Cancer Inst; 87: 1039±1040. POTTER JD, SLATTERY ML, BOSTICK RM, GAPSTUR SM, Colon cancer: a review of the epidemiology. Epidemiol Rev; 15: 499±545. REDDY B, ENGLE A, KATSIFIS S, SIMI B, BARTRAM HP, PERRINO P, MAHAN C, (1985) Biochemical epidemiology of colon cancer: effect of types of dietary fiber on fecal mutagens, acid, and neutral sterols in healthy subjects. Cancer Res; 49: 4629±4635. RISCH HA, JAIN M, MARRETT LD, HOWE GR, (1994) Dietary fat intake and risk of epithelial ovarian cancer. J Natl Cancer Inst; 86: 1490±1415. ROHAN TE, HOWE GR, BURCH JD, JAIN M, (1995) Dietary factors and risk of prostate cancer: a case-control study in Ontario, Canada. Cancer Causes Control; 6: 145±154. ROSE DP, GOLDMAN M, CONNOLLY JM, STRONG LE, (1991) High-fiber diet reduces serum estrogen concentrations in premenopausal women. Am J Clin Nutr; 54: 520±525. ROSE DP, (1992) Dietary fiber, phytoestrogens, and breast cancer. Nutrition; 8(1): 47±51. ROSE DP, (1993) Diet, hormones, and cancer. Annu Rev Publ Health; 14: 1±17. WEISSFELD JL, FLOOD A, SCHATZKIN R, HAYES RB,

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GROUP,

23 Phytoestrogens and the prevention of cancer Y. Ungar and E. Shimoni, Israel Institute of Technology

23.1

Introduction

Phytoestrogens are compounds produced by plants that may mimic or interact with estrogenic hormones. Their bioactivity makes these compounds an exciting development in the field of nutraceuticals and functional foods. Benefits of phytoestrogens include reducing cancer risk, modifying cholesterol homeostasis, and possible role in hormone replacement therapy treatments (Setchell 1998). Phytoestrogenic compounds were identified in numerous plant sources, however, the focus of this chapter is mostly isoflavones from soy. Soy isoflavones have been of great interest in the past decade due to their potential protective or preventive activity against a number of diseases such as cancers, cardiovascular diseases, and osteoporosis (Clarkson et al. 1995; Adlercreutz et al. 1995; Kurzer and Xu 1997; Anderson and Garner 1997; Setchell 1998). Several isoflavones from soy may bind to estrogen receptors to induce the expression of the genomic estrogen-response element. In bone cells this response can help maintain bone mass and density (Anderson and Garner 1998). They also act as tyrosine kinase inhibitors (Akiyama et al. 1987) and as antioxidants (Ruiz-Lerrea et al. 1987). In light of these studies and many others, the popularity of soy foods has increased dramatically in the past few years. In this chapter, we will review the occurrence of soy isoflavones and other phytoestrogens in foods, present their putative role in the prevention of hormone dependent and independent cancers, and point out some proposed mechanisms.

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23.2 Phytoestrogens in food: the effects of food processing and storage 23.2.1 Phytoestogens in food Phytoestrogen content in foods depends on the specific product, its solids content, and how it is affected by processing and storage conditions. It has been suggested that the threshold intake of dietary estrogens such as soy isoflavones to achieve a biological effect in humans is 30±50 mg/d (Setchell 1998). For the food-conscious consumer it is therefore very important to have readily available data on the typical content of these compounds in various foods. A comprehensive database for isoflavones was developed by a unique collaborative effort between the Food Composition Laboratory (FCL), and the Nutrient Data Laboratory (NDL) of ARS/USDA and the Department of Food Science and Human Nutrition of the Iowa State University (ISU). This database contains the most prominent isoflavones, daidzein, genistein, and glycitein as well as coumestrol, formononetin, and biochanin A. Phytoestrogen content in foods may vary markedly. No genistein or daidzein were found in raw materials such as alfalfa seeds (Franke et al. 1995, Murphy et al. 1999), flax seed (Mazur et al. 1996), lentils and lima beans (Franke et al. 1995, Mazur et al. 1998), as well as canola and soybean oils (Murphy et al. 1999). Similarly, processed bread and crackers contained extremely low isoflavone levels (Mazur et al. 1996). In beans, levels of genistein and daidzein vary according to the bean type, ranging from less than 10 gr/100gr up to over 1 mg/100gr (Franke et al. 1995, Fukutake et al. 1996, Mazur et al. 1998). Much higher isoflavone content was reported in soybeans, ranging from 50 to over 150 mg/100gr (Carrao-Panizzi and Kitamura 1995, Choi et al. 1996, Coward et al. 1993, Eldridge and Kwolek 1983, Farmakalidis and Murphy 1985, Franke et al. 1995, 1998, Jones et al. 1989, Mazur et al. 1998, Pratt and Birac 1979, Seo and Morr 1984, Setchell and Welsh 1987, Wang et al. 1990, Wang and Murphy 1996, Wang and Murphy 1994a,b). Other phytoestrogens such as coumestrol are found in significant concentrations in clover and soy sprouts (Franke et al. 1995, Wang et al. 1990). High levels of formononetin were detected in alfalfa sprouts (Murphy et al. 1999) and red clover (Petterson and Kiessling 1984), which contains also high amounts of biochanin A (Murphy et al. 1999, Petterson and Kiessling 1984). Soy-based food ingredients such as soy flour and soy protein concentrate and isolate contain very high amounts of these phytoestrogens: 130±200 and 100 mg/100gr respectively (Barnes et al. 1994, Coward et al. 1993, Eldridge and Kwolek 1983, Franke et al. 1995, 1998, Mazur et al. 1996, Murphy et al. 1999, Naim et al. 1976, Nguyenle et al. 1995, Padgette et al. 1996, Petterson and Kiessling 1984, Setchell and Welsh 1987, Wang et al. 1998a, Wang and Murphy 1996, Wang and Murphy 1994a,b, Coward et al. 1996, Seo and Morr 1984). Once processed, even the traditional soy foods contain lower amounts of isoflavone phytoestrogens. Tofu and miso contain 20±70 mg/100gr (Coward et al. 1993, Dwyer et al. 1994, Franke et al. 1995, 1998, Fukutake et al. 1996,

Phytoestrogens and the prevention of cancer 647 Murphy et al. 1999, Wang and Murphy 1996, Wang et al. 1990, Lu et al. 1995a, Wang and Murphy 1994b), whereas soy-milk contains 7±10 mg/100gr (Barnes et al. 1994, Coward et al. 1993, Dwyer et al. 1994, Franke et al. 1998, Jones et al. 1989, Lu et al. 1995a,b, Murphy et al. 1999, Nguyenle et al. 1995, Wang et al. 1990, Wang and Murphy 1996). Processed foods often use soy ingredients and contain variable amounts of isoflavone phytoestrogens. Infant formulas content showed high variability from 2 to 26 mg/100mg isoflavones, with genistein derivatives having a concentration about twice that of the daidzein derivatives (Murphy et al. 1997, Nguyenle et al. 1995, Setchell et al. 1997, Setchell and Welsh 1987). In soy cheese products their concentration reaches 7±40 mg/100gr (Coward et al. 1993, Franke et al. 1998, Wang and Murphy 1994b), and in meat substitutes such as burgers and meatless chicken nuggets, hot dog, or bacon 3±29 mg/100gr (Murphy et al. 1999, Wang and Murphy 1994b). 23.2.2 Processing and storage induced changes in phytoestrogens in food The concentration and the composition of soy-isoflavones in soy foods and soycontaining foods vary markedly, and depend on the formulation, raw material, processing, as well as post-processing changes during distribution and storage. For example, isoflavones content in soy sauce increased when it was produced from whole soybeans rather than defatted beans (Kinoshita et al. 1998). Wang and Murphy (1994a) identified 12 isomers of isoflavones, three aglycons (daidzein, genistein, glycitein) and nine glucosides (daidzin, genistin, glycitin; 600 -Oacetyldaidzin, -genistin, -glycitin; 600 -O-malonyldaidzin, -genistin, -glycitin). While high-protein soy ingredients contained similar concentrations compared with unprocessed soybeans, traditional nonfermented soybean foods had greater levels of glycosides than fermented foods. Processed soy foods contained only 6± 20% of the isoflavones of whole soybeans. In a later study, Murphy et al. (1999) evaluated isoflavones in 63 retail and institutional soy foods, and reported levels from 1 mg/g in soy sauce to 540 mg/g in tempeh. Hence, the concentration and chemical structure of soy isoflavones in foods is highly dependent on the raw material, processing and storage conditions. Changes in the content, derivatives, and chemical structure of phytoestrogens are very likely to occur during storage and processing. Long-term storage of soybean showed a dramatic decrease in the concentration of 6 00 -Omalonyldaidzin and 600 -O-malonylgenistin (Kudou et al. 1991; Lee et al. 2003). Hence, the malonyl derivatives appear to be most labile in the soybean raw material. Processing techniques also induce changes in the chemical profile of soy isoflavones in tempeh, soymilk, tofu, and protein isolate (Wang and Murphy, 1996, Wang et al. 1998a). Significant losses of isoflavones were observed during soaking (12%) and heat processing (49%) in tempeh production; coagulation (44%) in tofu processing; and alkaline extraction (53%) in soy protein isolate production (Wang and Murphy 1996). Malonyldaidzin and malonylgenistin decrease in the production of tempeh,

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soymilk, and tofu, producing higher levels of acetyldaidzin and acetylgenistin. Alkaline hydrolysis during protein isolate processing released deglycosilated daidzein and genistein, and an overall loss of 20% of the isoflavones (Wang et al. 1998a). A high temperature reduced the total isoflavone content in soy mixtures (Singletary et al. 2000). Most isoflavone degradation is attributed to the loss of the malonyl derivatives (Coward et al. 1998, Mahungu et al. 1999, Lee et al. 2003, Xu et al. 2002). Recent studies simulating commercial sterilization processes found differences in the stability of genistein and daidzein (Ungar et al. 2003). Changes in isoflavone composition and content may also occur during storage of soy products. An interesting report by Hayes et al. (2001) describe a decrease in the malonyl derivatives and a continuous change in the isoflavone profile, which continued during storage and were affected by storage temperature. Eisen et al. (2003) reported a decrease in soy-milk genistin content during six-month storage experiments at ambient conditions. Barnes et al. (1994) showed that the composition of isoflavone conjugates in soybeans and soy products is complex, and pointed out the potential implication of the chemical form on isoflavone metabolism, bioavailability and biological activity. It was their conclusion that studies are urgently needed to address this issue. Indeed, Ungar et al. (2003) showed that the changes in antioxidant potential in model solutions were different for daidzein and genistein, and depend on the pH. Evidence for the significance of the deteriorative reactions was given by Gallaher et al. (1996). Feeding a diet based on isolated soy protein (ISP) was found to reduce the occurrence of colonic precancerous lesions in rats. However, the same ISP after storage 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

References

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(1994), `The natural tyrosine kinase inhibitor genistein produces cell cycle arrest and apoptosis in Jukat T-leukemia cells', Leuk Res, 18, 431±39. TALALAY P (1989), `Mechanisms of induction of enzymes that protect against chemical carcinogenesis', Adv Enzyme Regul, 28, 237±50. TALALAY P, FAHEY J W, HOLTZCLAW W D, PRESTERA T, ZHANG Y (1995), `Chemoprotection against cancer by phase 2 enzyme induction', Toxicol Lett, 82±83, 173±79. THIAGARAJAN D G, BENNINK M R, BOURQUIN L D, KAVAS F A (1998), `Prevention of precancerous colonic lesions in rats by soy flakes, soy flour, genistein, and calcium', Am J Clin Nutr, 68, 1394S±99S. THOMPSON L U, SEIDL M M, RICKARD S E, ORCHESON L J, FONG H H (1996a) Antitumorigenic effect of a mammalian lignan precursor from flaxseed. Nutr Cancer 26, 159±65. THOMPSON L U, RICKARD S E, ORCHESON L, SEIDL (1996b), `Flaxseed and its lignan and oil components reduce mammary tumor growth at a late stage of carcinogenesis', Carcinogenesis, 17, 1373±76. TOU J C L, THOMPSON L U (1999), `Exposure to flaxseed or its lignan component during different development stages influences rat mammary gland structure', Carcinogenesis, 20, 1831±35. TRAGANOS F, ARDELT B, HALKO N, BRUNO S, DARZYNKIEWICZ Z (1992), `Effects of genistein on the growth and cell cycle progression of normal human lymphocytes and human leukemic MOLT-4 and HL-60 cells', Cancer Res, 52, 6200±208. UNGAR Y, OSUNDAHUNSI O F, SHIMONI E (2003), `Thermal stability of genistein and daidzein and its effect on their antioxidant activity', J Agric Food Chem, 51, 4394± 99. VERNA S P, SALAMONE E, GOLDIN B (1997), `Curcumine and genistein plant natural products show synergistic inhibitory effects on the growth of human breast cancer MCF-7 cells induced by estrogenic pesticides', Biochem Biophys Res Commun, 233, 692± 96. VERNA S P, GOLDIN B R (1998), `Effect of soy derived isoflavonoids on the induced growth of MCF-7 cells by estrogenic environmental chemicals', Nutr Cancer, 30, 232±39. WANG W, HIGUCHI C M (2000), `Dietary soy protein is associated with reduced intestinal mucosal polyamine concentration in male Wistar rats', J Nutr, 130, 1815±20. WANG C, KURZER M S (1997), `Phytoestrogen concentration determines effects on DNA synthesis in human breast cancer cells', Nutr Cancer, 28(3), 236±47. WANG H, MURPHY P A (1994a), `Isoflavone composition of American and Japanese soybeans in Iowa: effects of variety, crop year, and location', J Agric Food Chem, 42, 1674±77. WANG H, MURPHY P A (1994b), `Isoflavone content in commercial soybean foods', J Agric Food Chem, 42, 1666±73. WANG H J, MURPHY P A (1996), `Mass balance study of isoflavones during soybean

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(1990), `Simplified HPLC method for the determination of phytoestrogens in soybean and its processed products', J Agric Food Chem, 38, 185±90. È KELA È T, HASE T, ADLERCREUTZ H, KURZER M S (1994), 'Lignans and flavonoids WANG C, MA inhibit aromatase enzyme in human preadipocytes', J Steroid Biochem Mol Biol, 50, 205±12. WANG T T, SATHYAMOORTHY N, PHANG J M (1996), `Molecular effects of genistein on estrogen receptor mediated pathways', Carcinogenesis, 17, 271±75 WANG C, MA Q, PAGADALA S, SHERRARD MS, KIRSHNAN PG (1998a), `Changes of isoflavones during processing of soy protein isolates'. J Am Oil Chem Soc 75, 337±41. WANG W, LIU, LQ, HIGUCHI, C M, CHEN, H (1998b), `Induction of NADPH:quinone reductase by dietary phytoestrogens in colonic Colo205 cells', Biochem Pharmacol, 56, 189± 95. WEI H C, BOWEN R, ZHANG X S, LEBWOHL M (1998), `Isoflavone genistein inhibits the intiation and promotion of two stage skin carcinogenesis in mice', Carcinogenesis, 19, 1509±14. WU A H, ZIEGLER R G, HORN-ROSS P L, WEST D W, KOLONEL L N, ROSENTHAL J F (1996), `Tofu and risk of breast cancer in Asian-American', Cancer Epidemiol Biomark Prev, 5, 901±06. XU Z, WU Q, GODBER S J (2002), `Stabilities of daidzin, glycitin, genistin, and generation of derivatives during heating', J Agric Food Chem, 50, 7402±06. YOKOSHIKI H, SUMII K, SPERELAKIS N (1996), `Inhibition of L-type calcium current in rat ventricular cells by the tyrosine kinase inhibitor genistein and its inactive analog daidzein', J Mol Cell Cardiol, 28, 807±14. ZAVA D T, DUWE G (1997), `Estrogenic and antiproliferative properties of genistein and other flavonoids in human breast cancer cells in vitro', Nutr Cancer, 27, 31±40. WANG G, KUAN S S, FRANCIS O J, WARE G M, CARMAN A S

ZHANG J X, HALLMANS G, LANDSTROM M, BERGH A, DAMBER J E, AMAN P, ADLERCREUTZ H

(1997), `Soy and rye diets inhibit the development of Dunning R3327 rostatic adenocarcinoma in rats', Cancer Lett, 114, 313±14. ZHENG W, DAI Q, CUSTER L J, SHU X O, WEN W Q, JIN F, FRANKE A A (1999) `Urinary excretion of isoflavonoids and the risk of breast cancer', Cancer Epidemiol Biomarkers Prev, 8, 35±40. ZHOU J R, MUKHERJEE P, GUGGER E T, TANAKA T, BLACKBUM G L, CLINTON S K (1998), `Inhibition of murine bladder tumorigenesis by soy isoflavones via alterations in the cell cycle, apoptosis, and angiogenesis', Cancer Res, 58, 523±38.

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).

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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

Food phenolics and cancer chemoprevention

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

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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

Sources of further information and advice

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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Ho, C-T., Osawa, T., Huang, M-T. and Rosen, R.T. eds 1994. Food Phytochemicals and Cancer Prevention II: Teas, Spices, and Herbs. ACS Symposium Series 547. American Chemical Society. Washington, DC. Shahidi, F. and Ho, C-T. 2000. Phytochemicals and Phytopharmaceuticals. AOCS Press. Champaign, IL. Shahidi, F. and Naczk, M. 2004. Phenolics in Food and Nutraceuticals. CRC Press. Boca Raton, FL.

24.6

References

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and HAMMAR S (1996), `Effects of a combination of 9-carotene and vitamin A on lung cancer and cardiovascular disease', New Engl J Med, 334, 1150±1155. PAN N H, LIANG Y-C, LIN-SHIAU S-Y, ZHU N-Q, HO G T and LIN J-K (2000), `Induction of apoptosis by the oolang tea polyphenol theasinemin. A through cytochrome C release and activation of caspage-9 and caspage-3 in human U937 cells', J Agric Food Chem, 48, 6337±6346. PERCHELLET J-P and PERCHELLET E M (1989), `Antioxidants and multistage carcinogenesis in mouse skin', Free Rad Biol Med, 7, 377±408. PETERSON G and BARNES S (1991) `Genistein inhibition of the growth of human breast cancer cells: independence from estrogen receptors and the multi-drug resistance gene', Biochem Biophys Res Commun, 179, 661±667. PETERSON G and BARNES S (1993) `Genistein and biochanin A inhibit the growth of human prostate cancer cells but not epidermal growth factor receptor tryposine auto phosphorylation', Prostate, 22, 335±345. SHAHIDI F (1997), Natural Antioxidants: Chemistry, Health Effects and Applications, AOCS Press, Champaign, IL. SHIMIZU M, YOSHIMI N, YAMADA Y, MATSURAGA K, KAWABATA K, HARA A, MORIWAKI H and MORI H (1999), `Suppressive effects of chlorogenic acid on N-methyl-Nnitrosourea-induced glandular stomach carcinogenesis in male F344 rats', J Toxicol Sci, 24, 433±439. SIMON A, ALLAIS D P, DUROUX J L, BASLY J P, DURAND-FONTAINIER S and DELAGE C (1998), `Amathcariatical model describing tannin-protein association', Anal Biochem, 263, 46±50. SO F V, GUTHRIE N, CHAMBERS A R and CARROLL K K (1997), `Inhibition of poliferation of estrogen receptor-positive MCF-7 human breast cancer cells by flavonoids in the presence and absence of excess estrogen', Cancer Lett, 112, 127±133. MEYSKENS F L, VALANIS B, WILLIAMS J H, BARNHART S

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(1998) `Resveratrol inhibits cycloxygenase-2 transcription and activity in phorbol ester-treated human mammary epithelical cells', J Biol Chem, 273, 21875±21882. SURH Y-J (2002), `More than spice: capaicin in hot chilli pepper makes cancer cells commit suicide', J Natl Cancer Inst, 94, 1263±1265. SURH Y-J (2003), `Cancer Chemoprevention with dietary phytochemicals', Nature Rev, 3, 768±780. SURH J-Y, LEE E and LEE J M (1998), `Chemopreventive properties of some pungent ingredients present in red pepper and ginger', Mutat Res, 402, 259±267. TANAKA T, NISHIKAWA A, SHIMA H, SUGRE S, SHINODA T, YOSHIMI N, IWATA H and MORI H (1990), `Inhibitory effects of chlorogenic acid, reserpine, polyprenoic acid (E5166) or coffee on hepato carcinogenesis in rats and hamsters', Basic Life Sci, 52, 429, 440. TANAKA T, YOSHIMI N, SUGIE S and MORI H (1992), `Protective effects against liver, colon and tongue carcinogensis by plant polyphenols' in Huang M-T, Ho C-T and Lee CY, Phenolic Compounds in Food and their Effects on Health II. Antioxidant and Cancer Prevention, ACS Symposium Series 507. American Chemical Society, Washington, DC, 326±337. TANAKA T, KOJIMA T, SUZUI M and MORI H (1993), Chemoprevention of colon carcinogenesis by natural product of a simple phenolic compound protocatechuic acid: suppressing effects on tumor development and biomarkers expression of colon tumorigenesis', Cancer Res, 53, 3908±3913. SUBBARAMAIAH K

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and OHIGASHI H (1998), `Chemoprevention of 4-nitroquinoline-1-oxide induced oral carcinogenesis by citrus auraptene in rats', Carcinogenesis, 19, 425±431. UEDA J, SAITO N, SHIMAZU Y and OZAWA T (1996), `A comparison of scavenging abilities of antioxidants against hydroxyl radicals', Arch Biochem Biophys 333, 377±384. WALASZEK Z (1990), `Potential use of D-glucaric acid derivatives in cancer prevention', Cancer Lett, 54, 1±8. WANG Z Y, AGARWAL R, BICKERS D R and MUKHTAR H (1991), `Protection against ultraviolet B-radiation-induced photocarcinogensis in hairless mice by green tea polyphenols', Carcinogenesis, 12, 1527±1530. WATTENBERG L W (1983), `Inhibition of neoplasia by minor dietary constituents', Cancer Res, 43, 2448S±24535. WATTENBERG L W (1985), `Chemoprevention of cancer', Cancer Res, 45, 1±8. WATTENBERG L W (1992), `Inhibition of carcinogensis by minor dietary constituents', Cancer Res, 52, 20855±20915. WATTENBERG L W, COCCIA J B and LAM L K T (1980), `Inhibitory effects of phenolic compounds on benzo (a) pyrene-induced neoplasia', Cancer Res, 40, 2820±2823. YANG S Y and CHUNG J E (2000), `Tea and tea polyphenolics in cancer prevention', J Nutr, 130, 472S±478S. YEN G-C and CHEN H-Y (1994), `Comparison of antimutagenic effect of various tea extracts (green, oolong, pouchong and black tea)', J Food Protec, 57, 54±58.

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 `optimise' 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 `optimal' 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 (<50% RDA) of each of these eight micronutrients. In this chapter we will examine the evidence for a possible role of the different vitamin deficiencies in the aetiology of cancer and also whether there is any evidence that nutrient intakes greater than the RDA might be more protective against cancers than normal dietary intakes.

25.2

The role of vitamins in the prevention of cancer

25.2.1 Vitamin A Aspects of vitamin A metabolism that might link to cancer Vitamin A is present in plasma mainly as all-trans-retinol bound to retinol binding protein. In healthy adults the concentration is about 2 mol/L and is homeostatically controlled. Daily intakes of up to 7.5 mg vitamin A (25,000 IU) in vitamin A-sufficient persons have little or no effect on steady state

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concentrations (Willett et al. 1983). Variable amounts of vitamin A are stored in the liver but as stores decrease, plasma concentrations are reduced only when stores of vitamin A are almost exhausted. However, plasma retinol concentrations are depressed by illness, even in subclinical disease (Thurnham et al. 2003). Plasma retinol is therefore not a very useful biomarker of vitamin status as it does not reflect dietary intake until vitamin A reserves are critical and plasma concentrations can be depressed by occult illness, which could be wrongly interpreted as marginal vitamin A deficiency. Also present in plasma are very small amounts of retinoic acid metabolites bound to albumin (1±14 nmol/L) (Blaner and Olson 1994). High doses of vitamin A will increase plasma concentrations (4±10-fold) of these metabolites for a few hours (Buss et al. 1994). Vitamin A functions in vision, cell differentiation, immune response and embryonic development but the function most closely associated with cancer is cell differentiation (DeLuca et al. 1970). Most actions of vitamin A in cell differentiation are induced by retinoic acid by two classes of nuclear retinoid receptors (Kliewer et al. 1994). Carcinogenesis is characterised by aberrant differentiation, which is manifested either by blocking of cells at an early stage of differentiation or by redirection of differentiation towards an abnormal pathway. Retinol and retinol esters have been reported to maintain proper differentiation in many epithelial tissues, to reverse abnormal differentiation in premalignant and malignant cells and maintain normal mucocilliary epithelium for several months in organ culture of tracheal explants (IARC Working Group 1998b). Evidence of the importance of vitamin A in the maintenance of normal epithelial development, may be inferred from the results of several studies which used intermediate biomarkers of oropharyngeal cancer to examine the benefits of retinol and -carotene supplements against oral premalignancy. Positive results were obtained in several studies using the frequency of micronuclei in buccal cells and/or the grade of oral leukoplakia (Prasad et al. 1995; Stich et al. 1988, 1989) to monitor the effects of vitamin A, but none or equivocal results using oesophageal dysplasia (Wahrendorf et al. 1988). Evidence for dietary deficiency and associations with increased risk of cancer Dietary vitamin A is obtained in two forms, as preformed retinol in retinyl esters and as provitamin compounds, namely, the carotenes of which the most important is -carotene. Many of the early studies on vitamin A status and cancer risk did not distinguish between these two forms with the result that protective effects attributed to vitamin A should more correctly have been attributed to plant sources of provitamin A. The largest body of evidence associating vitamin A deficiency with specific cancer sites exists for lung cancer. Most case-control studies have reported an inverse association between total vitamin A intake and lung cancer. However, in general, where studies have examined preformed vitamin A and carotenoid intake separately, no association or only a weak inverse association with vitamin A was evident whereas most studies report an inverse association with carotenoid consumption. There is less

Vitamins and the prevention of cancer 685 evidence but a similar situation also appears to exist in the case of breast cancer. Overall the data suggest that preformed vitamin A does not influence lung or breast cancer risk (IARC Working Group 1998b). Likewise, with gastric, prostate, bladder, cervical, skin and colon cancer, there is no data to suggest that preformed vitamin A is protective but with upper aerodigestive tract cancer, in general, results from both case-control and intervention studies suggest preformed vitamin A is protective (IARC Working Group 1998b). Evidence for benefits from `optimal' nutrition Overall, there is no consistent evidence that four- to six-year supplementation of retinol reduces the risk of any type of cancer. In the CARET study, where retinol and -carotene together were compared with placebo, the risk of carcinoma of the lung was significantly elevated in the group supplemented vitamin A compared with the placebo group (Omenn et al. 1996). Among asbestos workers in the Wittenoom trial who received either retinol or -carotene (no placebo group), there was a lower rate of lung cancer in comparison to historical controls. Lung cancer was less common in the retinol than the -carotene group, but the difference was not significant (Musk et al. 1998). In two intervention trials in China where cancers of the stomach and oesophagus are common, retinol was administered either with zinc or a cocktail of micronutrients but there were no apparent benefits (Blot et al. 1993; Li et al. 1993). Likewise, in two randomised, double-blind, controlled trials done in Arizona to examine the efficacy of retinol supplementation on the incidence of the first new non-melanoma skin cancer there was very little obvious benefit from retinol. In one study, the retinol group showed a lower RR (0.7) of developing cutaneous squamous cell carcinoma (Moon et al. 1997) but in the second the proportion of participants who developed cancers during the study was slightly higher in the retinol than the placebo group (Levine et al. 1997). There was no difference in the basal cell carcinoma risk in response to vitamin A treatment in either study. In conclusion, there is some evidence that pre-formed retinol provides protection against cancers of the aero-digestive tract and retinol may exert anti-inflammatory effects in these tissues (Stich and Anders 1989; Thurnham et al. 1988; Wahrendorf et al. 1988) 25.2.2 Carotenoids Aspects of carotenoid metabolism that might link to cancer Over 600 carotenoids from natural sources have been characterised. In wellnourished human tissue, carotenoids are primarily present in adipose (80±85%) liver (10%) and muscle (2±3%) tissues but the highest concentrations are found in corpus luteum (60 g/g = 180 mol/L) and adrenal gland (20 g/g) while the concentration in the adipose and liver tissue is about 10 g/g. Total body pool of carotenoids is about 100±150 mg in a well-nourished person and approx 1% is found in the serum where the concentration is 0.08±8 mol/l (Bendich and Olson 1989). The carotenoids comprise 40 carbon molecules with a

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polyisoprene structure whose array of conjugated double bonds make them particularly effective at quenching free radicals. The antioxidant potential has been demonstrated in various in vitro systems (IARC Working Group 1998a) and in vivo systems (Collins 2001) and maintaining the redox status of the cell is the most likely way that carotenoids protect against cancer. Evidence for dietary deficiency and associations with increased risk of cancer There is considerable epidemiological evidence for a low risk of many cancers being associated with a high intake of carotenoids from fruit and vegetables (Hennekens 1986). In general two kinds of observational cohort study have assessed relationships between dietary intake of carotenoids and cancer; those based on blood concentrations of carotenoids as a proxy for dietary intake and those usually based on food frequency questionnaires to measure carotenoid intake. Results for prospective blood studies and lung cancer are remarkably consistent with almost all studies showing an inverse association between blood -carotene or total carotenoid concentrations and cancer risk (Connett et al. 1989; IARC Working Group 1998a). Likewise analysis of case-control studies that use food frequency data found similar results (IARC Working Group 1998a). The results for other sites are less compelling with the possible exception of cervical, oral and pharyngeal cancers (IARC Working Group 1998a). Databases for the major carotenoids in fruits and vegetables have now been published for a number of countries but most studies relied on food tables which provided only provitamin carotenes, i.e., mainly -carotene. Studies mainly looked at lung, digestive tract and breast cancer and while in the majority of cases the relative risk of cancer was below 1.0, the lower results were not significant. One of the few results which did achieve significance was the observation by Giovannucci et al. (1995) who found, at several time points, that the more frequent consumption of the dietary component lycopene, and of tomato intake as a food, in the Health Professions Follow-up Study, was associated with a lower risk of prostate cancer (Giovannucci 2002). Evidence for benefits from `optimal' nutrition The effects of `treatment' with -carotene (and to a lesser extent canthaxanthin) on possible antecedents of malignancy have been addressed in a substantial number of investigations. They provide evidence of the lack of efficacy of shortterm supplementation with -carotene in preventing colorectal adenomas, however, they do suggest that -carotene treatment (with or without retinol) brought about the regression of oral leukoplakia. It should be noted that many of the trials were not randomised or double-blinded and assessments of leukoplakia were often subjective. In addition, the results of other studies of possible relevance to carcinogenesis which assessed cytogenetic or immunological abnormalities have also been inconsistent (IARC Working Group 1998a). Five intervention trials have examined the effects of -carotene supplements, alone or in combination with selenium, a-tocopherol or retinol, on cancer

Vitamins and the prevention of cancer 687 incidence. In the studies in Linxian, China, -carotene (15 mg) was given with selenium and -tocopherol (Blot et al. 1993) or a multi-micronutrient mixture (Li et al. 1993) for five years. In the first study there were 21% lower incidence and mortality rates of gastric cancer (P<0.05) but no effect on oesophageal cancer (Blot et al. 1993). In the ATBC (Heinonen et al. 1994) and CARET (Omenn et al. 1996) studies, -carotene (20 and 30 mg and given with tocopherol or retinol respectively) was associated with 16 and 36% higher rates of lung cancer. Finally, in the Physicians Health Study, 50 mg -carotene or aspirin was given on alternate days for 11±12 years. There was a 22% nonsignificant reduction in the prevalence of lung cancer in the non-smokers and there were also no significant effects in the smokers (Hennekens et al. 1996). In conclusion, some benefits were associated with -carotene supplements against pre-malignant lesions of oral cancers, but there were no apparent benefits to be gained against cancer from supplementing human diets with large amounts of -carotene either singly or in combination with other nutrients. The reason for the apparent increased risks of cancer in persons who smoke tobacco is still being studied. Post hoc analysis of the results from the ATBC and CARET studies suggest that the excess mortality was associated with the heavy smokers and heavy drinkers (IARC Working Group 1998a) and in connection with this it is important to note that -carotene in rat lungs is a powerful indicator of carcinogen bioactivating enzymes (Paolini et al., 1999). Finally, it should be said that all the human intervention studies have been done with carotene. Analysis of dietary intake and blood data suggests that both intake and blood concentrations of the different carotenoids are inter-correlated but whether different effects on cancer risk would have been obtained if a different carotenoid had been used in the intervention studies is impossible to tell. 25.2.3 Vitamin D Aspects of metabolism that might link to cancer Vitamin D is largely obtained by conversion of 7-dehydrocholesterol to cholecalciferol through the action of ultraviolet light on the skin. Cholecalciferol is distributed in the lipid deposits in the body and the first stage of metabolism is by hydroxylation in the liver to 25-hydroxy-cholecalciferol (25-OHD). 25-OHD is a good biomarker of vitamin D status and plasma concentrations can vary from those associated with deficiency (<20) to over ~140 nmol/L in those exposed to generous amounts of sunlight (Holdsworth et al. 1984). The active form of vitamin D is 1,25-dihydroxy-cholecalciferal (calcitriol) and this is synthesised mainly in the kidney though it is now known that there are several other tissues where synthesis of calcitriol occurs, e.g., macrophages, bone, keratinocytes, brain glial cells, etc., but probably only for local distribution. The effects of calcitriol on immune cells are especially important (Casteels et al. 1995) and the potential impact of calcitriol on brain function may explain some of the effects of sunlight on moods and emotions (Stumpf 1988) but these are but a few of the many actions calcitriol is now known to have. The most thoroughly researched function

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of vitamin D is its role in maintaining adequate calcium concentrations in the blood and supporting healthy bone growth. It is a failure of proper bone mineralisation which results in the vitamin D deficiency disease rickets. It is now known that there are vitamin D receptors in most if not all cells throughout the body and there are many vitamin D responsive genes with a whole range of activities (Hannah and Norman 1994). Experimental studies have shown that vitamin D at physiological concentrations protects cell proteins and membranes against oxidative stress by inhibiting the peroxidative attack on membrane lipids (Wiseman 1993). In addition, experimental evidence suggests that hepatic glutathione content of calcitriol-treated rats increased by 50% (Chatterjee 2001), an effect which would reduce the risk of sister chromatid exchanges (Weitberg et al. 1985). Vitamin D, at a concentration range of 2 10ÿ8 to 5 10ÿ8M, induces apoptosis in most cancer cells, stabilises chromosomal structure and prevents DNA double strand breaks induced either by endogenous or exogenous factors. Vitamin D is also effective in stimulating DNA synthesis in adult alveolar II cells and provides a novel mechanism of modulating epithelial cell proliferation in the context of lung development and repair against injury. Experimental work has also shown that vitamin D can regulate various proto-oncogenes (c-myc, c-fos, c-jun), has differentiationinducing properties, anti-proliferative effects on keratinocytes and by enhancing gap-junction communication may inhibit malignant cell transformation (reviewed in Chatterjee 2001). Thus a lot of evidence has accumulated to suggest that vitamin D is important for DNA stability and therefore potentially for protection against cancer. Evidence for dietary deficiency and associations with increased risk of cancer As indicated above, dietary data alone is inadequate to assess vitamin D status and even sunlight exposure varies considerably in a population and is difficult to quantify hence observational studies rarely refer to vitamin D. Epidemiologically, carcinomas of colon, breast and prostate have been linked via sunlight exposure with vitamin D status (Garland et al. 1989, 1990; Schwartz and Hulka 1990) and there is some experimental data to support mechanisms of action (reviewed in Chatterjee 2001). Evidence for benefits from `optimal' nutrition There are numerous health benefits from adequate vitamin D status but little human evidence yet to suggest that vitamin D status protects against cancer. In the temperate latitudes of northern Europe, elderly people and women of reproductive age, particularly those whose cultural habits prevent much skin exposure to sunlight, are the persons most at risk of vitamin D deficiency. It seems appropriate that vitamin D status in the elderly should receive greater attention since, in any population, cancer rates are highest in this group and immobility is the main cause of poor vitamin D status. Maintaining vitamin D status may halt or reduce cancer progression and enable quality of life to be maintained for longer.

Vitamins and the prevention of cancer 689 25.2.4 Vitamin C Aspects of metabolism that might link to cancer Vitamin C, a water-soluble glucose derivative, has considerable antioxidant activity in vitro, in part because of its ease of oxidation and because the semidehydroascorbate radical derived from it, is of low reactivity. Vitamin C in vivo is an essential cofactor for a range of enzymes involved in diverse metabolic pathways, but much recent literature has focused on its antioxidant effects. Consumption of food rich in vitamin C (fruits and vegetables) is associated with lower risk of cardiovascular disease, of many types of cancer and possibly of neurodegerative disease, but the extent to which vitamin C contributes to these effects is uncertain (Halliwell 2001). Ascorbate is present in human plasma at concentrations in the range 20 to 90 mol/L while intracellular concentrations may be in the molar range. It is interesting to note that concentrations of gastric juice ascorbate are three to four times higher than those in plasma (Rathbone et al. 1989). The role of gastric ascorbate may serve at least two functions. Ascorbate will convert insoluble ironIII to the more easily absorbed to ironII, but gastric ascorbate may also protect against intra-gastric nitrosamine formation. It is noteworthy that while the major case-control studies of gastric cancer and carotene intake suggested a consistently lower risk of cancer with higher consumption of foods containing carotenes, the effect of carotene was attenuated when vitamin C intake was introduced in the statistical model (Risch et al. 1985). Many nitrosamines are potent carcinogens and while there is no proof that they cause cancers in man, they do occur in human tissues and have been implicated in the causation of gastric cancer (Correa et al. 1975). Elevated bacterial colonisation associated with gastritis or achlorhydria would favour increased nitrate to nitrite conversion and the formation of nitrosamines. Ohshima and Bartsch (1981) subsequently reported that nitrosamines could be formed in man when they showed the increase in urinary nitrosoproline excretion following the consumption of nitrate and proline and that the formation could be suppressed by simultaneous administration of vitamin C or E. Gastric cancer tends to be more common in poorer than in the industrialised countries and thus a contributory factor may be the greater fluctuation in vitamin C availability due to seasonal variability in supply. It is widely believed that a significant contributor to the age-related development of cancer is the relentless attack of reactive oxygen species upon DNA (Ames et al. 1985, 1993). Much of this damage is repaired but a low level of oxidatively modified bases remains in DNA. The concentration of 8-hydroxy20 -deoxyguanosine (8OHdG), one of the base damaged products whose mutagenicity has been established may be around 1 per 106 guanine residues or even higher (Halliwell 2000). Halliwell (2001) has pointed out that levels of oxidised bases are elevated in cells at sites of chronic inflammation, consistent with the idea that chronic inflammation raises the risk of cancer development, and that they also appear elevated in some pre-neoplastic lesions. Genotoxic agents seem to accelerate oxidative DNA damage, the prime example of human

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relevance being cigarette smoking (Asami et al. 1997). Inflammation and smoking are frequently associated with depressed plasma concentrations of vitamin C (Thurnham 1997) which may increase the risk of oxidative DNA damage but ascorbate supplements given to people who smoked did not decrease urinary excretion rates of 8OHdG (Prieme et al. 1997). In contrast, several studies using fruit and vegetables reported decreased levels of 8OHdG in lymphocytes, total white cells or urine, suggesting decreased oxidative damage to DNA but in a recent review it was concluded that there was no compelling evidence to date that ascorbate supplements can decrease the levels of oxidative damage in vivo except perhaps in subjects with very low vitamin C intakes. Similarly there is no conclusive evidence from studies of strand breaks, micronuclei, or chromosomal aberrations for a protective effect of vitamin C (reviewed in Halliwell 2001). Evidence for dietary deficiency and associations with increased risk of cancer Vitamin C is one of the important components of fruits and vegetables and these foods are strongly associated with lower risks of stomach, mouth and pharynx, oesophagus, lung and colon cancer and more weakly associated with protection against cancers of the larynx, pancreas, breast and bladder (World Cancer Research Fund 1997). A review mainly of case-control studies in 1944 also found that high intakes of vitamin C and or fruit and vegetable consumption were associated with lower risks of cancer of the aerodigestive system (Sauberlich 1994). Many studies also report lower concentrations of vitamin C in the blood of cases compared to the controls. However, as pointed out elsewhere, the importance of such data is difficult to assess. Plasma ascorbate is strongly influenced by dietary intake and appetite may well be reduced in some cases, and also inflammation depresses blood ascorbate concentrations (Thurnham 1997). However, in spite of the large amount of data suggesting that dietary vitamin C reduces cancer risks, it is difficult separating the effects of vitamin C from those of the potentially very large number of other `protective' substances in fruit and vegetables. Evidence for benefits from `optimal' nutrition There is no evidence either from studies in patients with severe vitamin C deficiency (Fraga et al. 1991) or healthy subjects (Rehman et al. 1998) that replenishment of the diet with vitamin C above current RDA values for the UK or USA, has any greater effect on 8OHdG concentrations (Halliwell 2001). Most intervention trials with vitamin C are targeted at cardiovascular disease (CVD). However, the recently reported randomised, double-blind, MRC/BHF Heart Protection Study which provide 600 mg vitamin E, 250 mg vitamin C and 20 mg -carotene daily for five years to patients with early evidence of CVD, found no benefit on either CVD or cancer risk (Heart Protection Study Group 2002). Likewise, the Chinese study which provided a multi-nutrient supplement containing vitamin C (180 mg/day) for five years found no effects on the two major cancers in the area ± stomach or oesophageal cancer (Li et al. 1993).

Vitamins and the prevention of cancer 691 Taken together these data suggest that elevated daily intakes of vitamin C offer no extra protection against cancer than that provided by current RDAs. 25.2.5 Vitamin E Aspects of metabolism that might link to cancer Vitamin E circulates in the blood bound to lipoproteins. It is incorporated into lipoproteins in the liver at the time of synthesis of the very low density lipoproteins (VLDL). On average there are 6 mol of vitamin E per lipoprotein molecule. As lipoproteins transport most of the lipid in the body, the amount of vitamin E in blood tends to be related to lipid load. Blood cholesterol concentrations are the most stable biomarker of lipid and the molar ratio of vitamin E to cholesterol is usually between 4±6 mol vitamin E per 1,000 of cholesterol (Thurnham et al. 1986). In the tissues, vitamin E is found in membranes of all cellular and sub-cellular structures. The amount present tends to be proportional to oxygen exposure thus lung and heart have the highest concentrations while brain is lowest and muscle and liver in between (Kornbrust and Mavis 1979). The importance of oxidation damage in the potential formation of products which might lead to cancer is outlined in the section on vitamin C. -Tocopherol is regarded as the most reactive chain-breaking antioxidant vitamin found in the body (Burton and Ingold 1981). In the presence of chain-breaking phenolic antioxidants (ArOH) like -tocopherol, oxidation damage is minimised by terminating the chain at an earlier stage and generating a semi-stable free radical ArO . For vitamin E to retain its antioxidant properties, it must be reduced back to ArOH, that is, it must react with a hydrogen donor. The localisation of vitamin E in membranes means that the donor is highly likely to be watersoluble and can easily be transported away to be regenerated itself. Vitamin C and cellular glutathione are the most likely reductants (Niki et al. 1982). Vitamin E also has another function that can be viewed as antioxidant although it is not known to involve quenching of radicals. Vitamin E is reported to inhibit protein kinase C (PKC) activity in many cell types (Azzi et al. 1993; Keaney et al. 1996) and suppress activation of the transcription factors, nuclear factor B (NF-B) and activator protein-1 (AP-1) (Eugui et al. 1994). PKC is an important signalling molecule situated on outer membrane of cells (Nishizuka 1984). Physiologically it is activated by extracellular signals through an increase in calcium and diacylglycerol but it can also be stimulated directly by tumour promotor phorbol myristate (Nishizuka 1984). NF-B is critical for the inducible expression of many genes involved in the immune and inflammatory responses IL-1, IL-2, IL-2Ra, IL-6, IL-8, TNFa, TNFb, bIFN, GM-CSF, serum amyloid, 1±acid glycoprotein. Activation of NF-B is achieved by a variety of agents: virus, UV-light, ROS, phorbol esters, cytokines and mitogens. All these agents signal situations of stress. A characteristic feature of the NF-B activation is the rapidity with which this protein is induced. TNF- can cause significant activation of NF-B

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within minutes. The rapidity of action allows NF-B to function as an effective signal transducer, connecting events occurring in the cytoplasm to responses in the nucleus (Kopp and Ghosh 1995). Phorbol esters are potent tumour promoters and initiate the effects on the cell by binding to protein kinase C (Nishizuka 1984). More recently, it was shown that a saturating dose of -tocopherol succinate (40 mol/L) inhibited oxidant production by neutrophils which were stimulated with phorbol myristate acetate (PMA). The tumour promotor directly activates PKC by binding to a specific site on the regulatory domain of the enzyme. This binding displaces an inhibitory pseudosubstrate and unfolds the kinase over the membrane surface. It is suggested that the action of -tocopherol on PKC activity is related to its property as a stabiliser of lipid bilayer membranes and hence prevents the unfolding of the PKC on the cell surface (Chan et al. 2001). The physiological interpretation of the effects of vitamin on PKC are open to question. The effects of vitamin E on neutrophil activity followed the uptake of saturating doses of the vitamin. Supplementation of human volunteers with large quantities of the vitamin (30±90 times the daily intake of 10 mg) has transient effects on plasma concentrations of -tocopherol (Esterbauer et al. 1992) and only increased tissue concentrations in heart muscle approximately 2±3 fold (Mickle et al. 1991). Whether such an increase in -tocopherol could influence signalling pathways in vivo is not known nor is it known whether blocking of the signalling pathway would be advantageous. As indicated above, NF-B is a critical control factor involved in the immune and inflammatory responses and is self-regulated to prevent over-response. If the initial response of NF-B should be blunted by excess vitamin E, this could be a disadvantage. Evidence for dietary deficiency and associations with increased risk of cancer Although vitamin E is believed to be a highly important antioxidant in the body, fewer studies have reported relationships between dietary intakes or blood concentrations of vitamin E and risks of cancer than for the carotenoids or vitamin C (Carroll and Kritchevsky 1994). The reasons may be because intake of vitamin E is difficult to quantify because much comes from vegetable oils used in food preparation. In addition, both animal and plant foods tend to contain vitamin E as it is an integral part of the tissue structure. Thus intakes of vitamin E are usually fairly homogeneous within a population (World Cancer Research Fund 1997) and the plasma concentration of vitamin E tends to reflect the amount of lipid in blood rather than vitamin E in the diet (Thurnham et al. 1986). Persons who are sick will frequently have depressed appetites and depressed plasma lipids. Thus either the lack of useful biomarkers of vitamin E status or indeed the lack of vitamin E deficiency may be the reason why the World Cancer Research Fund report (World Cancer Research Fund 1997) concluded that there was only possible evidence of a decreased risk of lung and cervical cancer and insufficient evidence to make any conclusions on colon and rectum. Human studies have shown that dietary vitamin E very effectively blocks nitrosamine formation (see section on vitamin C (Ohshima and Bartsch 1981)).

Vitamins and the prevention of cancer 693 However, reviews on the subject highlight the potential importance of vitamin C but not vitamin E in the prevention of cancer (Chen et al. 1988; Mirvish 1986). Again, difficulties in identifying differences between subjects in their intake of vitamin E might be the reason or it may be that in the aqueous environment of the stomach, vitamin E is less important than vitamin C. In fact, there is no evidence to suggest any benefit against cancer from elevated dietary intakes of vitamin E. 25.2.6 Riboflavin Aspects of metabolism that might link to cancer Riboflavin is a water-soluble B vitamin, also known as vitamin B2. It has a fundamental role in energy metabolism and the generation of adenosine triphosphate. Riboflavin is present in a variety of both plant and animal foods but it is richest in dairy products. Deficiency of the vitamin is not associated with any characteristic disease. Early evidence of deficiency can often be detected by cracks in the skin at the corners of the mouth (angular stomatitis) and lips (cheilosis) which can also be accompanied by a swollen red beefy tongue (glossitis). More severe deficiencies are associated with scrotal dermatitis. From the point of view of cancer, the involvement of riboflavin in glutathione, folate and vitamin B12 metabolism is probably of greatest relevance. Riboflavin in the form of flavin adenine dinucleotide (FAD) is an essential cofactor for the enzyme glutathione reductase. The function of glutathione reductase is to regenerate reduced glutathione (GSH) from oxidised glutathione (GSSG). GSH is a highly important cellular antioxidant found in all aerobic tissues and assists in maintaining a reducing environment within the cell (Meister 1983), is an essential substrate for glutathione peroxidase (Adelekan and Thurnham 1996) and probably assists in maintaining vitamin C in the reduced, i.e., active form (Mrtensson and Meister 1991). Riboflavin is also a precursor of the cofactor flavin mononucleotide (FMN). FMN and FAD both serve as cofactors in the metabolism of the vitamins pyridoxine, folate and B12 and potentially influence homocysteine metabolism (see section on folate) (Selhub 1999). Evidence for dietary deficiency and associations with increased risk of cancer Dairy products are the richest dietary sources of riboflavin but in developing countries, unless milk consumption is a part of the culture, most people obtain most riboflavin from cereal grains and the intake is barely adequate (Thurnham et al. 1971). There is some evidence to suggest that deficiencies of riboflavin are associated with increased risks of aerodigestive cancers especially oesophageal cancer (Larsson et al. 1975). In certain parts of the world such as north-eastern Iran, Kazakstan, Southern Transkei and Henan Province in China, agestandardised incidence rates for oesophageal cancer in men can exceed 100 per 100,000 per annum (Day and Munoz 1982; Li et al. 1980). In these areas

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poor vitamin A, riboflavin and zinc status are commonly found (Kmet et al. 1981; Thurnham et al. 1982) and treatment of patients with inflammatory lesions in the lower two-thirds of the oesophagus with riboflavin appeared to improve the condition (Crespi et al. 1979). In industrialised countries, aerodigestive cancers are strongly linked to alcohol abuse and smoking but alcohol consumption is not practised widely in the middle-eastern countries or China (Cook-Mozaffari et al. 1979) and smoking habits were also very different (Thurnham et al. 1988). Intervention studies done with a mixed supplement of retinol palmitate, riboflavin and zinc over 12 months, failed to demonstrate any convincing link between any of the nutrients and the early lesions (Munoz et al. 1985). However, while the riboflavin results clearly showed that only the vitamin-treated group were influenced by riboflavin, there were improvements in vitamin A status and to a lesser extent zinc in both vitamin and placebo groups after the 13.5 month intervention. A re-analysis of the data, showed that a histologically normal oesophagus was more likely to be present in those who showed large increases in retinol and zinc blood concentrations (Wahrendorf et al. 1988). The possible involvement of the main component of placebo capsule, the antioxidant mannitol, and environmental factors in the improvement in vitamin A and zinc status in the placebo group, is discussed elsewhere (Thurnham et al. 1988). Evidence for benefits from `optimal' nutrition It was already being noted in the 1970s that improving nutritional status in northern European countries was leading to the disappearance of aerodigestive cancers (Larsson et al. 1975). The predisposing factors for aerodigestive cancers in northern Europe may have been different to those in the Middle East and China for there was clearly no evidence that the riboflavin supplement reduced the prevalence of oesophageal lesions in the Chinese study (Munoz et al. 1985; Wahrendorf et al. 1988). The Chinese subjects received 200 mg riboflavin weekly but blood tests showed that riboflavin status was not optimal either at 2 or 13.5 months post-treatment (Thurnham et al. 1988). 200 mg of riboflavin per week is equivalent to ~18 mg/day which is more than ten times the RDA for riboflavin (Department of Health 1991). However, the dose was given on one day per week and probably much was lost through malabsorption and urinary excretion following plasma overload, hence status never normalised. There is also evidence that riboflavin status may not be optimal in some industrialised countries. In Norwegian blood donors, low plasma riboflavin concentrations were associated with slightly elevated homocysteine concentration, and although the relationship was mainly confined to subjects with a particular polymorphism of the MTHFR gene (Hustad et al. 2000) (see Fig. 25.1 and section on folate), it does suggest there is some evidence of room for improvement in riboflavin status in the adult population.

Vitamins and the prevention of cancer 695

Fig. 25.1 Folate metabolism and DNA synthesis, methylation and repair.

* B-vitamin-dependent enzymes: 5-methyltetrahydrofolate:homocysteine methyl transferase (methionine synthase, vitamin B12 dependent), 5,10-methylenetetrahydrofolate reductase (vitamin B2 dependent) and serine hydroxymethyl transferase (vitamin B6 dependent). ** Other abbreviations: dUMP is deoxyuridine-50 -phosphate; dTMP is deoxythymidine-50 phosphate; THF is tetrahydrofolate. Figure modified from Fenech (2001).

25.2.7 Folate, vitamin B12 and vitamin B6 Aspects of metabolism that might link with cancer Folic acid is a water-soluble vitamin. It is present in fruits and vegetables but because it is sensitive to heat and oxidation, overcooking will destroy the vitamin. Vitamin B6 is widely distributed in vegetable, cereal and animal foods and deficiencies are rare. Vitamin B12 is found mainly in foods of animal origin and vegetarians and especially vegans will have a high risk of vitamin B12 deficiency. Liver is a rich source of all three vitamins. An early sign of deficiency of folic acid and vitamin B12 is megaloblastic anaemia as the lack of folic acid reduces DNA synthesis and cell multiplication. Deficiency of vitamin B6 results in hypochromic anaemia as deficiency interferes with the synthesis of haemoglobin. Both the riboflavin cofactors FAD and FMN can influence vitamin B12 metabolism as they can influence the supply of 5-methyl tetrahydrofolate for the enzyme methionine synthase (EC 2.1.1.135). FMN is a cofactor for pyridoxine-50 -phosphate oxidase (EC 1.4.3.5) through which the active form of vitamin B6 is generated, pyridoxal-50 -phosphate, whereas FAD is a cofactor for the enzyme 5,10-methylenetetrahydrofolate reductase (MTHFR, EC 1.7.99.5) (Selhub 1999). MTHFR catalyses the formation of 5± methyltetrahydrofolate, the methyl donor for methionine synthase. Thus all three vitamins and riboflavin are potentially involved in the regulation of homocysteine (Northrop-Clewes and Thurnham 2002) (Fig. 25.1).

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The methionine cycle is a major source of methyl groups for purine nucleotides and thymidylate for DNA synthesis. Vitamin B12 is especially important for it occupies a key position in the remethylation of homocysteine to methionine by which newly synthesised, folate-bound, methyl groups can replenish the supply of 1±carbon fragments consumed in methylation reactions. When methionine synthase activity falls, the concentration of S-adenosyl methionine (SAM) diminishes DNA methylation, 5±methyl tetrahydrofolate concentrations accumulate and cause folate to become unavailable for the conversion of deoxyuridine-50 -phosphate (dUMP) to deoxythymidine-50 -phosphate (dTMP). A deficiency of vitamin B12 or methionine will ultimately affect folate metabolism (Fig. 25.1). Elevated DNA damage and altered methylation of DNA are important risk factors for cancer (Blount and Ames 1995). When dUMP accumulates, uracil is incorporated into DNA instead of thymine (Eto and Krumdieck 1986). The most plausible explanation for the chromosome breaking effect of low folate is excessive uracil misincorporation into DNA, a mutagenic lesion that leads to strand breaks in DNA during repair. Both in vitro and in vivo studies with human cells clearly show that folate deficiency causes expression of chromosomal fragile sites, chromosome breaks, excessive uracil in DNA, micronucleus formation and DNA hypomethylation (reviewed in Fenech 2001). Evidence for dietary deficiency and associations with increased risk of cancer The earliest evidence of chromosomal damage in human cells in vivo from folate and vitamin B12 deficiency was obtained from studies linking the expression of Howell-Jolly bodies in erythrocytes with magaloblastic anaemias. Howell-Jolly bodies are whole chromosomes or chromosome fragments left behind during the production and maturation of the erythrocyte. The bodies are the same as micronuclei seen in other cells and are most readily observed in splenectomised subjects, as the spleen normally filters micronucleated erythrocytes from the blood. A case study (Everson et al. 1988) reported a 30-year-old male with Crohn's disease to have a high erythrocyte micronuclei count (67/1000 red cells) in association with low serum folate of 4.3 nmol/L (normal >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 <9 nmol/L and plasma B12 concentrations <147 pmol/L (i.e. marginal or deficient). Vitamin C and E and -carotene concentrations did not show strong inverse association with the micronucleus index (MacGregor et al. 1997). Studies on blood samples from the same individuals also showed a 70±fold higher uracil concentration in DNA in individuals with low serum folate (<9 nmol/L) in comparison with those whose serum folate was higher. Thus the above and other evidence reviewed in Fenech (2001), suggest that there is an increased risk of DNA and chromosomal damage associated with low

Vitamins and the prevention of cancer 697 dietary folate intakes. Of course the work with splenectomised subjects has to be interpreted cautiously since the scavenging action of the spleen to remove micronucleated red cells is part of the repair process but the association between DNA damage and low dietary folate intakes is clearly seen and the risk of cancer will increase, the greater the risk of DNA damage. It has also been shown that a higher frequency of lymphocyte micronuclei was present in apparently healthy men (50±70 years) with elevated homocysteine concentrations and low folate and B12 status. The micronucleus index strongly correlated with homocysteine concentrations in men with either high or low vitamin status and with vitamin B12 concentrations (r 0.315) but not with folate (r 0.013) (Fenech et al. 1997). Others have also shown that blood homocysteine concentrations respond to folate supplements (Ward 2001) but that the amount of dietary folate needed to reduce the risk of neural tube defects is approximately 400 g per day which is not easy to obtain without supplementation (Cuskelly et al. 1996). Evidence for benefits from `optimal' nutrition Ames (1998) has suggested that vitamin deficiencies sufficient to cause strand breaks are present in 10% (folate), 14% (B12, elderly) and 10% (B6) of the US population and a higher percentage of the poor. He also suggests that dietary intakes above the current RDI may be particularly important in those with extreme defects in the absorption and metabolism of these vitamins, for which ageing is a contributory factor. Anaemia is a problem in both industrialised and non-industrialised countries and deficiency of both folate and vitamin B12 contribute to the problem. The absorption of vitamin B12 presents more problems in the elderly with a rise in both pernicious anaemia due to defects in intrinsic factor production and achlorhydria (Denham and Chanarin 1985; Pennypacker et al. 1991). Functional foods might help to redress the problem unless of course they were more expensive. It has been clearly shown that folate supplements reduce the risk of neural tube defects in high-risk patients (Smithells et al. 1976) and others have shown that even in the general population of mothers presenting in the ward, the risk of NTD is inversely related to their folate status. Furthermore, studies have shown that it is difficult meeting suggested intakes of 400 g folate to avoid NTD, in the absence of foods containing dietary supplements (Cuskelly et al. 1996). Aside from NTD however, several studies suggest that folate intakes protect against colon (Giovannucci et al. 1998) and breast cancer (Zhang et al. 1999). Likewise, in a recent prospective study of 88,758 women over 16 years, there were 535 cases of colon cancer (Fuchs et al. 2002). They found that women with no family history of colon cancer who consumed more than 400 g folate/d had an age-adjusted RR of colon cancer of 0.48 (95% CI 0.28±0.83) in comparison with those consuming 200 g or less. On the basis of the cellular studies and in vivo studies using micronuclei described above, Fenech (2001) argues that an RDI level of 700 g/day for folic acid and 7 g/day for vitamin B12 would be appropriate for genomic stability in young adults. Current RDIs are below 200 g folic acid per day and ~2 g vitamin B12/day which would mean at least

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trebling our intakes of folate and more than trebling the intake of vitamin B12. Clearly, supplements would be needed to achieve such intakes. 25.2.8 Niacin Aspects of metabolism that might link to cancer Niacin is another water-soluble B vitamin, sometimes known as vitamin B3. There are two main forms in the diet, nicotinic acid (pyridine-3-carboxylic acid) and nicotinamide (nicotinic acid amide). Both forms are substrates for the synthesis of the nicotinamide dinucleotide coenzymes, NAD+ and the phosphorylated derivative, NADP+. The amino acid tryptophan is also an important precursor form of nicotinic acid. Diets that contain both meats and cereals should contain sufficient amounts of the vitamin to meet dietary requirements. Deficiencies are known to occur in impoverished populations where meat is often a luxury and the cereal consumed is corn (Zea maize) or some forms of millet in India (Sorghum vulgare). In corn, niacin occurs in an unavailable form, niacytin, unless pretreated by alkaline hydrolysis. The deficiency disease resulting from niacin deficiency is pellagra in which there are visible changes in skin pigmentation and sunburn-like rashes on areas exposed to the sun. There are also inflammatory changes in the mucosa of the mouth, tongue and gastrointestinal tract sometimes producing diarrhoea. It is also frequently accompanied by dementia, and insomnia is also common (Thurnham et al. 2000). The deficiency is rare in industrialised countries though it can accompany alcohol abuse and in patients treated for tuberculosis with hydrazine-related drugs (Darvay et al. 1999). Interest in the potential role of niacin in genetic stability revolves around the finding that NAD+ is the sole substrate for the enzyme poly(ADP-ribose) polymerase-1 (PARP-1, EC 2.4.2.30). This enzyme is associated with DNA repair, cellular differentiation, gene expression and cell death. The enzyme PARP-1 is activated by DNA strand breaks, and although it is not directly involved in the repair process, it is a component of the base-excision repair pathway and believed to facilitate the repair. Cells and animals lacking the enzyme appear to be more sensitive to the lethal and genotoxic effect of ionising radiation and alkylating reagents. Nicontinamide (one of the dietary forms of niacin) is a specific inhibitor of the enzyme, and cellular studies show that its incorporation into the medium in amounts which inhibit PARP-1, the frequency of sister chromatid exchanges increases. As a result of PARP-1 activation, cellular NAD+ is consumed and the size of the NAD+ pool and supply of dietary niacin via the diet are considered important for genomic stability (reviewed in Hageman and Stierum 2001). The importance of PARP-1 activity in genomic stability and preservation of cellular functions is supported by the finding that differences in longevity between species is associated with PARP activity measured in lymphocytes (Burkle et al. 1994). Of interest also was the report that PARP-1 activity in lymphoblastoid cell lines from centenarians was significantly elevated in

Vitamins and the prevention of cancer 699 comparison with those derived from younger controls, 20±70 years (Muiras et al. 1998). As far as we know however, such observations do not relate to differences in dietary niacin. Evidence for dietary deficiency and associations with increased risk of cancer Hageman and Stierum ( 2001) report several short-term studies in cells, rats and mice where genomic stability was studied after increasing nicotinamide concentrations or studies in humans where either nicotinamide or niacin was supplemented. Generally speaking, the addition of nicotinamide to experimental systems showed increased evidence of genomic instability. However, when 100 mg/day of nicotinamide (plus zinc and carotenoids) was given to humans for seven weeks, there was no effect on blood concentrations of NAD+ and there was evidence of increased resistance against oxidative DNA damage and enhanced DNA repair in peripheral blood leukocytes (PBL). Supplementing humans with 100 mg nicotinic acid per day for eight weeks showed similar beneficial effects to nicotinamide in PBL. However, in a separate study on 21 male smokers given doses ranging from 25 to 100 mg nicotinic acid daily for 14 weeks, there was a small but significant increase in sister chromatid exchanges in PBL in those who received the top dose, and no effects on micronuclei or hprt variant frequencies at any of the doses tested. Because of the location of deficiency signs in pellagra and the inflammatory damage to the mucosa, marginal niacin deficiency has been associated with an increased risk of oesophageal cancer. However, a controlled trial in which 40 mg niacin and 5.2 mg riboflavin was taken daily for 5.25 years did not reduce the incidence of oesophageal cancer or of cancer mortality during the period of supplementation (Blot et al. 1993). There is some experimental evidence that niacin supplementation may protect against UV-induced skin cancer since the number of skin cancers were reduced in mice by topical application of nicotinamide together with niacin in the diet (Gensler et al. 1999). Evidence for benefits from `optimal' nutrition Although there is evidence that niacin has a role in maintaining genomic stability, there is no evidence for increased risks of cancer associated with currently consumed diets. The recommended intake for adults in the UK is 12 to 17 mg/day (Department of Health 1991) and dietary surveys suggest that intakes are probably more than double the RNI (Gregory et al. 1990). 25.2.9 Thiamin, pantothenic acid, biotin and vitamin K None of these vitamins has been associated with any protective effects against cancer. There were reports that the administration of vitamin K to new-borns increased the risk of childhood cancers but further research has not confirmed this. Dietary deficiencies of pantothenic acid and biotin are very rare since both are widely distributed in animal and plant foods (Department of Health 1991). Thiamin is obtained both from animal and plant foods but the milling of cereals

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removes the thiamin-rich endosperm. However, wheat flour is fortified with thiamin in the UK and this considerably reduces any risk of deficiency. In industrialised countries alcohol abuse is most likely to be the cause of thiamin deficiency as ethanol inhibits the active transport of thiamin across the gut (Cathcart and Thurnham 1998). Vitamin K is mainly obtained from dark green leafy vegetables but fruits, dairy produce, cereals and meats can also provide significant amounts. In addition, the synthesis of menaquinones in the gut and their storage in the liver would also appear to benefit vitamin K status. Thus deficiencies of these vitamins are rare and there is no convincing data to associate them with any protection against cancer.

25.3

Future trends

Many of the vitamins, particularly those with antioxidant properties like vitamins E and C and the pro-vitamin, -carotene, have in recent years been credited with beneficial properties far in excess of scientific fact because disease was an oxidative process and oxidant damage was a potentially important cause of cancer. In addition, a considerable amount of experimental work supported the idea that the antioxidant vitamins, and some of the others, could protect against experimentally induced cancers and the formation of lesions believed to be early evidence of malignancy. Thus the hypothesis developed that antioxidants must modify these oxidative processes. However, human trials of specific nutrients have been disappointing with as much disease generally appearing in the placebo as in the vitamin-treated group. Indeed, in two studies where -carotene or control was given to smokers, there was an actual increase in lung cancer in the -carotene group. What many scientists overlooked, however, was the fact that antioxidants did indeed have a role in modulating disease processes but they were just single components in the whole immune defence mechanism. The probability is that most people in industrialised countries already have optimal concentrations of most nutrients in their tissues and trying to boost these concentrations by increasing intakes 100-fold or more has very little metabolic effect. It is important to have optimal concentrations but these are not necessarily amounts which `saturate' tissues. At worst, saturation may cause an imbalance in the delicately regulated pathways of our defence mechanisms and aggravate a disease process. Before the -carotene intervention trials, it was widely recognised that the plasma -carotene concentrations in smokers were lower than those in non-smokers. Researchers interpreted the low concentrations as a need for more -carotene by smokers. The possibility that low -carotene concentrations were a protective adaptation to the oxidative stress of smoking was not considered. There is, however, a lot of evidence to suggest that low concentrations of vitamins A and C are protective responses to the presence of disease (Thurnham 1997) and -carotene and vitamin C show many similarities in smokers.

Vitamins and the prevention of cancer 701 Work will no doubt still continue to investigate the role of vitamins in cancer. However, attention is turning again to the impressive body of evidence that fruit and vegetables are protective against chronic disease and cancer. The polyphenols are being examined with increasing amounts of interest. However, it is to be hoped that the magic bullet approach has lost its attraction and that there will be more attention to the effects of lifestyle and dietary patterns on metabolism and the way our defences respond to toxins and pathogens. Our dietary patterns influence how we metabolise potentially toxic foreign compounds and our risks of cancer. We are too complicated for any one substance to provide overall protection. Fruits and vegetables are the best proved functional foods we have. Our objective for the future should be to market these more effectively.

25.4

Sources of further information and advice

Information on the vitamin composition of unprocessed food can be obtained from a variety of sources but in general in the United Kingdom they are based on McCance and Widdowsons', A Composition of Foods (Paul and Southgate 1978) which has been updated seven or eight times. Information on processed foods can often be obtained from the information contained on the packet or from the manufacturer. Information on vitamin metabolism can be obtained from a variety of textbooks for example Human Nutrition and Dietetics that is constantly being revised and updated (Thurnham et al. 2000). In addition there are many other food- or nutrition-orientated textbooks that provide information on vitamins, e.g., The Nutrition Handbook for Food Processors (2002) (Northrop-Clewes and Thurnham 2002) or The Dietary Enhancement of Human Immune Functions (2003) (Thurnham and Northrop-Clewes 2004). There is an enormous amount of information on diet and cancer in the World Cancer Research Fund report entitled Food Nutrition and the Prevention of Cancer: a Global Perspective (1997) (World Cancer Research Fund 1997). The International Agency for Research on Cancer (Lyon, France) also publishes much literature on cancer and its epidemiology and prevention. Two handbooks referred to in this chapter were those on Vitamin A (IARC Working Group 1998b) and Carotenoids (IARC Working Group 1998a).

25.5

References

and THURNHAM D I (1998), `Glutathione peroxidase (EC 1.11.1.9) and superoxide dismutase (EC 1.15.1.1) activities in riboflavin-deficient rats infected with Plasmodium berghei malaria', Brit J Nutr, 79, 305±309. AMES B (1998), `Micronutrients prevent cancer and delay aging', Toxicology Letters, 102/ 103, 5±18. ADELEKAN D A

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(2001), `DNA damage from micronutrient deficiencies is likely to be a major cause of cancer', Mutation Res, 475, 7±20. AMES B N, SAUL R L, SCHWIERS E et al. (1985), `Oxidative DNA damage as related to cancer and aging: Assay of thymine glycol, thymidine glycol, and hydroxymethyluracil in human and rat urine', in Molecular biology of aging: Gene stability and gene expression., Sohal R S et al., eds, Raven Press, New York. 137±144. AMES B N, SHIGENAGA M K, HAGEN T M (1993), `Oxidants, antioxidants and the degenerative diseases of aging', Proc Natl Acad Sci USA, 90, 7915±7922. ASAMI S, MANABE H, MIYAKE J et al. (1997), `Cigarette smoking induces an increase in oxidative DNA damage, 8OHdG, in a central site of the human lung', Carcinogenesis, 18, 1763±1766. AZZI A, BOSCOBOINIK D, CHATELAIN E et al. (1993), `D--Tocopherol control of cell proliferation', Mol Asp Med, 14, 265±271. BENDICH A and OLSON J A (1989), `Biological action of carotenoids', FASEB J, 3, 1927± 1932. BLANER W S and OLSON J A (1994), `Retinol and retinoic acid metabolism,' in The retinoids: biology, chemistry and medicine, 2nd edn, Sporn M B, Roberts A B, Goodman D S, eds, Raven Press Ltd, New York. 229±255. BLOT W J, LI J-Y, TAYLOR P R et al. (1993), `Nutrition intervention trials in Linxian, China: supplementation with specific vitamin/mineral combinations, cancer incidence, and disease specific mortality in the general population', J Natl Cancer Inst, 85, 1483±1492. BLOUNT B C and AMES B (1995), `DNA damage in folate deficiency', Bailliere's Clin Haematol, 8, 461±478. BURKLE A, MULLER M, WOLF I et al. (1994), `Poly(ADP-ribose)polymerase activity in intact or permeabilised leukocytes from mammalian species of different longevity', Mol Cell Biochem, 138, 85±90. BURTON G W and INGOLD K U (1981), `Auto-oxidation of biological molecules. 1. The antioxidant activity of vitamin E and related chain-breaking phenolic antioxidants in vitro', J Am Chem Soc, 103, 6472±6477. BUSS N E, TEMBE E A, PREDERGAST B D et al. (1994), `The teratogenic metabolites of vitamin A in women following supplements and liver', Hum Exp Toxicol, 13, 33±43. CARROLL K K and KRITCHEVSKY D (eds) (1994), Nutrition and disease update: Cancer, edn, AOCS, Champaign, Illinois. CASTEELS K, BOUILLON R, WAER M et al. (1995), `Immunomodulatory effects of 1,25± dihydroxyvitamin D3', Curr Opin Nephrol Hypertens, 4, 313±318. CATHCART A E and THURNHAM D I (1998), `Thiamin: physiology', in Encyclopedia of Human Nutrition, vol. 3 Sadler M J, Caballero B, Strain J J, eds, Academic Press, London. 1858±1863. CHAN S S, MONTEIRO H P, SCHINDLER F et al. (2001), `-Tocopherol modulates tyrosine phosphorylation in human neutrophils by inhibition of protein kinase C activity and activation of tyrosine phosphatases', Free Rad Res, 35, 843±856. CHATTERJEE M (2001), `Vitamin D and genomic stability', Mutation Res, 475, 69±88. CHEN L H, BOISSONNEAULT G A, GLAUERT H P (1988), `Vitamin C, vitamin E and cancer', Anticancer Res, 8, 739±748. COLLINS A R (2001), `Carotenoids and genomic stability', Mutation Res, 475, 21±28. CONNETT J E, KULLER L H, KJELSBERG M O et al. (1989), `Relationship between carotenoids and cancer. The multiple risk factor intervention trial (MRFIT) study', Cancer, 64, 126±134. AMES B

Vitamins and the prevention of cancer 703 COOK-MOZAFFARI P J, AZORDEGAN F, DAY N E et al. (1979), `Oesophageal cancer studies in the

Caspian littoral of Iran: results of a case-control study', Br J Cancer, 39, 293±309. et al. (1975), `A model for gastric cancer epidemiology', Lancet, ii, 58±60. CRESPI M, MUNOZ N, GRASSI A et al. (1979), `Oesophageal lesions in northern Iran: a premalignant lesion', Lancet, ii, 217±221. CUSKELLY G J, MCNULTY H, SCOTT J M (1996), `Effect of increasing dietary folate on red cell folate: implications for prevention of neural tube defects', Lancet, 347, 657± 659. DARVAY A, BASARAB T, MCGREGOR J M et al. (1999), `Isoniazid induced pellagra despite pyridoxine supplementation', Clin Exp Dermatol, 24, 167±169. DAY N E and MUNOZ N (1982), `Cancer of the oesophagus', in Cancer epidemiology and prevention, Schottenfeld D and Fraumeni J, eds, W.B. Saunders, Philadelphia. 596±623. DELUCA H F, SCHUMACHER M, WOLF G (1970), `Biosynthesis of fucose-containing glycoproteins from a rat small intestine in normal and vitamin A deficient conditions,', J Biol Chem, 245, 4551±4558. DENHAM M J and CHANARIN I (eds) (1985), Blood disorders in the elderly, Churchill Livingston, Edinburgh. DEPARTMENT OF HEALTH (eds) (1991), Dietary Reference Values for Food Energy and Nutrients for the United Kingdom. Report on Health and Social Subjects, No. 41, HMSO, London. ESTERBAUER H, GEBICKI J, PUHL H et al. (1992), `The role of lipid peroxidation and antioxidants in oxidative maodification of LDL', Free Rad Biol Med, 13, 341±390. ETO I and KRUMDIECK C L (1986), `Role of vitamin B12 and folate deficiencies in carcinogenesis', in Essential nutrients in carcinogenesis, Poirier L A, Newberne P M, Pariza M W, eds, Plenum Press, New York. 313±331. EUGUI E M, DE LUSTRO B, ROUHAFZA S (1994), `Some antioxidants inhibit, in a coordinate fashion, the production of tumor necrosis factor-, IL-1b and IL-6 by human peripheral blood mononuclear cell', Immunol, 6, 422. EVERSON R B, WEHR C M, EREXSON G L et al. (1988), `Association of marginal folate depletion with increased human chromosomal damage in vivo: demonstration by analysis of micronucleated erythrocytes', J Natl Cancer Inst, 80, 525±529. FENECH M (2001), `The role of folic acid and vitamin B12 in genomic stability of human cells', Mutation Res, 475, 57±67. FENECH M, DREOSTI I E, RINALDI J R (1997), `Folate, vitamin B12, homocysteine status and chromosome damage of lymphocytes in older men', Carcinogenesis, 18, 1329± 1336. FRAGA C, MOTCHNIK P A, SHIGENAGA M K (1991), `Ascorbic acid protects against endogenous oxidative DNA damage in human sperm', Proc Natl Acad Sci USA, 88, 11003±11006. FUCHS D, WILLETT W C, COLDITZ G A et al. (2002), `The influence of folate and multivitamin use on the familial risk of colon cancer in women', Cancer Epidemiol Biomark Prevent, 11, 227±234. GARLAND C F, COMSTOCK G W, GARLAND F C et al. (1989), `Serum 25-hydroxyvitamin D and colon cancer: eight year prospective study', Lancet, ii, 1176±1178. GARLAND F C, GARLAND C F, YOUNG J F (1990), `Geographic variation in breast cancer mortality in the United States: a hypothesis involving exposure to solar radiation', Prev Med 614±622. CORREA P, HAENSZEL W, CUELLO C

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et al. (1999), `Oral niacin prevents photocarcinogenesis and photoimmunosuppression in mice', Nutr Cancer, 34, 36±41. GIOVANNUCCI E (2002), `A prospective study of tomato products, lycopene, and prostate cancer risk', J Natl Cancer Inst, 94, 391±398. GIOVANNUCCI E, ASHERIO A, RIMM E B et al. (1995), `Intake of carotenoids and retinol in relation to risk of prostate cancer', J Natl Cancer Inst, 87, 1767±1776. GIOVANNUCCI E, STAMPFER M, COLDITZ G A et al. (1998), `Multivitamin use, folate and colon cancer in women in the Nurses Health Study', Ann Intern Med, 129, 517±524. GREGORY J R, FOSTER K, TYLER H, WISEMAN M (eds) (1990), The dietary and nutritional survey of British adults., HMSO, London. HAGEMAN G J and STIERUM R H (2001), `Niacin, poly(ADP-ribose) polymerase-1 and genomic stability', Mutation Res, 475, 45±56. HALLIWELL B (2000), `Why and how should we measure oxidative DNA damage in nutritional studies? How far have we come?', Am J Clin Nutr, 72, 1082±1087. HALLIWELL B (2001), `Vitamin C and genomic stability', Mutation Res, 475, 29±35. HANNAH S S and NORMAN A W (1994), `1,25±Dihyroxyvitamin D3-regulated expression of the eukaryotic genome', Nutr Rev, 52, 376±382. HEART PROTECTION STUDY GROUP (2002), `MRC/BHF Heart protection study of antioxidant vitamin supplementation in 20536 high-risk individuals: a randomised placebo-controlled trial', Lancet, 360, 23±33. HEINONEN O P, HUTTUNEN J K, ALBANES D et al. (1994), `The effect of vitamin E and beta carotene on the incidence of lung cancer and other cancers in male smokers', New Engl J Med, 330, 1029±1035. HENNEKENS C H (1986), `Micronutrients and cancer prevention', New Engl J Med, 315, 1288±1289. HENNEKENS C H, BURING J E, MANSON J E et al. (1996), `Lack of effect of long-term supplementation with beta carotene on the incidence of malignant neoplasms and cardiovascular disease', New Engl J Med, 334, 1145±1149. HOLDSWORTH M D, DATTANI J T, DAVIES L et al. (1984), `Factors contributing to vitamin D status near retirement age', Hum Nutr Clin Nutr, 38C, 139±149. HUSTAD S, UELAND P M, VOLLSET S E et al. (2000), `Riboflavin as a determinant of plasma total homocysteine: effect of modification by the methylenetetrahydrofolate reductase C677T polymorphism', Clin Chem, 46, 1065±1071. IARC WORKING GROUP (eds) (1998a), Carotenoids, WHO International Agency for Research on Cancer, Lyon. IARC WORKING GROUP (eds) (1998b), Vitamin A, International Agency for Research on Cancer, Lyon. KEANEY J F, GUO Y, CUNNINGHAM D et al. (1996), `Vascular incorporation of -tocopherol prevents endothelial dysfunction due to oxidised LDL by inhibition of protein kinase C stimulation', J Clin Invest, 98, 386±394. KLIEWER S A, UMESONO K, EVANS R M et al. (1994), `The retinoid X receptors. Modulators of multiple hormonal signalling pathways,' in Vitamin A in health and disease, Blumhoff R, ed., Marcel Dekker, New York. 239±255. KMET J, MCLAREN D S, SASSI F (1981), `Epidemiology of oesophageal cancer with special reference to nutritional studies among the Turkomen of Iran,' in Advances in modern human nutrition, Tobin R B and Mehlman M A, eds, Pathotox, New York. 343±365. KOPP E B and GHOSH S (1995), `NF-kB and rel proteins in innate immunity', Adv Immunol, 58, 1±27. GENSLER H L, WILLIAMS T, HUANG A C

Vitamins and the prevention of cancer 705 and MAVIS R D (1979), `Relative susceptibility of microsomes from lung, heart, liver, kidney, brain and testes to lipid peroxidation: correlation with vitamin E content', Lipids, 15, 315±322. LARSSON L-G, SANDSTROM A, WESTLING P (1975), `Relationship of Plummer-Vinson disease to cancer of the upper alimentary tract in Sweden', Cancer Res, 35, 3308± 3316. LEVINE N, MOON T E, CARTMEL B et al. (1997), `Trial of retinol and isotretinoin in skin cancer prevention: a randomised, double-blind, controlled trial. Southwest Skin Cancer Prevention Study Group', Cancer Epidemiol Biomark Prevent, 6, 957±961. LI M, LI P, LI B (1980), `Recent progress in research in oesophageal cancer in China', Adv Canc Res, 33, 173±249. LI J-Y, TAYLOR P R, LI B et al. (1993), `Nutrition intervention trials in Linxian, China: multiple vitamin/mineral supplementation, cancer incidence, and disease-specific mortality among adults with esophageal dysplasia', J Natl Cancer Inst, 85, 1492±1498. MACGREGOR J T, WEHR C M, HIATT R A et al. (1997), `Spontaneous genetic damage in man: evaluation of interindividual variability, relationship among markers of damage, and influence of nutritional status', Mutation Res, 377, 125±135. MEISTER A (1983), `Selective modification of glutathione metabolism', Science, 220, 472± 477. MICKLE D A G, WEISEL R D, BURTON G W et al. (1991), `Effect of orally administered alphatocopheryl acetate on human myocardial alpha-tocopherol levels', Cardiovasc Drugs Ther, 5, 309±312. MIRVISH S S (1986), `Effects of vitamin C and E on N-nitroso compound formation, carcinogenesis and cancer', Cancer, 58, 1842±1850. MOON T E, LEVINE N, CARTMEL B et al. (1997), `Effect of retinol in preventing squamous cell skin carcinoma in moderate-risk subjects: a randomized double-blind controlled trial. South-west Skin Cancer Prevention Study Group', Cancer Epidemiol Biomark Prevent, 6, 949±956. MRTENSSON J and MEISTER A (1991), `Glutathione deficiency decreases tissue ascorbate levels in newborn rats: ascorbate spares glutathione and protects', Proc Natl Acad Sci USA, 88, 4656±4660. MUIRAS M-L, MULLER M, SCHACHTER F et al. (1998), `Increased poly(ADP-ribose) polymerase activity in lymphoblastoid cell lines from centenarians', J Mol Med, 76, 346±354. MUNOZ N, WAHRENDORF J, LU J-B et al. (1985), `No effect of riboflavin, retinol and zinc on prevalence of pre-cancerous lesions of esophagus', Lancet, ii, 111±114. MUSK A W, DE KLERK N H, AMBROSINI G L et al. (1998), `Vitamin A and cancer prevention I: observations in workers previously exposed to asbestos at Wittenoom, Western Australia', Int J Cancer, 75, 355±361. NIKI E, TSUCHIYA J, TANIMURA R et al. (1982), `Regeneration of vitamin E from chromanoxyl radical by glutathione and vitamin C', Chem Lett, 27, 798±792. NISHIZUKA Y (1984), `The role of protein kinase C in cell surface signal transduction and tumor promotion', Nature, 308, 693±696. NORTHROP-CLEWES C A and THURNHAM D I (2002), `Vitamins', in The nutrition handbook for food processors, Henry C J K and Chapman C, eds, CRC & Woodhead Publishing Ltd, Cambridge. 34±96. OHSHIMA H and BARTSCH H (1981), `Quantitative estimation of endogenous nitrosation in humans by monitoring N-nitrosoproline excreted in the urine', Cancer Res, 41, 3658±3662. KORNBRUST D J

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et al. (1996), `Effects of a combination of beta carotene and vitamin A on lung cancer and cardiovascular disease', New Engl J Med, 334, 1150±1155. PAOLINI M, CANTELLI-FORTI G, PEROCCO P, PEDULLI G F, ABDEL-RAHMAN S Z (1999), `Cocarcinogenic effect of -carotene', Nature, 398, 760±761. PAUL A A and SOUTHGATE D A T (eds) (1978), McCance and Widdowson's The composition of foods, 4th edn, HMSO, Oxford. PENNYPACKER L C, ALLEN R H, KELLY J P et al. (1991), `High prevalence of cobalamin deficiency in elderly outpatients', J Am Geriatr Soc, 39, 1155±1159. PRASAD M P, MUKUNDAN M A, KRISHNASWAMY K (1995), `Micronuclei and carcinogen DNA adducts as intermediate end points in a nutrient intervention trial of precancerous lesions', Eur J Cancer,B Oral Oncol, 31B, 155±159. PRIEME H, LOFT S, NYYSSONEN K et al. (1997), `No effect of supplementation with vitamin E, ascorbic acid or coenzyme Q10 on oxidative DNA damage estimated by 8-oxo7,8-dihydro-2'-deoxyguanosine excretion', Am J Clin Nutr, 65, 503±507. RATHBONE B J, JOHNSON A W, WYATT J I et al. (1989), `Ascorbic acid: a factor concentrated in human gastric juice', Clin Sci, 76, 237±241. REHMAN A, COLLIS C S, YANG M et al. (1998), `The effect of iron and vitamin C cosupplementation on oxidative damage to DNA in healthy volunteers', Biochem Biophys Res Comm, 246, 293±298. RISCH H A, JAIN M, CHOI N W et al. (1985), `Dietary factors and the incidence of cancer of the stomach', Am J Epidemiol, 122, 947±959. ROSS A C and TERNUS M E (1993), `Vitamin A as a hormone. Recent advances in understanding the actions of retinol, retinoic acid and beta-carotene', J Am Diet Assoc, 93, 1285±1290. SAUBERLICH H E (1994), `Vitamin C and cancer', in Nutrition and disease update, cancer, 1st edn, Carroll K K and Kritchevsky D, eds, AOCS Press, Champaign, Illinois. 111±172. SCHWARTZ G G and HULKA B S (1990), `Is vitamin D deficiency a risk factor for prostate cancer (hypothesis)?', Anticancer Res, 10, 1307±1312. SELHUB J (1999), `Homocysteine metabolism', Ann Rev Nutr, 19, 217±246. SMITHELLS R W, SHEPPARD S, SCHORAH C J (1976), `Vitamin deficiencies and neural tube defects', Arch Dis Child, 51, 944±950. STICH H F and ANDERS F (1989), `The involvement of reactive oxygen species in oral cancer of betel quid/tobacco chewers', Mutation Res, 214, 47±61. STICH H F, ROSIN M P, HORNBY A P et al. (1988), `Remission of oral leukoplakias and micronuclei in tobacco/betel quid chewers treated with beta-carotene and with beta-carotene plus vitamin A', Int J Cancer, 42, 195±199. STICH H F, BRUNNEMANN K D, MATHEW B et al. (1989), `Chemopreventive trials with vitamin A and b-carotene: some unresolved issues', Prev Med, 18, 732±739. STUMPF W E (1988), `Vitamin D-soltriol. The heliogenic steroid hormone: somatotropic activator and modulator. Discussion from histochemical studies led to new concepts', Histochemistry, 89, 209±220. THURNHAM D I (1997), `Impact of disease on markers of micronutrient status', Proc Nutr Soc, 56, 421±431. THURNHAM D I and NORTHROP-CLEWES C A (2004), `Effects of infection on nutritional and immune status,' in Dietary enhancement of human immune functions, Hughes D A, Bendich A, Darlington L G, eds, Humana Press, New Jersey, 35±64. THURNHAM D I, MIGASENA P, VUDHIVAI N et al. (1971), `A longitudinal study on dietary and OMENN G S, GOODMAN G E, THORNQUIST M D

Vitamins and the prevention of cancer 707 social influences on riboflavin status in pre-school children in northeast Thailand', SE Asian J Trop Med Publ Hlth, 2, 552±563. THURNHAM D I, RATHAKETTE P, HAMBIDGE K M et al. (1982), `Riboflavin, vitamin A and zinc status in Chinese subjects in a high-risk area for oesophageal cancer in China', Hum Nutr Clin Nutr, 36C, 337±349. THURNHAM D I, DAVIES J A, CRUMP B J et al. (1986), `The use of different lipids to express serum tocopherol:lipid ratios for the measurement of vitamin E status', Ann Clin Biochem, 23, 514±520. THURNHAM D I, MUNOZ N, LU J-B et al. (1988), `Nutritional and haematological status of Chinese farms: the influence of 13.5 months treatment with riboflavin, retinol and zinc', Eur J Clin Nutr, 42, 647±660. THURNHAM D I, BENDER D A, SCOTT J et al. (2000), `Water-soluble vitamins,' in Human nutrition and dietetics, 10th edn, Garrow J S, James W P T, Ralph A, eds, Churchill Livingstone, Edinburgh. 249±286. THURNHAM D I, MCCABE G P, NORTHROP-CLEWES C A et al. (2003), `Effect of sub-clinical infection on plasma retinol concentrations and assessment of prevalence of vitamin A deficiency: a meta-analysis', Lancet, 362, 2052±2058. WAHRENDORF J, MUNOZ N, LU J-B et al. (1988), `Blood retinol, zinc and riboflavin status in relation to precancerous lesions of the esophagus: Findings from a vitamin intervention trial in the People's Republic of China', Cancer Res, 48, 2280±2283. WARD M (2001), `Homocysteine, folate and cardiovascular disease', Int J Vit Nutr Res, 71, 173±178. WEITBERG A B, WEITZMAN S A, CLARK E P et al. (1985), `Effects of antioxidants on oxidantinduced sister chromatid exchange formation', J Clin Invest, 75, 1835±1841. WILLETT W C, STAMPFER M J, UNDERWOOD B A et al. (1983), `Vitamins A, E and carotene: effects of supplementation on their plasma levels', Am J Clin Nutr, 38, 559±566. WISEMAN H (1993), `Vitamin D is a membrane antioxidant. Ability to inhibit irondependent lipid peroxidation in liposomes compared to cholesterol, egosterol and tamoxifen and relevance to anticancer action', FEBS Lett, 326, 285±288. WORLD CANCER RESEARCH FUND (1997), `Vitamins', in Food, nutrition and the prevention of cancer: a global perspective, American Institute for Cancer Research, Washington. 404±416. ZHANG S, HUNTER D J, HANKINSON S E et al. (1999), `A prospective study of folate intake and the risk of breast cancer', J Am Med Assoc, 281, 1632±1637.

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

Inflammatory bowel disease incorporates two major diseases, ulcerative colitis and Crohn's disease. These two diseases, though sharing similar features of gut mucosal inflammation, are distinct entities. Their pathogenesis remains incompletely understood. Both diseases are commonest in the Western, developed world, with highest incidence in northern climates.1,2 Reported incidences for ulcerative colitis from North America and northern Europe have been in the range of 10±20 new cases per 100,000 population (associated prevalence rates are about 150±250/100,000). There is a marked rise in the frequency of Crohn's disease in the developed world in the past fifty years, with a prevalence of approximately 100 per 100,000 population in North America and northern Europe. Genetic factors are known to play a role in the pathogenesis of inflammatory bowel disease. This is demonstrated by concordance in monozygous twin studies. Concordance rates are 40±50% for Crohn's disease and <10% for ulcerative colitis. Also, 10±25% of affected patients have a first-degree relative with the disease. However, the incomplete concordance seen in twin studies suggests that environmental factors also contribute to the pathogenesis of the disease. Equally, the rise in incidence in Crohn's disease points to the importance of environmental factors in its aetiology. The marked increase in the incidence of Crohn's disease has occurred as countries become more developed and industrialised. With changes in lifestyle and environment, improving levels of sanitation have altered the microbial environment. This means altered exposure to microbes and infections during childhood.3 Inflammatory bowel disease may be a disorder of the mucosal immune system due to lack of

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stimulation and education of the immune responses.4 It is interesting that parallel to an increase in Crohn's disease, other chronic inflammatory disorders, including allergies, asthma, multiple sclerosis and insulin-dependent diabetes mellitus have also increased in incidence. Environmental changes associated with industrialisation may alter immune system development and pose a risk factor for inflammatory bowel disease in the genetically susceptible individual.4

26.2 Managing inflammatory bowel disease: the role of probiotics 26.2.1 Role of enteric flora in the healthy intestine Underpinning the probiotic concept is the role of the normal intestinal microflora in health and disease.4 The importance of the intestinal microflora makes sense if we consider that the intestine contains a wide variety (>400 different species) of non-pathogenic bacteria numbering up to 1011 cells per gram of intestinal content.5 Experiments with germ free animals reveal several beneficial effects of the resident microflora.6 The commensal bacteria act as a defence against infection using several mechanisms, including displacement, competition for nutrients and the production of antimicrobial factors against pathogens. But in addition, the integrity of the mucosa requires cell signalling among the microflora, epithelium and mucosal immune system.7 The intestinal microflora primes the mucosal immune response and keeps it in a state of `controlled physiological inflammation'. Without the microflora, mucosal associated lymphoid tissue is under developed and cell mediated immunity is defective.8 Induction and/or maintenance of oral tolerance to ingested antigens also requires microbial colonisation of the gastrointestinal tract in early life. Understanding the influence of the gastrointestinal flora has prompted interest in the therapeutic modification of the enteric flora with probiotics or prebiotics. 26.2.2 The role of the enteric flora in inflammatory bowel disease The role of the enteric microflora in driving the inflammatory response in genetically susceptible individuals has been emphasised as a key component in the pathogenesis of inflammatory bowel disease and is based on several lines of evidence.7,9±11 First, lesions of mucosal inflammation in inflammatory bowel disease are seen in areas of the gut with highest bacterial numbers. Second, the importance of the continuity of the faecal stream in predisposing to relapses is well recognised and surgical diversion of the faecal stream is therapeutically beneficial to the distal bowel. Third, therapeutic efficacy is seen with the use of antibiotics. Fourth, immune reactivity to intestinal bacteria is detectable in patients with inflammatory bowel disease which suggests a loss of immunological tolerance to components of the microflora.12,13 Fifth, there have

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been reports of an increased number of bacteria within the mucosa of patients with inflammatory bowel disease compared with that of non-inflamed intestine and healthy controls.14,15 The highest numbers have been seen in Crohn's disease with the numbers increasing with severity of disease. Finally, the description of the first susceptibility gene for Crohn's disease, CARD15/NOD2, has provided a basis for explaining the interaction between bacteria and the immune response. CARD15/NOD2 encodes a protein that is involved in the recognition of bacterial products and initiates the inflammatory cascade via activation of the transcription factor nuclear factor kappa B (NFkB).16 Evidence from animal models Evidence for the role of bacteria in inflammatory bowel disease has also been seen in experimental animal models of both Crohn's and colitis. Genetically engineered knockout and transgenic animal models of inflammatory bowel disease have been reported in which the influence of the enteric flora has been directly demonstrated by comparing animals raised in germ free versus conventional facilities.17,18 Colonisation with normal enteric flora is necessary for development of the disease. The normal flora is a common factor in these models driving the inflammatory process regardless of the genetic makeup or immune mechanism. Several different micro-organisms have been demonstrated to induce colitis in animal models. These include enterococcus faecalis, causing colitis in the IL-10 knockout mice, and Bacteroides vulgatus, which induced inflammation in the HLA-B27 rat model.19,20 The above evidence has prompted the therapeutic modification of the enteric flora in inflammatory bowel disease. Specific micro-organisms in inflammatory bowel disease Despite the importance of bacteria in the pathogenesis of colitis and Crohn's disease, no specific micro-organism has been implicated in causing the intestinal inflammation. The roles of Mycobacterium paratuberculosis, measles virus, Listeria monocytogenes and adherent E. coli in the pathogenesis have been examined. Strains of adherent-invasive E. coli have been isolated in the mucosa of patients with Crohn's disease.21 Mycobacterium Paratuberculosis has been cultured from the intestine of patients with Crohn's disease and detected by molecular methods in the granulomas of resected tissue from patients.22 Possible disease modifying mechanisms of transient pathogens are the disruption of the mucosal barrier (allowing increased uptake of luminal antigens), mimicry of self-antigens and activation of the mucosal immune system via modulation of transcription factors such as NFkB. However, a direct cause and effect relationship has not been established for any of these organisms. Indeed, conditions favouring transmission of infection (low socio-economic status, overcrowding, poor sanitation) appear to protect against inflammatory bowel disease, arguing against an infectious aetiology.23

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26.2.3 Role for probiotic bacteria in inflammatory bowel disease Probiotics and functional foods Hippocrates, the father of modern medicine, said: `Let food be thy medicine and medicine be thy food'.24 The term `functional food' has been coined to include `any food or food ingredient that may provide a health benefit beyond the traditional nutrients it contains'.25 Probiotic bacteria are forms of functional food that are of particular interest and relevance to gastroenterologists, particularly in the setting of inflammatory bowel disease. Along with other biologic therapies for inflammatory bowel disease, probiotics are an exciting option for the treatment of ulcerative colitis and Crohn's disease. Our understanding of the importance of the enteric flora in the pathogenesis of inflammatory bowel disease and the interest of the public in `natural' treatments mean that probiotics are attracting growing attention as an option to therapeutically modify the enteric flora in this disease. Terminology The term probiotic describes `live microbial food supplements which beneficially affect the host animal by improving its intestinal microbial balance'.26 The concept of probiotics had been introduced as far back as the early 1900s by Metchnikoff. He reported a group of Bulgarian peasants who drank large amounts of fermented milk and had notable longevity.26,27 Following his report, he began to modify colonic microflora with sour milk. The gram positive rod he used was Bulgarian bacillus and it is probable that this organism is now that known as L. delbrueckii subsp. Bulgaricus which, with S. thermophilus, is responsible for the traditional fermentation of milk into yoghurt. Probiotics are non-pathogenic microbial organisms which survive passage through the gastrointestinal tract and are believed to have potential beneficial health effects. Bacteria associated with probiotic activity are most commonly lactobacilli or bifidobacteria, but other nonpathogenic bacteria, including certain Escherichia coli strains and non-bacterial organisms such as Saccharomyces boulardii have been used. A probiotic bacterium is required to fulfil certain selection criteria including being of human origin, acid and bile tolerance, adherence to intestinal cells, survival in the gut, production of anti-microbial substances and modulation of the immune response.28 However the current definition of a probiotic may now be too limited. The emphasis of this definition is on the use of live, whole microbial organisms. However, preliminary studies have already used bacterial constituents such as DNA instead of live bacteria with beneficial effect.29 Genetically modified bacteria have also been tested and a genetically engineered lactobacillus secreting the anti-inflammatory cytokine IL-10 has attenuated colitis in an animal model of colitis.30 Therefore, future use of the organisms may be outside the definition of probiotics. For this reason the term `pharmabiotics' may be more appropriate. This umbrella term includes live and dead organisms and constituents thereof, and encompasses genetically engineered microbes.

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26.2.4 Mechanisms of probiotic effect in inflammatory bowel disease The cellular and molecular mechanisms by which probiotics affect inflammation have not been elucidated and are probably multiple. Proposed mechanisms focus on how probiotics influence the immune response. Commensal flora are known to contribute to immune homeostasis.7,8 There are several molecular pathways which are suggested as candidates for the site of probiotic immune effects. Gut epithelium and dendritic cells Intestinal epithelial cells form the first point of contact for bacteria within the gut lumen and have an important role in bacteria-host communication.31 It is believed that the microflora influences the mucosal immune response through signalling with the gut epithelium.9,32,33 The intestinal epithelial cells distinguish between commensal and pathogenic bacteria and distinct signalling pathways may be activated. Interactions with Toll-like receptors and dendritic cells in the gut are believed to be involved in this communication.34,9 Dendritic cells inhabiting the gut mucosa are mostly immature and potentially prone to modulation by the environment, containing micro organisms. Dendritic cells are responsible for the stimulation of T cells and seem to have an important role in the balance between inducing TH1, TH2 and TH3 cytokine profiles35 It has been demonstrated that the Th1/Th2/Th3 cytokine profiles induced by gut dendritic cells can be modulated by the administration of lactobacilli.36 In a separate study, the probiotic bacteria Bifidobacterium infantis and Lactobacillus salivarius have been seen to induce dendritic cells to produce IL10 rather than IL-12.37 Several other studies have confirmed the ability of probiotic bacteria to induce an anti-inflammatory or regulatory cytokine profile by in vitro immunocompetent cells, including the promotion of systemic and mucosal TH2 responses and the reduced production of mucosal TNFalpha.7,38,39 Nuclear factor kappaB pathway The Nuclear Factor kappaB (NFkB) pathway, a nuclear factor involved in the transcriptional regulation of inflammatory genes, mediates responses to invasive pathogenic bacteria. Indeed, certain non-pathogenic organisms have been shown to counterbalance epithelial responses to invasive bacteria via an effect on the inhibitor kappaB/nuclear factor kappaB pathway.40 A recent study has demonstrated that a commensal bacteria, Bacteroides thetaiotaomicron, also acted on NFkB to attenuate pro-inflammatory cytokine expression, but via a unique mechanism. The mechanism involved limiting the duration of action of NFkB by promoting its nuclear export through a peroxisome proliferator activated receptor- -dependent (PPAR- ) pathway.41 Bacterial DNA It has been suggested that the whole, live bacteria may not be necessary for the immune responses seen with probiotics. In a recent study, bacterial DNA has been shown to have potent immuno-stimulatory effects and was used to

Probiotics in inflammatory bowel disease

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attenuate colitis in a number of murine models.28 The DNA sequences used are termed immunostimulatory sequences (ISS) or CpG motifs, and it is suggested that the attenuation of colitis occurs by anti-apoptotic mechanisms. Intestinal permeability Apart from immune mechanisms, it is also suggested that probiotic bacteria may have a beneficial effect on permeability of the gut barrier. There is evidence to suggest that the epithelial barrier function is reduced in inflammatory bowel disease.42 Several probiotic strains have demonstrated an ability to enhance the epithelial barrier function, based on measurements of intestinal permeability in excised mucosal tissue from animal models and humans.43,44

26.3 Analysing the effectiveness of probiotics in inflammatory bowel disease 26.3.1 Probiotics in animal models of IBD The efficacy of probiotics in attenuating colitis has been demonstrated in experimental animal models. These models include the interleukin-10 knockout murine model,45±48 methotrexate induced colitis49 and the CD45Pbhi transfer model50 (Table 26.1). The IL-10 knock-out mice develop colitis when colonised with normal enteric flora but remain disease-free if kept in germ-free conditions. In a study of IL-10-/- mice, colonisation with L. plantarum 299v was performed two weeks before transferring from a germ-free environment to a specific pathogen-free environment.48 This treatment led to a reduction in disease activity and a significant decrease in mesenteric lymph node IL-12 and IFN- production. A role for Lactobacillus reuteri in prevention of colitis in IL-10-/- mice was also demonstrated.45 In this study, the oral administration of the prebiotic lactulose (shown to increase the levels of Lactobacillus species) and rectal swabbing with L. reuteri restored Lactobacillus levels to normal in neonatal mice, originally found to have low levels of lactobacilli species. This effect was associated with the attenuation of colitis. In a placebo controlled trial, orally administered Lactobacillus salivarius UCC118 reduced the incidence of colon cancer and the severity of mucosal inflammation in IL-10-/- mice.46 L. salivarius was also shown to modify the gut microflora in these animals as C. perfringens, enterococci and coliform levels were significantly reduced in the probiotic group. A further trial confirmed the efficacy of L. salivarius UCC118 and demonstrated efficacy for Bifidobacterium infantis 35624 in attenuation of colitis in the IL-10-/- mouse model.47 The amelioration of disease activity in this study was associated with modulation of the gut microflora as investigated by culture-independent 16sRNA direct gradient gel electrophoresis. In addition, mucosal pro-inflammatory cytokine production was significantly reduced. Indeed, the oral route of administration may not be essential for certain probiotic effects. Reduced inflammatory scores and reduced production of

Table 26.1 Summary of probiotic efficacy in animal models Probiotic micro organism

Type of study

Trial outcome

Reference

Lactobacillus reuteri

IL-10-/- mice. N = 4±8 per group. Placebo controlled trial

Prebiotic lactulose and probiotic L. reuteri attenuated colitis and improved mucosal barrier function.

Madsen et al., 199945

Lactobacillus salivarius UCC118

IL-10-/- mice. N = 10 per group. Placebo controlled.

Reduced incidence of colon cancer and mucosal inflammation. Modulation of faecal flora.

O'Mahony et al., 200146

Lactobacillus salivarius UCC118 and Bifidobacterium infantis 35624

IL-10-/- mice. N = 10 per group. Placebo controlled

Attenuation of disease. Modulation of gut microflora. Reduction in in vitro production of IFN- , TNF- and IL-12. TGF- levels maintained.

McCarthy et al., 200347

Lactobacillus salivarius UCC118

L-10-/- mice. CIA model N = 10 per group. Placebo controlled

Attenuation of colitis and arthritis following subcutaneous administration of probiotic. Reduction in proinflammatory cytokine production.

Sheil et al., in press51

Lactobacillus plantarum 299v

IL-10-/- mice. Placebo controlled

Attenuation of colitis. Reduction in IL-12 and IFN produced by stimulated mesenteric lymph node cells.

Schultz et al., 200248

Lactobacillus rhamnosus GG

HLA-B27 transgenic rats.

Prevented recurrence of colitis.

Dieleman et al., 200152

Combination of L. acidophilus La-5, L. delbruÈckii subsp. bulgaricus, Bifidobacterium Bb-12, and Streptococcus thermophilus.

HLA-B27 transgenic rats.

Attenuated colitis following treatment with the prebiotic inulin and a combination of probiotic organisms.

Schultz et al., unpublished data

Probiotics in inflammatory bowel disease

715

proinflammatory cytokines have been observed in IL-10-/- mice which had been injected subcutaneously with L. salivarius UCC118.51 Modified probiotics in animal models Combinations of probiotic treatment with prebiotics or antibiotics have been used to increase the beneficial effect. The combination of the prebiotic inulin, and the probiotic organisms L. acidophilus La-5, L. delbruÈckii subsp. bulgaricus, Bifidobacterium Bb-12, and Streptococcus thermophilus significantly decreased inflammation in HLA-B27 rats.52 Furthermore, genetically modified probiotics have been developed. Lactococcus lactis was engineered to secrete biologically active IL-10 and a significant reduction in inflammation was observed in both IL10-/- and DSS murine models.30 The investigators concluded that genetically engineered bacteria for local administration of a therapeutic agent, such as Il-10, may be a useful strategy in the treatment and prevention of mouse models of IBD and have potential for human use. Live versus dead bacteria It may not even be necessary to administer live bacteria to achieve benefit. Bacterial DNA has been shown to have potent immuno-stimulatory effects. In a trial by Rachmilewitz et al. (2002), bacterial DNA was used to attenuate colitis in a number of murine models suggesting an anti-inflammatory effect for bacterial DNA that warrants further study.29 26.3.2 Human trials of probiotics in patients with inflammatory bowel disease Evidence that the enteric flora play a role in the pathogenesis of IBD and results from models of IBD which have demonstrated beneficial effects for probiotics has prompted clinical studies examining the effect of these organisms in patients with inflammatory bowel disease. Trials in ulcerative colitis (Table 26.2) A number of studies have examined the use of a non-pathogenic E. coli, E. coli strain Nissle 1917, in the setting of ulcerative colitis. Kruis et al. (1997) first performed a randomised, double blind clinical trial where 120 patients with inactive ulcerative colitis were randomised to receive oral E.coli strain Nissle 1917 or mesalazine.53 They reported that there was no difference in relapse rates in the probiotic treated group compared to patients on mesalazine. Relapse rates were 11.3% for the mesalazine treated group and 16.0% for the E. coli group. Life table analysis showed a relapse free time of 103+/-4 days for mesalazine and 106+/-5 days for E. coli. From the results of this preliminary study, probiotic treatment appeared to offer another option for maintenance therapy of ulcerative colitis.53 Further beneficial results were described by Rembacken et al. (1999) in a study where a total of 116 patients with active ulcerative colitis were recruited. 75% and 68% of the mesalazine and E.coli groups achieved remission,

Table 26.2 Summary of human trials of probiotic therapy in ulcerative colitis (N = number of subjects in trial) Study type

Organism used

Trial outcome

Reference

Randomised controlled trial

E. coli strain Nissle 1917 N=120

Patients with active colitis demonstrated similar relapse rates compared to patients on mesalazine.

Kruis et al., 199753

Randomised, controlled trial

E. coli strain Nissle 1917 N = 116

Confirmed result from Kruis et al., 1997

Rembacken et al., 199954

Open labelled trial

VSL#3. N = 20.

Maintenance of remission in patients

Venturi et al., 199956

Randomised controlled trial

E. coli strain Nissle 1917. N = 327

Remission maintained in patients receiving probiotic

Kruis et al., 200155

Open labelled trial

Saccharomyces boulardii N = 25.

Treatment given in combination with mesalazine for relapse of U.C. Remission achieved in 17 patients.

Guslandi et al., 200357

Probiotics in inflammatory bowel disease

717

respectively. In the second maintenance part of this study, the relapse rate in both groups was markedly higher than the investigators anticipated, 73% for the mesalazine group and 67% for the E.coli group. The time to relapse was not significantly different between the groups.54 These results suggested that the non-pathogenic E. coli was equivalent to mesalazine in maintaining remission, however these relapse rates are similar to those of placebo-treated patients. In a larger, 1 year multi-centre, randomised, double-blind, remission maintenance study of 327 patients, E. coli was shown to be as effective as mesalazine in maintaining remission with relapse rates of 45% for the E. coli group and 36 % in the mesalazine group, therefore offering an alternative to mesalazine in maintenance of remission in ulcerative colitis patients (Kruis et al., 2001).55 The probiotic cocktail VSL#3, a mixture of 4 lactobacilli strains (Lactobacillus plantarum, Lactobacillus casei, Lactobacillus acidophilus, Lactobacillus delbrueckii ssp. bulgaricus), three bifidobacteria strains (Bifidobacterium infantis, Bifidobacterium breve, Bifidobacterium longum) and 1 strain of Streptococcus salivarius ssp. thermophilus, has been studied in ulcerative colitis. There is a high concentration of bacteria in this mixture with potential synergistic relationships to enhance suppression of potential pathogens. The effect of VSL#3 on maintenance of remission in UC patients was evaluated using an open label design.56 In this pilot study, 20 patients in remission were treated for 12 months. At the end of the trial 15 out of 20 patients (75%) remained in remission. A recent study has investigated the use of Saccharomyces boulardii in the setting of ulcerative colitis. In an open, non-placebo controlled study, 25 patients with a relapse of ulcerative colitis were treated with mesalazine in combination with S. boulardii. 17 patients achieved remission.57 Trials in pouchitis (Table 26.3) Convincing evidence for beneficial probiotic effects in inflammatory bowel disease is seen in the treatment of pouchitis. In an open labelled study (Friedman et al., 2000), patients with pouchitis were treated with Lactobacillus GG and fructooligosaccharide. Patients reported a beneficial effect when the probioticprebiotic mix was administered as an adjuvant to antibiotic therapy. Remission was documented by suppression of symptom scores and reversal of endoscopic findings.58 Gionchetti et al. (2000) have studied VSL#3 in the setting of pouchitis and have demonstrated the efficacy of this probiotic mix in maintenance of remission in patients with chronic pouchitis.59 In a randomised, double blind, placebo-controlled trial, 40 patients with pouchitis received one month of antibiotic treatment and were in clinical and endoscopic remission. Patients were then randomised to receive VSL#3 or placebo for 9 months. At the end of the study 3 patients (15%) had relapsed in the VSL#3 group compared to 20 (100%) in the placebo group. In a follow up study, this group has also used VSL#3 as prophylaxis in patients after ileo-anal pouch formation surgery to prevent pouchitis. 40 patients were randomised to receive VSL#3 or placebo. At 1 year follow up, 10% of probiotic treated patients had developed pouchitis,

Table 26.3 Summary of human trials of probiotic therapy in pouchitis (N = number of subjects in trial) Study type

Organism used

Trial outcome

Reference

Open labelled trial

Prebiotic fructooligosaccharide and probiotic. N = 10

Effective in inducing remission in combination with antibiotic

Friedman et al., 200058

Randomised controlled trial

VSL#3. N= 40.

Maintenance of remission in chronic pouchitis after antibiotic induced remission. 15% relapse rate compared with 100% in control group.

Gionchetti et al., 200059

Randomised controlled trial

VSL#3. N = 40.

Prevention of acute pouchitis in patients after ileoanal pouch surgery. 10% pouchitis rate in probiotic group compared with 40% in control group.

Gionchetti et al., 200360

Randomised controlled trial

VSL#3 (6g). N=36.

Maintenance of remission in recurrent or refractory pouchitis after antibiotic induced remission. 85% remained in remission at one year, compared with 6% in placebo group.

Mimura et al, 200461

Probiotics in inflammatory bowel disease

719

compared with 40% of the placebo treated group.60 A recent study has again examined the role of VSL#3 in maintaining remission following treatment of refractory or recurrent pouchitis. Thirty-six patients with recurrent pouchitis (at least twice in the past year) or requiring continuous antibiotics, in whom remission was induced by 4 weeks of antibiotics, were randomised to receive 6 g ofVSL#3 or placebo daily for one year or until relapse. 85% of the VSL#3 treated group remained in remission at one year compared with 6% (one patient) in the placebo group.61 Trials in Crohn's disease (Table 26.4) In Crohn's disease, an early study involved the use of Sacccharomyces boulardii.62 In a double-blind study, 20 patients with moderately active Crohn's disease were randomised to treatment with this organism or placebo for 7 weeks. The probiotic treated patients had a significant decrease in Crohn's disease activity index (CDAI) compared with the control group. More recently, a randomised double blind trial randomised 32 Crohn's disease patients in clinical remission to receive either mesalamine alone or mesalamine plus S. boulardii. Clinical relapse was observed in only 6.25% of patients receiving mesalamine plus S.boulardii, while 37.5% relapse rate was observed in the group receiving mesalamine alone.63 Studies have examined the efficacy of Lactobacillus GG in the treatment of Crohn's disease. Malin et al. (1996) reported that in paediatric Crohn's disease, consumption of Lactobacillus GG was associated with increased gut IgA levels which could promote the gut immunological barrier.64 Gupta et al. (2000) also reported improved clinical scores and improved intestinal permeability in an open-labelled pilot study in a small study involving 4 paediatric Crohn's disease patients.65 A double-blind study investigated the use of the E. coli Nissle 1917 strain in Crohn's disease.66 Malchow et al. randomised 28 patients in remission to receive either E. coli or placebo. At 1 year follow up, the relapse rates were significantly reduced in the group that received E. coli (30%) compared with 70% in the placebo group. In a large double blind, randomised study the efficacy of VSL#3 combined with antibiotic treatment on the post-operative recurrence of Crohn's disease (CD) was compared to treatment with mesalamine alone. 40 patients were randomised to receive rifaximin for 3 months followed by VSL#3 for 9 months or mesalamine for 12 months. At the end of the trial 20% of the patients had recurrent CD in the probiotic/antibiotic group while 40% of patients in the mesalamine group relapsed.67 In an open study of patients with mild active Crohn's disease despite 5±ASA therapy, patients were offered either steroids or a trial of Lactobacillus salivarius subsp. salivarius UCC118 for 6 weeks. Of the 25 patients enrolled, 19 successfully completed the study and avoided steroids for a 3 month follow up period. The mean CDAI at enrolment was 217, falling to 150 at the end of the study period.68

Table 26.4 Index.)

Summary of human trials of probiotic therapy in Crohn's disease (N= number of subjects in trial CDAI = Crohn's Disease Activity

Study type

Organism used

Trial outcome

Reference

Randomised controlled trial

S. boulardii. N = 20.

Decrease in CDAI in probiotic group

Plein et al., 199362

Open labelled trial

Lactobacillus GG. N = 14.

Increase in gut IgA response

Malin et al., 199664

Randomised controlled trial

E. coli strain Nissle 1917. N = 28.

Remission achieved in patients on probiotics and steroids greater than with steroids alone

Malchow et al., 199766

Open labelled trial

Lactobacillus GG in children. N = 4.

Improved intestinal permeability and CDAL

Gupta et al., 200065

Randomised controlled trial

VSL#3 with antibiotic. N = 40

Patients with CD had 20% remission when given antibiotic and VSL#3 compared to 40% in mesalazine treated group.

Campieri et al., 200067

Randomised controlled trial

Saccharomyces boulardii. N = 32

Maintenance of remission in treatment group superior as relapse observed in 6.25% of patients receiving probiotic plus mesalazine compared to 37.5% on mesalazine alone.

Guslandi et al., 200063

Open labelled trial

Lactobacillus salivarius 118. N = 25.

Reduction of mean CDAI and induction of IgA in patients with relapse.

McCarthy et al., 200168

Randomised controlled trial

Lactobacillus rhamnosus GG

No difference seen in rate of controlled trial recurrence 1 year after surgery between group given probiotic or control

Prantrera et al., 200269

Probiotics in inflammatory bowel disease

721

Finally, a recent study of 45 CD patients who underwent curative surgery, compared the recurrence rate 1 year after surgery in patients treated with Lactobacillus rhamnosus GG or placebo. No difference was seen between the patients receiving probiotic (16% recurrence rate) or the placebo group (10%).69 While the trials summarised above are promising, a number of larger controlled trials are necessary before the use of probiotics can be recommended in the routine therapy of IBD.

26.4

Future trends

Although preliminary studies are clearly promising, large placebo-controlled, randomised, double-blinded clinical trials are required to clarify the role of probiotic bacteria in the treatment of inflammatory bowel disease. The aim of studies of probiotics in inflammatory bowel disease in the future will also be to increase our knowledge of how probiotics exert their effect. We need to expand our understanding of how best to use probiotics and which probiotic to use in each disease. Optimal doses will need to be determined, as will dosing schedules. Up to now, these parameters have varied between studies and a more consistent approach is desirable. The route of administration also requires more study, in particular to determine whether the oral route is always essential. The issue of live versus dead bacteria remains unclear. The beneficial effects of bacterial DNA argue against the need for probiotics to be always viable in order to have an influence on immune mechanisms. However, irrespective of the mechanism of action, there are reasons which might favour therapeutic usage of live over dead bacteria. Live bacteria may be more reliable for enteric transit and occupation of microbial niches. Secondly, live bacteria offer the advantage of elaborating biological molecules other than immunomodulatory DNA Detailed strain characterisation is also required for all potential probiotic strains before the use of combinations can be recommended. The potential exists for synergistic and even antagonistic effects between bacterial strains and this requires further study. Data need to be accumulated on individual probiotics because varying responses are seen between different probiotics, even within bacterial species. Finally, disease-specific probiotic organisms designed to target particular diseases or particular patients, (the `designer probiotic'), may become a possibility as we increase our understanding of molecular mechanisms behind the anti-inflammatory effects of individual probiotics. What is already clear is that there will be an increasing role for bacteria or bacterial products in a therapeutic setting alongside more conventional treatments for inflammatory bowel disease. Microbial therapeutics is an expanding field inviting further investigation, and we should not allow ourselves to become captives of the definition of probiotics.

722

Functional foods, ageing and degenerative disease

26.5

Source of further information

http://proeuhealth.vtt.fi is the website of PROEUHEALTH, which is a scientific/ medical/commercial collaboration between research groups from sixteen European countries.

26.6

Acknowledgements

The authors are funded in part by Science Foundation Ireland, the Health Research Board, the Higher Education Authority and the European Union (prog id QLK 2000-00563).

26.7 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

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of an oral Escherichia coli preparation and mesalazine in maintaining remission of ulcerative colitis. Aliment Pharmacol Ther 1997; 11: 853±858. REMBACKEN BJ, SNELLING AM, HAWKEY PM, AXON ATR. Non-pathogenic Escherichia coli versus mesalazine for the treatment of ulcerative colitis: a randomised trial. Lancet 1999; 354: 635±639. KRUIS W, FRIC P, STOLTE M. The Mutflor Study Group. Maintenance of remission in ulcerative colitis is equally effective with Escherichia coli Nissle 1917 and with standard mesalazine. Gastroenterology 2001; 120: A680 (Abstract). VENTURI A, GIONCHETTI P, RIZZELLO F, JOHANSSON R, ZUCCONI E, BRIGIDI P et al. Impact on the composition of the faecal flora by a new probiotic preparation: preliminary data on maintenance treatment of patients with ulcerative colitis. Aliment Pharmacol Ther 1999; 13: 1103±1108. GUSLANDI M, GIOLLO P, TESTONI PA. A pilot trial of Saccharomyces boulardii in ulcerative colitis. Eur J Gastroenterol Hepatol 2003; 15: 697±698. FRIEDMAN G, GEORGE J. Treatment of refractory `pouchitis' with probiotic and probiotic therapy. Gastroenterology 2000; 118: A4167. GIONCHETTI P, RIZZELLO F, VENTURI A, BRIGIDI P, MATTEUZZI D,BAZZOCHI G et al. Oral bacteriotherapy as maintenance treatment in patients with chronic pouchitis: a double-blind, placebo-controlled trial. Gastroenterology 2000; 119: 305±309. GIONCHETTI P, RIZZELLO F, HELWIG U, VENTURI A, LAMMERS KM, BRIGIDI P et al. Prophylaxis of pouchitis onset with probiotic therapy: A double-blind, placebocontrolled trial. Gastroenterology 2003; 124: 1202±1209. MIMURA T, RIZZELLO F, HELWIG U et al. Once daily high dose probiotic therapy (VSL#3) for maintaining remission in recurrent or refractory pouchitis. Gut 2004; 53: 108±114. PLEIN K, HOLZ J. Therapeutic effects of Saccharomyces boulardii on mild residual symptoms in a stable phase of Crohn's disease with special respect to chronic diarrhoea-a pilot study. Z Gastroenterol. 1993; 31: 129±134. GUSLANDI M, MEZZI G, SORGHI M, TESTONI PA. Saccharomyces boulardii in the maintenance of Crohn's disease. Dig Dis Sci 2000; 45: 1462±1464. MALIN M, SUOMALAINEN H, SAXELIN M et al. Promotion of IgA immune response in patients with Crohn's disease by oral bacteriotherapy with Lactobacillus GG. Annals nutrition Metab 1996; 40: 137±145. GUPTA P, ANDREW H, KIRSCHNER BS et al. Is Lactobacillus GG helpful in children with Crohn's disease? Results of a preliminary open-label study. J Pediatr Gastroenterol Nutr 2000; 31: 453±457. MALCHOW HA. Crohn's disease and Escherichia coli. A new approach in therapy to maintain remission of colonic Crohn's disease? J Clin Gastroenterol 1997; 25: 653±658. CAMPIERI M, RIZZELLO F, VENTURI A, POGGIOLI G, UGOLINI F, HELWIG U et al. Combination of antibiotic probiotic treatment is efficacious in prophylaxis of postoperative recurrence of Crohn's disease: a randomised controlled study vs mesalamine. Gastroenterology 2000; 118: A4179 (Abstract). MCCARTHY J, O'MAHONY L, DUNNE C, et al. An open trial of a novel probiotic as an alternative to steroids in mild/moderately active Crohn's disease (Abstract). Gut 2001; 49(supp. III): 2447. PRANTRERA C, SCRIBANO ML, FALASCO G, ANDREOLI A, LUZI C. Ineffectiveness of probiotics in preventing recurrence after curative resection for Crohn's disease: a randomised controlled trial with Lactobacillus GG. Gut 2002; 51: 405±409.

27 Assessing the effectiveness of probiotics, prebiotics and synbiotics in preventing diseases G. C. M. Rouzaud, The University of Reading, UK

27.1

Introduction: diet and gastrointestinal diseases

Acute gastrointestinal infections are a major draw on health funding in the developing world. They are among the most common disorders and account for a significant proportion of General Practitioner's time. Symptoms can range from mild short-term discomfort such as in the case of food poisoning to lifethreatening conditions such as bowel cancer. For instance, the number of food poisoning cases reported annually in the UK is generally greater than 100,000 and the actual occurrence may be much higher as a vast number of cases remain unreported. Moreover, the aetiological agents of infection quite often remain undetermined. Diet is one of the major environmental factors involved in the onset of various gastro-intestinal or systemic diseases. Colo-Rectal cancer (CRC) and Irritable Bowel Diseases (IBD), for instance, are mainly observed in Europe and North America where a Western diet predominates. A prophylactic approach whereby the diet is manipulated to manage or prevent such disorders and other systemic disorders known to be affected by diet may be both easily applicable for the consumers/patients and cost-effective for the health funding bodies. The gut microflora plays an active role in the health of the host. Its fermentative activities allow utilisation of substrates that have not been broken down in the upper part of the intestinal tract. The release of nutrients in the large bowel, although secondary to the digestion process in the small intestine, determines the maintenance of intestinal tract integrity and the intake of

Assessing probiotics, prebiotics and synbiotics in preventing diseases 727 micronutrients such as vitamin B and K. Fermentation of undigested carbohydrates and proteins releases organic acids such as Short Chain Fatty Acids (SCFA). These are known to be beneficial for epithelial proliferation and exert bactericidal effects on potentially pathogenic organisms. Additionally, the gastrointestinal tract is the primary system involved in immune responses as it is the first port of entry of xenobiotic compounds and opportunistic pathogens. The gut microflora stimulates the immune system in a non-inflammatory manner, priming the response to possible colonisation of pathogens. On the other hand, the release of toxins or deleterious compounds as a product of fermentation from pathogens and indigenous bacteria may trigger detrimental effects. For example, bacteria such as Escherichia coli ETEC, Salmonella enteritidis, and Vibrio cholerae produce endotoxins that severely damage the mucosae of the small intestine resulting in active secretion of ions from mucosal cells into the intestinal lumen. This consequently results in chronic diarrhoea and dehydration for the patient. Another example is the release of carcinogenic compounds as a result of proteolytic activities by autochthonous bacteria such as bacteroides and eubacteria (Carman et al., 1988; Van Tassell et al., 1982). Furthermore, the production of toxic gases such as H2S by sulphate reducing bacteria is thought to be associated with IBD and ulcerative colitis. The gut microflora is extremely diverse and accounts for a whole array of functions. Until fairly recently it was extremely difficult to measure such diversity but the refinement of molecular techniques has confirmed the presence of approximately 500 bacterial species in the intestinal community (Suau et al., 1999). Bacterial cells inhabiting the lower intestine considerably out-number eukaryotic cells present in our body. In this community, major genera have been identified but at the species level, the knowledge on colonisation of intestinal tract is still not complete (Fig. 27.1). The major groups within the intestinal community are commonly from Bacteroides spp., Enterobacteria or Clostridia spp. These populations are present in high numbers in the commensal microbiota but they are also closely related to pathogenic strains such as Clostridium difficile and Escherichia coli. These populations may furthermore be associated with potentially detrimental processes affecting the health of the host as mentioned earlier. On the other hand, bifidobacteria and lactobacilli are considered beneficial in the microbiota and their prevalence is generally a good indicator of health promotion. For instance, the gut microflora of breast-fed infants is primarily constituted of Bifidobacteria (Favier et al., 2002). The protective effect of bifidobacteria against enteropathogens has been demonstrated and they are essential for the constitution of the infant gastrointestinal defence barrier and for stimulation of early immunological responses (Sudo et al., 1997). Current management strategies are targeted towards improving the resistance to infections, and alleviating conditions such as Irritable Bowel Syndrome (IBS), chronic gut disorders (IBD and CRC), lactose intolerance, atopic and food allergies, recurrent vaginal thrush and mineral bioavailability. It is evident that not all probiotics or prebiotics will be able to achieve these goals. There is,

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Fig. 27.1 The composition of the adult gastro-intestinal microflora. Numbers are approximate concentrations of each bacterial group per millitre of faecal sample (adapted from Gibson and Roberfroid, 1995).

however, promising evidence that some success can be achieved. Overall further research and clinical trials should prove the efficacy of these approaches. 27.1.1 Economic and medicinal relevance of pro-, pre- and synbiotics Pharmaceutical costs for human use have exponentially risen in the past decade. The trend is likely to progress as a result of the past indiscriminate use of antibiotics which led to a reduction in their potency, efficiency and emergence of antibiotic resistant pathogens. Parallel to the development of new generation drugs, alternative routes are currently being explored such as functional food materials that may offer improved health without the need for drug administration. Public and industrial perceptions of the importance of gut microbiology in human health and nutrition have led to a major increase in probiotic and prebiotic product development and application.

Assessing probiotics, prebiotics and synbiotics in preventing diseases 729 It is estimated that well over one million consumers in the UK regularly (each day) consume probiotics. The European market value is well in excess of one billion euros per annum (Young, 1998). For the full market value to be realised, however, it is imperative that dietary modes such as probiotic, prebiotic and synbiotics are based upon sound scientific principles that provide irrefutable claims on efficacy and function. Currently in Europe, there is no legal requirement to produce a portfolio of data on human effects before launching new pro- and prebiotic products. However the use of probiotics and prebiotics in target populations such as infant or elderly or in disease states requires a level of investigation equal to the development of a new pharmaceutical. Consumers and clinicians should be provided with an assessment of physiological, microbial and safety aspects especially if these management tools are to be used as alternatives or complementary to traditional drug therapy. Similarly, development of synbiotic products (combination of pre- and probiotics) require comparative data to assess whether the combination is of additional value relative to the intake of compounds in separate form.

27.2

Definitions of probiotics, prebiotics and synbiotics

27.2.1 Overview of probiotic concept Current definition restricts probiotic classification to live microorganisms which upon ingestion elicit beneficial effects on the intestinal balance of the host (Fuller, 1989). Evidence supporting prophylactic action of characterised probiotic strains such as Lactobacillus rhamnosus and Lactobacillus fermentum in the vagina (Reid et al., 2001) may lead to extension of the definition to their use for topical application on tissues other than the digestive tract. A point that is sometimes argued is the necessity for the probiotic to be alive not only at the time of ingestion but also at the site of action, namely the large bowel. This implies a resistance of the probiotic strains to chemical changes and secretions occurring during transit through the stomach and the upper intestine. Some studies have shown that a stimulation of the immune system could be displayed by dead probiotic cells (Kaila et al., 1995; Ouwehand and Salminen, 1998). This passive mechanism, although desirable, is less potent than active healthpromoting effects elicited by live microorganisms. For example, live probiotics strains may adhere more strongly to the intestinal mucosa (Tuomola et al., 2000) or produce bacteriocins against potential pathogens (Talarico et al., 1989). As live micro-organisms, probiotics must have a proved safety record. It has been suggested that strains only be classified as probiotic once their beneficial effects have been established in vivo (Reid et al., 2003). This accounts for the fact that probiotic traits are generally specific to a particular strain and may not be applicable to all probiotics (Jacobsen et al., 1999). Currently most of the microorganisms corresponding to the definition of probiotics, stricto sensu, are from bifidobacteria and lactobacilli genera (Table 27.1).

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Table 27.1 Examples of bacterial strains currently used as probiotics in humans Bacterial genus

Strain

Lactobacillus species

Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus bulgaricus Lactobacillus Lactobacillus Lactobacillus

Bifidobacterium species

GG reuteri plantarum johnsonii gasseri delbrueckii subsp. casei shirota rhamnosus fermentum

Bifidobacterium Bifidobacterium Bifidobacterium Bifidobacterium

bifidum longum infantis lactis BB12

27.2.2 Overview of prebiotic concept In contrast to probiotics that have been the subject of research for a hundred years (Chen and Chen, 1989), the development of prebiotics as functional food is recent and rapidly expending. The notion of prebiotic stemmed from the observation that resistant carbohydrates such as oligosaccharides are selectively fermented by bifidobacteria and can contribute to human health by inducing changes to the indigenous intestinal microflora without the need for ingestion of live microorganisms (Wang and Gibson, 1993). The latest definition accepts as prebiotic any `non-viable food component which evades digestion in the upper gut, reaches the colon intact and is selectively fermented by beneficial bacteria in the gastro-intestinal tract' (Roberfroid et al., 1998). Currently, most of the food components classified as prebiotics are low to medium molecular weight carbohydrates. High molecular weight carbohydrates such as dietary fibres are not classified as prebiotic as they are not selectively fermented (Crittenden et al., 2002). Recognised prebiotics are primarily built from glucose, galactose, xylose and fructose (Table 27.2). Inulin, fructo-oligosaccharides (FOS) and lactulose are prebiotics with the most documented effects in vivo due to their widespread commercial availability. Oligosaccharides containing other monosaccharides such as arabinose, rhamnose, glucosamine, galacturonic acid are also under study (Table 27.2). The mechanism underlying the selective fermentation process is still unclear. For example, the relationship between molecular weight and selectivity has not been clarified yet. An increase of selectivity is seen when polysaccharides decrease in size, for instance with xylan to xylooligosaccharides (Okazaki et al., 1990), dextran to isomaltose (Olano-Martin et al., 2000) and pectins to pectic oligosaccharides (Olano-Martin et al., 2002). A better understanding of the relation between structure of oligosaccharides and

Table 27.2 Examples of exiting prebiotic oligosaccharides Oligosaccharides

Structure

Natural occurrence

Inulin Fructo-OS

Fru 2!(1Fru)n n 20 Fru 2!(1Fru)n n 2-5

Chicory, onion Inulin from chicory.

Lactulose Lactosucrose

Gal 1!4Fru Gal 1!4Glc1$2 Fru

None None

Trans-galacto-OS

Tri to penta saccharides with Gal 1!6Gal and Gal 1 !3Gal linkages Gal1 !6Glc1$2 Fru Glc1 !(6Glc)n n 1±4

Human and cow milk

Soybean OS Isomalto-OS Gluco-OS Gentio-OS Chito-OS Xylo-OS Arabino-xylo-OS Oligodextrans Pectic-OS Arabino-galacto-OS Arabino-OS Rhamno-galacturo-OS Galacturonic-OS Sialic acid OS

Di to Heptasaccharides with Glc1!2Glc Glc1!6Glc linkage Gln 1-4Glu Gln 1-4Glu Xyl !4Xyl Glc1!(6Glc)n n 1±4

N-acetyl neuraminic acid

Soybean whey Corn starch Oat glucans

mucopolysaccharides Corn cobs, oat spelt wheat dextran Pectins Soybeans Sugar beet Apple Polygalacturonic acids Human milk, -casein, lactoferrin

Key: Glu = glucose; Gal = galactase; Fru = fructose; Xyl = xylose; OS = oligosaccharide

Manufacturing · Hydrolysis of inulin by inulinase · Synthesis from sucrose by - fructosyl-transferase Chemical isomerisation of lactose Synthesis from lactose and sucrose by -fructofuranosidase Synthesis from lactose syrup by -galactosidase Extraction from whey Synthesis from starch using -amilase, pullulanase, -glucosidase Sucrose + maltose/glucosyl transferase

xylanase endodextranase endoglucanase endoarabinanase rhamnogalacturonase endogalacturonase

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functional benefits in the gut may lead to the manufacture of prebiotics with enhanced selectivity that could be targeted toward particular strains in the commensal flora (Rastall and Gibson, 2002). 27.2.3 Overview of synbiotic concept The concept of synbiotic is a combination of both probiotic and prebiotic approaches. A synbiotic aims at stimulating the growth and/or activity of indigenous bifidobacteria and lactobacilli by using an appropriate carbohydrate in conjunction with probiotic strain. Prebiotic may provide additional protection during intestinal transit to ensure persistence of the probiotic strain to the lower intestinal tract. Prebiotic may enhance the growth of the probiotic strain and of the targeted commensal populations. Dual advantages of both approaches may thus be realised. 27.2.4 Types of products and comparison of ease of use In the design of new product or dietary management strategy, the array of probiotic strains currently available is a notable advantage. Several strains can be combined in the same product. Food vehicles range from fermented milks, dairy products and fruit juices to various forms of freeze-dried supplements (capsule, pills). Survival of live bacteria is however an issue. Probiotics are usually anaerobic bacteria and do not survive well at temperature changes. Fermented milks, for example, need refrigeration which is a burden to their distribution. Strain efficacy after freeze-drying is debatable (Heyman, 2000). Survival after ingestion may also be uncertain (Kailasapathy and Chan, 2000). Prebiotics by-pass this burden as they are non-viable and reach the colon intact. Their structure confers additional food manufacturing properties such as thickening agent or sweeteners. Prebiotics are therefore more amenable to industrial process, with a longer shelf-life and can be incorporated to a larger variety of food such as infant formulae, weaning food, cereals and confectionery as well as beverages, dairy products and dietary supplements. Their versatility is a major potential but is still under-represented in countries other than Japan. 27.2.5 Target populations Prebiotics, probiotics and synbiotics may exert their purported effects in the large bowel at three different levels. They increase bifidobacteria and lactobacilli populations from the indigenous microbiota, and increase immuno-protection of the host. They also act by preventing colonisation by potential pathogens such as Listeria monocytogenes, Clostridium difficile, Esherichia coli, Helicobacter pylori, Salmonella typhimerium or Candida albicans (Nomoto et al., 1985; Hudault et al., 1997; Kabir et al., 1997; Mack et al., 1999; Payne et al., 2002; Hopkins and Macfarlane, 2003). Consequently, these prevention strategies should reveal their efficacy in populations with an

Assessing probiotics, prebiotics and synbiotics in preventing diseases 733 imbalanced microflora (as in IBD or IBS or during long-term antibiotherapy), in individuals where the immune system is fragile (neonates, infants, ederly and allergic patients) or when the colonization by a pathogen is likely. For instance, optimised probiotic or prebiotic could be used prophylactically in immunocompromised patients in intensive care units or in conditions where the occurrence of pathogen translocation is high, (e.g. patients undergoing abdominal surgery, chemo- or antibiotherapy). Populations in developing countries with prevalence of infectious agents may also benefit from the pre- and probiotic strategies. In healthy individuals, the effect may not be elicited as clearly. An example is the response of healthy individuals to ingestion of FOS or lactulose. Bifidogenesis induced by the consumption of prebiotic was inversely correlated to the level of bifidobacteria present in the faecal microflora at the start of the study (Tuohy et al., 2001; Tuohy et al., 2002). Clear investigation in the target populations are now required to assess efficacy.

27.3

Safety issues in the use of probiotics and prebiotics

27.3.1 GRAS status Existing probiotics and prebiotics are granted the status of Generally Regarded As Safe (GRAS status) products. Unlike new drugs or pharmaceuticals that are intensively screened for safety, rigorous safety assessments of pro- and prebiotics are lacking. The natural occurrence of prebiotic compounds in commonly consumed food products and the long history of use of fermented milk containing probiotic bacteria have so far justified the absence of risk assessment for these types of functional food. With the emergence of less well known probiotic microorganisms and the design of functional carbohydrates, potential risks of probiotics and prebiotics may need to be more carefully examined. The theoretical risk is bacteremia developing from either overgrowth of probiotic organisms or from the intestinal populations enhanced by prebiotic consumption. Bifidobacteria and lactobacilli are indigenous inhabitants of the gut microflora and also the major organisms targeted by pro- and prebiotics dietary management strategies. Bifidobacteria and lactobacilli are not closely related to recognised human pathogenic bacteria (Tuohy et al., 2003). Cases of infections by lactobacillus and bifidobacteria are extremely rare (Borriello et al., 2003). Cases of translocation of lactobacilli have rarely been reported as a result or as complications of existing severe medical conditions (Borriello et al., 2003). Given the safe record of use generally observed throughout the world, a recent committee of experts has deemed the risk of death by probiotics of the lactobacilli or bifidobacteria genera as negligible (Borriello et al., 2003). By extension, the risk of bacteremia associated with prebiotic is probably even lower. Consequently probiotics, prebiotics and synbiotics are dietary management tools that are recognised as harmless and have little contraindications or precautions of use. There is however a need for establishing standard operating procedures for safety assessment of new probiotics and prebiotics. A collection

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of probiotic strains recognised by the International Depository Authority would allow a more efficient control worldwide and set the safety standards for novel strains (Reid et al., 2003). For instance, it is proposed that databases containing molecular profile and genomic identification of well characterised probiotic strains be available to the scientific community (Borriello et al., 2003). This database could be used as reference when new probiotic strains are isolated. Human feeding trials and clinical trials comparable to phase 2 development of novel drugs must also be undertaken to eliminate any potential adverse effects of emerging pro- and prebiotic compounds before their launch on the market. 27.3.2 Anti-microbial resistance transfer There is some evidence that Enterococcus spp. could be applied as a probiotic therapy (Cremonini et al., 2002). However, caution must be maintained because some strains of enterococci have been associated with nosocomial and antibiotic associated infections. One example is the emergence of a pathogenic vancomycin resistant enterococcus, which is known to cause severe infections in patients who have catheters, intravenous devices or undergoing dialysis (Warren et al., 2003). Vancomycin is a last resort drug in the treatment of many antibiotic resistant pathogens including methicillin-resistant Staphylococcus aureus and such spread of resistant genes is concerning (Jones et al., 2001). Moreover, it has been reported that a tetracycline resistant gene (tet S) was exchanged easily from Listeria monocytogenes to a strain of Enterococcus faecalis (Charpentier et al., 1994; Francois et al., 1997). This demonstrates that the use of enterococci as a probiotic may raise health concerns. Such antibiotic resistant genes could be spread widely throughout the gut microbiota to susceptible recipient commensal bacteria (Tuohy et al., 2003). It has been suggested that strains used as probiotic should be susceptible to at least two of the common molecules used in human antibiotherapies (Borriello et al., 2003). 27.3.3 Side effect Although harmless, there are possible side effects encountered when using proand prebiotics. Due to the manipulation of the intestinal microflora, the volume of material arising in the colon and the volume of fermentation end-products are increased, this is particularly relevant when prebiotic oligosaccharides are used. An increase in stool frequency and stool weight is often reported in human feeding trials (Ito et al., 1995; Bouhnik et al., 1996; Chen et al., 2001). Absorption of a large dose of prebiotic such as inulin or lactulose (greater than 20 g/day) may lead to laxative effect (Bouhnik et al., 1999). Bifidobacteria and lactobacilli are not gas producers in the colon so appropriate doses of prebiotic should not increase the volume of gas in the large bowel (Probert and Gibson, 2002). Gas distension is a possible side effect that may discourage the intake of pro- and prebiotics, it is therefore critical that newly developed products are selective toward non-gas producer bacteria.

Assessing probiotics, prebiotics and synbiotics in preventing diseases 735 27.3.4 Analysis of risk versus benefit There is no upper limit in the amount of probiotics that can be ingested at each dose. Concentrated doses of 1010 Colony Forming Units (CFU) per day of Lactobacillus rhamnosus GG and Lactobacillus johnsonii La1 have been administrated to healthy volunteers with no adverse effect (Saxelin et al., 1995; Schiffrin et al., 1995). A dose response study using Lactobacillus johnsonii La1 showed that a minimum amount of 1010 CFU per day was required to observe an immune response (Donnet-Hughes et al., 1999). A highly concentrated probiotic preparation (VSL#3) containing eight strains of probiotics is currently raising interest (Gionchetti et al., 2000a). When a dose of 3*1012 per day of VSL#3 was administered to IBD patients in clinical trials, recorded side-effects were deemed minor to inexistent (Gionchetti et al., 2000b; Venturi et al., 1999; Gionchetti, 2003). The administration of lower doses of probiotics in the range of 106±109 CFU per day may limit the viability of the probiotic in the colonic environment unless an appropriate delivery vehicle is administered (Lee and Salminen, 1995). Excessive consumption of prebiotic oligosaccharides may lead to undesirable effects such as mentioned in section 27.3.3. Optimum doses of prebiotics have been determined for common prebiotics such as FOS and trans-galactooligosaccharides in various populations. Doses of FOS administered in feeding and clinical trials range from 3g to 20g per day in adults and 0.4g to 3.0g per day in infants (Bouhnik et al., 1999; Moro et al., 2002; Moore et al; 2003). These doses were found in accordance with the amount of naturally-occurring oligosaccharides ingested in a diet rich in vegetables (Van Loo et al., 1995). A minimal intake between 4±10 g per day for induction of a bifidogenic effect is often suggested but there is currently no recommended intake available (Roberfroid et al., 1998). The persistence of pro- and prebiotic effects when their intake is stopped has not been well established. In many feeding studies, colonic microbial changes are observed after treatment with pre-and probiotic but the effect generally ceased with the interruption of treatment (Tuohy et al., 2002; Gionchetti et al., 2000b). Long-term daily intake seems to be necessary to achieve optimum efficiency, however few studies have looked at long-term consequences of these dietary approaches.

27.4 Methods for determining mode of action and effectiveness 27.4.1 In vitro systems Various in vitro model systems have been validated and allow a reproduction of the physico-chemical events encountered in the different parts of the gastrointestinal tract. Available models are of various degrees of complexity from single stage fermenters to a cascade of bioreactors simulating the physiological difference between each part of the colon (Rumney and Rowland, 1992; Molly et al., 1994; Minekus et al., 1995; Macfarlane et al., 1998). Generally,

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temperature, pH, redox potential and transit time are controlled. Reproduction of peristaltic movements may vary from stirring to differential pulse movements. The gastro-intestinal secretion and digestive absorption are also simulated with some models (Minekus et al., 1995). The latest advances in the area are models simulating the attachment of microbial cells to an artificial intestinal membrane (biofilm reactor) (Probert and Gibson, 2004). In vitro models allow the study of the human gut ecosystem in controlled laboratory conditions whilst preserving the diversity of gut microbiota. Fermentative activities of a large array of substrates can be tested, these models have thus been proved useful in the initial steps of development of new/ emerging pre- and probiotics. 27.4.2 Animal models Animal models are a more realistic representation of the mammalian intestinal tract (Rumney and Rowland, 1992). These models allow detailed study of the systemic effects and host-response resulting from the manipulation of the gut microflora. Gastro-intestinal disorders can be induced in some animal models such as ulcerative colitis (Cummings et al., 2003), colo-rectal cancers (Hambly et al., 1997), necrotising enterocolitis (Butel et al., 2002). As immune response and microbial effects of probiotics, prebiotics and synbiotics are often speciesspecific, human flora associated animals are of preferential use for assessing the effectiveness of organisms or carbohydrates under study (Mallett et al., 1987; Djouzi et al., 1997; Edwards et al., 2003). 27.4.3 Ex vivo model Biopsies of intact and pathologic tissues allow an investigation of the ecological niches present in the gut and a characterisation of the microflora attached to the intestinal epithelium (Schultsz et al., 1999). Most of the in vitro or animal models use faecal microflora as starting inoculum. Although faecal microflora is a good representation of the luminal microflora, bacteria adhering to the epithelium are likely to differ (Macfarlane et al., 1998). The development of molecular tools has greatly improved the possibilities of exploring microflora from biopsies tissues. Characterisation of the epithelium-adhering microflora may consequently advance greatly in the next few years. Tissue culture is another system often employed to characterise attachment properties of emerging pro-, and prebiotics. Tissue cultures, although validated systems, encounter limitations as models are often derived from cancer cell lines. Limitations are also seen as tissue cultures need to be kept aerobic whereas most of the probiotics are anaerobic strains. Tissue culture may give indication of the immune response after exposure to prebiotics or probiotics but the models often need optimisation to reflect the real conditions encountered at the intestinal site, particularly in pathological phase (such as ulceration in IBD).

Assessing probiotics, prebiotics and synbiotics in preventing diseases 737 27.4.4 Human trials Definitive assessment of pro- and prebiotic effects are only achieved by results of well-designed human feeding studies. Ideally, trials should be double-blind, randomised and placebo-controlled (similar to phase 2 trials in drug development procedure). Comparative studies in multiple centres are advantageous. In the case of prebiotic, the choice of placebo is not always clarified, some studies using non-degradable polysaccharides (starch, maltodextrin) or readily digested saccharides (glucose/lactose) (Tuohy et al., 2002). Record of food intake and bowel habit during the trial period brings generally useful information. If the product is designed to be used as replacement therapy or as a complement therapy in a particular disease state, sample size, exclusion criteria and primary end-points must be well defined. Criteria such as history of drug administration, genetic susceptibility and family history must be taken into account. End-points such as extension of remission period (in IBD) or restoration of normal bowel function (in IBS) may be considered. There may also be a need for comparative studies with standard therapy (phase 3 study). Effectiveness of probiotic studies may involve comparison between well established strains and new strains or combination of strains. Similarly, synbiotic trials may require specific design measuring the potential synergistic effect between the synbiotic components. Follow-up studies (phase 4) are useful to determine the long-term effect of probiotic and prebiotic use (Reid et al., 2003). 27.4.5 New molecular tools for analysing gut microflora (biomarkers) Modern molecular techniques have led to the possibility of characterising the complete gut microflora in situ. It has enabled both the qualitative and quantitative monitoring of phylogenetically related bacterial groups without the need for traditional cultivation techniques which only select for those bacteria which are culturable in the laboratory (viable but non-culturable VBNC). Analysis of 16S rDNA gene profiles obtained directly from faeces has greatly expanded estimates of species diversity within the microflora. About 70% of clones correspond to novel bacterial lineages, whereby the majority fell in the three dominant groupings: Bacteroides spp., Clostridium coccoides, and Clostridium leptum (Suau et al., 1999). In feeding trials a range of 16S rDNA gene probes designed to target the most important groups of bacteria present in the gut microflora are applied to monitor the changes in bacterial numbers. These techniques known as Fluorescent in situ Hybridisation (FISH) allow a quantification of microbial populations. With the increase in the isolation and identification of novel bacterial species, however, the number of probes available is rapidly expanding and so is the number of species being monitored. With an increasing level of analysis, there is a need for high throughput techniques allowing an integrated analysis of these changes whilst conserving the information on the microbial diversity.

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Bacterial community analysis using 16s rDNA gene fingerprinting techniques such as T/DGGE, allows a qualitative whole community analysis of samples. T/DGGE separates PCR amplicons according to their sequence variation. The profiles obtained can distinguish a change in a specific species or indeed monitor the overall changes in diversity in response to the application of a functional food or probiotic. Another approach that is being developed is Real Time quantitative PCR, which though expensive is less time consuming than FISH and ultimately will be more robust in quantifying bacterial numbers (Matsuki et al., 2002). A combination of both quantitative and qualitative molecular approaches is extremely useful in evaluating the efficacy of pro-, pre- and synbiotics in boosting human health. Further to monitoring of bacterial change following probiotic or prebiotic ingestion, there is a need to understand how these changes affect the expression of genes both in the microbial population and in the intestinal epithelium. Only this kind of information will lead us to understand the mechanisms whereby probiotic and prebiotic are effective. More and more bacterial genomes are being sequenced and with the rapid development of DNA microarray technology, the crosstalk (transcriptomics; gene expression) between probiotics and human mucosa cells is very much on the horizon. Such data can be subsequently used to predict both the proteomics and the metabolomics of the effects of pre-, pro- and synbiotics on gut health. 27.4.6 Survival of probiotics strain Most commercial probiotics have been tested in vitro for their resistance to gastric acidity and bile salts but few data are available relative to the survivability in situ (Morelli, 2000; Fernandez et al., 2003). Survival varies considerably between strains belonging to identical genera. Lactobacillus strains, for example, generally survive well in vitro in the presence of acidic pH and bile acid, confirming their strong potential as probiotics (Fernandez et al., 2003). These specific traits may however not be sufficient to determine survivability in situ. The environment from which the probiotic strain originates may also affect chances of survival. In a study in vitro comparing the survival of 47 strains of Lactobacillus spp., strains isolated from the human gut were found to display a better survivability than probiotic strains isolated from food or dairy products (Jacobsen et al., 1999). Subsequently, five strains selected from the screening in vitro were studied in a feeding experiment. Findings confirmed that intestinal strains L. rhamnosus, L reuterii and L. GG were persisting in the intestinal tract in higher numbers than dairy strains L casei subsp. alactus and L. delbrueckii subsp. lactis (Jacobsen et al., 1999). Determination of survivability in vivo still requires clarification. Confusion is often made between transient, persistent or colonising effect of studied strains. Gastric acid, bile salts and pancreatic secretions are all barriers towards longterm persistence of probiotic. Additional properties may however be needed for effective competition of probiotic strains against the diversity of the indigenous

Assessing probiotics, prebiotics and synbiotics in preventing diseases 739 microflora. Cell-attachment and antimicrobial activities of the probiotic candidate may contribute greatly to the bacterial survival in vivo. Finally, the design of synbiotics should improve the survivability of probiotic strains as the presence of prebiotic compounds influence positively the number of viable cells in the preparation. Resistant starch was found to enhance the survival of Bifidobacterium lactis in simulated gastric and intestinal content (Crittenden et al., 2001). A combination of Fructo-oligosaccharide and Lactobacillus acidophilus increased the persistence of the probiotic in a model in vitro of the human gut (Gmeiner et al., 2000). 27.4.7 Labelling issues and health claims Probiotics and prebiotics, as other functional foods, rely on their specific properties and potential health claims for their promotion on the consumer market. Limitations exist regarding health claims that can be made on available probiotic, prebiotic and synbiotic products. In the United States, the Food and Drug Administration has not issued any regulatory category for this type of functional food. The Dietary Supplements Health Education Act allows general structure/function claims relative to the maintenance of normal metabolic functions. Mentions of treatments or cures of diseases or illnesses are not permitted. In Europe, new recommendations edited by the European Commission may allow the use of food claims when the food is classified as `novel food'. This legislation applies only if safety and efficacy of the products are clearly substantiated with relevant scientific and clinical studies. In Japan, a restricted range of health claims are permitted when the product is labelled `Food Specified for Health Use' (FOSHU). FOSHU status is regulated by the Japanese authorities and only allocated upon scientific evidence of health efficacy. Labels of commercial products should stipulate the identity of active ingredients and their concentrations. Information on the viable cell concentration at the end of the shelf life should appear on products containing probiotic strains. Sensitivity of microbial strain to storage conditions may however induce a greater variation in the number of viable cells recovered from products designed for long shelf life (e.g. freeze-dried supplement) than from those intended for short term consumption (e.g fermented milks). 27.4.8 Purity and content The prospective use of pre-and probiotics as preventive or therapeutic foods requires high quality standards of production particularly when the targeted population has lower immune defence. Good manufacturing practice and handling procedure should be in place. To avoid contamination, starter cultures and biotechnological tools used for probiotic production should be carefully monitored. Due to limitations in the manufacturing process, current prebiotic preparations are generally mixtures of polysaccharides of various chain lengths.

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Functional foods, ageing and degenerative disease

The presence of mono- and di-saccharides may hinder the specificity of the prebiotic. Chemical extraction of oligosaccharides from food may also result in undesirable colour or flavour. To overcome these issues, new enzymatic technologies providing higher oligosaccharide selectivity and more palatable properties are developed (Rastall and Maitin, 2002).

27.5 Evidence for the effects of pro-, pre- and synbiotics on acute and chronic diseases 27.5.1 Acute disorders Traveller's diarrhoea Attempts to prevent or reduce the occurrence of traveller's diarrhoea have involved the intake of probiotic agents such as L. acidophilus, B. bifidum, L. bulgaricus, L. GG (Black, 1989; Hilton et al., 1997). In a placebo-controlled study of two cohorts travelling at two different holiday destinations L. GG has proved efficacious in reducing the incidence of diarrhoea in one cohort but no significant changes were seen in the second cohort. Aetiological agents involved in this study were not identified but were likely to be different at both sites. This may explain the unsuccessful prophylactic action. A specific probiotic may not elicit anti-pathogenic activities toward a wide range of pathogens. Multibiotherapy or symbiotic product may increase the efficacy of the dietary strategy. More structure-function studies are required to identify pro- or prebiotics with anti-pathogenic activities Antibiotic associated diarrhoea Diarrhoea often occurs as a side effect of antibiotherapy. The disruption of the intestinal microbial balance induces an attenuation of the natural defence barrier against pathogens. Opportunistic pathogens such as Clostridium difficile may then proliferate and pseumembranous colitis may be seen as complications (Fooks and Gibson, 2002). L. GG and B. longum ingested solely have led to a significant decrease of the diarrhoeal episode in erythromycin-induced subjects (Marteau et al., 2001). A combination of L. acidophilus and L. bulgaricus has also reduced the incidence of diarrhoea induced by ampicillin, neomycin and amoxicillin-clavulanate (Witsell et al., 1995; Clements et al., 1983; Gotz et al., 1979). An association of B. longum and L. acidophilus was also successfully used in the prevention of clyndamicin-induced diarrhoea (Nord et al., 1997). Rotaviral diarrhoea in children Rotaviruses are a common cause of gastro-enteritis in infants. L. GG and B. bifidum have been used in clinical trials for prevention and shedding of rotaviruses (Isolauri et al., 1994; Saavedra et al., 1994). L. GG showed a drastic reduction of the duration of rotaviral diarrhoea in several studies whereas the same probiotic seemed less effective in diarrhoea unrelated to rotaviruses (Marteau et al., 2001).

Assessing probiotics, prebiotics and synbiotics in preventing diseases 741 The putative mechanism of action is a stimulation of the immune response specific to rotaviruses and reinforcement of the mucosal integrity. Necrotising enterocolitis (NEC) Enterocolitis is a gastro-intestinal disorder of pre-term neonates treated in intensive care units and receiving enteral feeds. Infectious agent is unclear but bacteria from the commensal microflora such as Clostridium difficile or Clostridium butyricum may be involved (Butel, 2003). Bifidobacterial population in the intestinal lumen of neonates fed enterally is abnormally low. This may result in poor immunity and favourable conditions for translocation of bacteria to the systemic environment (Dai and Walker, 1999). Preventive administration of L. acidophilus and B.infantis to newborns admitted in intensive care units significantly reduced the occurrence of NEC in comparison to historical cohort (Hoyos, 1999). Animal models of NEC have also shown an effect of fructo-oligosaccharide in the reduction of necrosis and ulceration. Additional clinical trials are needed to confirm observations in animals and underpin the mechanism of action. 27.5.2 Chronic disorders Autistic children Gastroenteritis is commonly associated with autistic children spectrum disorders. Studies of faecal samples from autistic children have highlighted an alteration of the intestinal microflora (Finegold, 2002). Microbial metabolites released in the gut may play a psychoactive role in autistic pathology (Bingham, 2003). Probiotic approach may contribute to the relief of gastrointestinal symptoms and help toward the normalisation of the autistic intestinal flora. Clinical practices and carers of autistic individuals have circumstantially reported an improvement of autistic symptoms upon probiotic intake but no appropriate epidemiological and feeding trials are currently available to confirm these observations (Bingham, 2003). Ulcerative colitis (UC) Ulcerative colitis is characterised by an acute inflammation of the intestinal tract with no relation to infection. Individuals affected by the disease experience periods of relapse and remission throughout their lives. The location of main symptoms in the large intestine suggests a link between the local microflora and the disease. Animal models suggest that the normal microflora is needed for the disease to occur but no specific aetiological agent has been identified. One hypothesis is that UC is due to a partial breakdown of tolerance to the normal commensal colonic flora (Macpherson et al., 1996). Several pathogens have been suggested as causative agents of the disease: Escherishia coli (DarfeuilleMichaud et al., 1998), sulphate-reducing bacteria (Gibson et al., 1991), mycobacteria, pseudomonas and helicobacter species. The control of the disease involves heavy anti-inflammatory medication or surgery. The use of antibiotics is often not conclusive. Manipulation of the diet

742

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of colitis sufferers has shown an improvement at least in the maintenance of remission periods. Randomised clinical trials have confirmed that pro-and prebiotic use may be effective in this pathology by prolonging the microbial composition of the remission state (Table 27.3). The mode of actions of probiotic and prebiotic in UC are thought to be manifold and complementary. Probiotic studies in UC animal models have demonstrated a probiotic interaction with colonocytes leading to a possible down-regulation of the inflammatory response (Steidler et al., 2000). Prebiotics such as germinated barley are a convenient and efficacious way of stimulating the production of butyrate at the colitis site. Butyrate produced by fermentation of germinated barley promotes the proliferation of colonocytes and restores the integrity of the intestinal mucosa in animal models of colitis (Kanauchi et al., 2001). Colo-rectal cancer The human microflora has an important role in the development of CRC. Microbial enzymes expressed by bacteria of the clostridium and bacteroides groups are able to convert dietary constituents to genotoxic or carcinogenic compounds (Table 27.4). Probiotics are thought to act as preventive agent of the carcinogenesis by inhibiting the activity of these enzymes. Most of the experimental evidences in vivo are based on animal models (Tuohy et al., 2003). Rodents fed a diet supplemented with several probiotic strains of Lactobacillus spp. and Bifidobacterium spp. developed less colonic DNA damage and fewer tumours than placebo-fed counterparts (Pool-Zobel et al., 1996). An epidemiological study reported a negative correlation between the consumption of dairy probiotics and colonic adenomas (Burns and Rowland, 2000). Probiotic effect is likely to take place over a long time frame. Results of prospective studies investigating the long term preventive effect of probiotics against CRC are lacking. Prebiotics such as inulin, fructo-oligosaccharides, lactulose and galacto-oligosaccharides have shown effective protection in CRC animal models. Results of human feeding trials are however less consistent. In a parallel, placebo controlled study of 20 volunteers, the administration of lactulose at half the pharmacological dose (10g/day) did not influence significantly the level of faecal genotoxicity (Tuohy et al., 2002). The production of butyrate, a potent regulator of epithelial cell is one of the mechanisms thought to underlie the preventive effect of prebiotics. A synbiotic combination containing L. rhamnosus, B. bifidum Bb12 and fructooligosaccharide have shown encouraging results in vitro and in animal models (Van Loo and Jonkers, 2001). The synbiotic is currently tested in a randomised clinical trial involving CRC patients and individuals at high risk of developing CRC (Syncan project). Irritable bowel syndrome (IBS) Irritable bowel syndrome defines a range of symptoms including abdominal pain, flatulence, constipation and diarrhoea (Mercenier et al., 2003). Unlike

Table 27.3 Randomised clinical trials testing the effectiveness of probiotic and prebiotic agents in patients suffering from Ulcerative Colitis Probiotic treatment

Type of trials

Measured response

Reference

Probiotic supplement VSL#3 (L. acidophilus, L. bulgaricus, L. casei, L. plantarum, B. breve, B. infantis, B. longum, S. thermophilus) E. coli (Nissle 1917)

Feeding trial

Gionchetti, 2003

Placebo controlled trial

· Long-term maintenance of colitis remission · Increase in faecal counts of bifidobacteria and lactobacilli Maintenance of colitis remission

L. GG

Feeding trial

Increase in the immune response

Rembacken et al., 1999 Malin et al., 1996

Prebiotic treatment

Type of trials

Measured response

Reference

Germinated barley foodstuff

Placebo controlled trial

· Increase Short Chain Fatty Acid production · Increase faecal counts of bifidobacteria · Maintenance of colitis remission

Bamba et al., 2002

Wheat fibre and ispanghula

Long term double blind cross-over feeding trial

No effect of treatment on faecal counts of bifidobacteria and lactobacilli

Edjerhamn et al., 1991

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Functional foods, ageing and degenerative disease

Table 27.4 Bacterial enzymes involved in the production of carcinogenic compounds Example of enzymes involved in development of CRC -glucuronidase -glycosidase Azoreductase Nitroreductase IO hydratase-deshydrogenase Nitrate/nitrite reductase

IBD, IBS is characterised by the absence of intestinal inflammation or pathology of the intestinal mucosa. The chronic stage occurs after gastro-enteritis or a course of antibiotics. Women from Western countries are the major group of sufferers. The recurrence of yeast colonisation by Candida albicans and a low prevalence of lactobacilli and bifidobacteria have been observed in IBS sufferers (Smejkal et al., 2003). Probiotics may help the alleviation of syndrome but the effect is difficult to measure in vivo. L. plantarum 299v, L. GG and VSL#3 were tested in separate randomised placebo-controlled trials and results showed only limited effect on IBS symptoms (Kim et al., 2003; O'Sullivan and O'Morain, 2000). The development of a synbiotic product may be a more satisfactory strategy in IBS management as it allows a dual action on the bowel motility and composition of intestinal microflora (Smejkal et al., 2003).

27.6

Sources of further information and advice

27.6.1 Ongoing research programmes: European Frame programmes V and VI In Europe, the PROEUHEALTH cluster is a large international research network which aims to assess the medical efficacy of pre-, pro- and synbiotics and to understand the mechanistic principles underlying these dietary management tools. The cluster includes eight complementary European projects and covers all aspects of the development of pro-, pre- and synbiotic (http:// proeuhealth.vtt.fi). Research interests focus on the development and validation of molecular methodologies allowing the production of biomarkers and in situ molecular probes. Emphasis is also given to the improvement of technological capabilities for the production of novel safe probiotics and prebiotics. Combinations of technical developments are geared to the conduction of clinical trials and to the study of the interactions between intestinal microflora and host cells. 27.6.2 WHO/FAO documentation The World Health Organisation in conjunction with the Food Agriculture Organisation has recently held a consultation of experts on the evaluation of

Assessing probiotics, prebiotics and synbiotics in preventing diseases 745 health and nutritional properties of powder milk with live lactic acid bacteria (http://www.who.int/foodsafety/publications/fs_management/probiotics/en). The aim of this consultation was to review the latest information and scientific evidence available on the functional and safety aspects of probiotics present in dairy products. The Committee of experts laid out recommendations for the use of the term probiotic in food. Guidelines were also provided regarding levels of evidence necessary to make a health claim. The recommendations will be considered in the Codex Alimentarius in terms of labelling and claims for food (http://www.codexalimentarius.net). 27.6.3 New scientific association dedicated to this area (ISAPP) Scientists throughout the world have felt the need to improve the level of scientific integrity associated with the study and application of probiotics and prebiotics. The International Scientific Association for Probiotics and Prebiotics (ISAPP) is a non-profit organisation comprised of international scientists whose intent is to develop approaches and products that are optimally designed for the improvement of health. (http://www.isapp.net). The association aims at identifying gaps in knowledge, at promoting scientific and multidisciplinary studies, and at defining standards required to assess novel pro- and prebiotics. ISAPP in its first publication has listed the traits for which genomic loci may be relevant to study (Reid et al., 2003). In its second publication, ISAPP will give a view on the impact of probiotic and prebiotic in the management of diseases. 27.7.4 Standard reference list and book Additionally to material published by the afore-mentioned international Institutions, readers may be referred to recent comprehensive publications: Tannock G, Probiotic and Prebiotic: Where Are We Going? Wymondham, Caister Academic Press, 2002 Gibson G, Ottaway P and Rastall R, Prebiotics: New Developments in Functional Foods, Oxford, Chandos Publishing, 2000

27.7

Conclusion

Pre-, pro- and synbiotics offer good opportunities for reducing impact of food related diseases. A strong body of in vitro and in vivo evidence now exists. A growing number of well designed clinical trials have highlighted the positive outcomes against specific disease conditions. As the number of pro-, pre- and synbiotic candidates increases, concerted international actions are taken to assess their efficacy, to elevate manufacturing standards and to regulate their use on the consumer market. The application of newly developed molecular techniques in the field and the development of physiological biomarkers open up possibilities for screening

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Functional foods, ageing and degenerative disease

new probiotic strains and for refining their target site in the gut. Multi-functional prebiotics designed with specific anti-adhesive properties may inhibit the binding of specific pathogens to the intestinal mucosa thereby allowing an increased resistance to infection. Probiotics engineered to carry specific immune-enhancing molecules may be used as vaccine vectors or as regulators of the immune response. However, in many disorders and diseases, the mode of action of pro- and prebiotics is still speculative and under explored. Future studies will need to establish the interaction between host cells and pro- and prebiotic agents. Identification of bacterial metabolites, cells receptors sites and gene activated by the pro/prebiotic agent will provide strong basis to ascertain the specificity of action and efficacy in vivo. Long term and large scale human studies examining clinical and molecular parameters should be undertaken to determine the relationship between maintenance of a beneficial gut microflora and human health.

27.8

References

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Index

abdominal obesity 245 aberrant crypt foci (ACF) 554±5 acetate 532±3, 633 acetyl-CoA carboxylase (ACC) 262, 264±5 achlorhydria 177 acute disorders 740±1 added fibre 629 adenomatous polyposis coli (APC) gene 534, 535 adenomatous polyps 535, 545 screening patients after removal of 545 adenosine triphosphate (ATP) 119, 486 adequate intake (AI) for total fibre 630 adhesion 367, 403 prevention by probiotics and prebiotics 427, 428, 441±4, 462 adhesion molecules 353±4 soluble adhesion molecule concentrations 353±4, 369±70 adipose tissue 230, 231, 260±1 adjuvant chemotherapy 550±1 adjuvant radiotherapy 551 age-related diseases, diet and 66±8 age-specific intestinal microflora 315±16 ageing and degenerative diseases 490 determinants of healthy ageing 57±8 effects on oral health 187±8 and the immune system 352±3 ageing population 394

ajoene 65 alcohol 282, 283, 542 alendronate 118±19 alkaline elution method 595 allergic inflammation 352 allergy 315, 377±8 allicin 65±6 allochthonous flora 528±9 -linolenic acid (ALA) 355, 357, 360, 371±2, 381 2-macroglobulin (2M) 502 -tocopherol 691 Alzheimer's disease 67 Ames test 559, 589 amino acids 26, 305±6 AMP-activated protein kinase (AMPK) 265 anaemia 31, 697 aneuploidy 588, 589 angiogenesis anti-angiogenic functional foods 501±12 body function and degenerative disease 495±500 angiopoietin-2 (Ang2) 496, 498 animal diets, enriched 380 animal models/studies 14, 237, 631, 736 antibacterial activity of probiotics 430±50, 456±9, 463 foetal origin of adult diseases 20±1 glucosinolates and prevention of cancer 618±20

754

Index

IBD probiotics 713±15 role of bacteria 710 phyto-oestrogen action on bone 124±5 phyto-oestrogens and cancer prevention 651±2, 653±4 sucrose and dental caries 215±16 anoxia 492 anthocyanins 670 anti-angiogenic functional foods 501±12 dietary restriction 511 future trends 511±12 range and mechanism of action 501±2 types of compound 502±10 antibacterial activity of probiotics 429±61 antibiotic-associated diarrhoea (AAD) 404±5, 740 antibiotics 404, 533 anticarcinogens 672±3 antigens 349 CEA 547±8 presentation and n-3 PUFAs 368 tumour-associated 541 anti-microbial resistance transfer 734 antimutagenicity 581±614 future trends 603±4 limitations of identification methods 600±3 dose-effect relationships 603 predictive value of endpoints and test systems 600±1 specificity of protection 601±2 time-kinetics of protection 602±3 methods of identification 583±600 mechanism of DNA protection 583±6 methodological aspects 586±600 synbiotics 554±6 antioxidant effects 71, 592, 595 glucosinolates 622±3 antiproliferative activity 656±7 antitumourigenic activities 554±6 apatite 185 apigenin 509 apolipoprotein E (Apo E) 101 interaction with vitamin K 103±4 apoptosis 486, 488, 489±90, 501±2 induction by glucosinolates 622 osteoclast apoptosis 122±3 apples 192 approval of health claims 13±14 arachidonic acid 357, 358, 365±6 as an eicosanoid precursor 361±3 Arctiin 652

arginine 306 ascorbate 689 asthma 377 atherosclerosis 378 ATR-IR technique 69 atrophic gastritis 402±3 Australia 156±7 autistic spectrum disorders 741 autochthonous flora 528 automated microscopy-based image analysis 334±5 azoreductase 557 azoxymethane (AOM) 555±6 B-cells 302 bacteremia 733 bacterial DNA 712±13, 715 bacterial mutagenicity tests 589±94 bacterial succession 302±3, 395±9, 529 bacteriocins 428±9, 431±4, 444±5 Bacteroides 395, 727 barrier effect 427±8 basic fibroblast growth factor (bFGF) 498 Belgium 12, 157 benign prostatic hyperplasia (BPH) 62 benzoic acid derivatives 670, 671 benzyl ITC (BITC) 618, 619, 620 Berberis 71 beta amyloid plaques 67 -carotene 684, 685, 686±7, 700 -glucosidases 556±7 -glucuronidases 556±7 bifidin 451 bifidobacteria 315, 401, 406, 419, 727, 733 adhesion 403 microbiota succession 302±3, 395, 398 probiotics 422±3, 425±6, 526, 729±30 antibacterial activity 451±61 Bifidobacterium adolescentis 316 Bifidobacterium longum 315±16, 426 bifidocin B 455 bifunctional enzyme inducers 585 bioavailability 158, 176, 177 biochemical markers bone turnover 85±6 colorectal cancers 547±8, 560±1 biomarkers 737±8 colorectal cancers 548, 558±61, 562 satiety 284±5 using to assess weight-control foods 286 biomonitoring studies 597±600 biophenols 64

Index biopsies 736 biotechnology 39, 70, 316±17, 463, 715 biotin 699±700 biphosphonates (BPs) 118±19 blocking agents 673 blood, satiety biomarkers in 285 bone formation and calcium fortification 174±7 growth 85±7 metabolism 120±1 phyto-oestrogen action on 122±4 animal studies 124±5 human studies 125±6 turnover/remodelling 85±6 bone health 174±83 TCC and 174±83 vitamin D fortification and 139±73 see also osteoporosis bone mass 85±7 bone mineral density (BMD) 100±1, 116, 145±6 bowel diseases 312 see also inflammatory bowel disease (IBD) Bowman-Birk inhibitor (BBI) 503 brain 285 bran 35, 633±4 breast cancer 19 fibre and 639±40 phyto-oestrogens and prevention 649±52 breast milk, human 27±30, 395 butyrate 427, 507, 532±3, 633, 742 butyric acid 504±5 C-reactive protein (CRP) 370 CA 19.9 tumour marker 548 CA 242 tumour marker 548 caffeine 103, 585 calciferol see vitamin D calcification 174±5 calcitonin (CT) 117±18 calcitriol 687 calcium 88±90, 234, 309, 544 calcium-to-phosphate ratio 91, 177 fortification 174±7, 180±1 choosing a calcium fortifier 176±7 key issues 175±6 optimal daily intake 174, 175 oral health 191, 195 role in calcification process 174±5 VDR genotype-calcium interactions 102

755

calcium carbonate 178±9, 180 calcium citrate (TCC) 174±83 as a calcium supplement 177 fortification 180±1 measuring effectiveness 178±80 calcium phosphates 179 calciuria 92±3, 94, 99 calciuric factors 103 caloric restriction 493, 511 Canada 157 cancer 67±8, 315 dietary fibre and prevention 628±44 glucosinolates and prevention 617±20 hormonally related 637±8, 640 oral 184±5 phenolics and chemoprevention 669±80 phyto-oestrogens and prevention 649±55 vitamins and prevention 681±707 see also under individual types of cancer candidate genes nutrient-sensitive 234±5 obesity 226±30, 231 osteoporosis 100±1 candidiasis 459 cannabinoid terpenes 510 capers 66 capsaicin 62, 506 capsaicinoids 676 caramels, hard 204±5, 206 carbohydrate response elements (ChoRE) 268 carbohydrates 527 anti-angiogenic 503±4 dental caries and 185, 186, 200±1 reduction of fermentable carbohydrates 188±9 infant nutrition 27±8 maternal nutrition 24±5 and satiety 282, 283 carboxylase 96±7 carcinoembryonic antigen (CEA) 547±8 carcinogens 672±3 detoxification through induction of phase II enzymes 621 inactivating potential carcinogens 554 modulation of carcinogen activating phase I enzymes 620±1 mutagens as 581±2 cardiovascular disease 378 CARD15/NOD2 710 caries, dental see dental caries

756

Index

cariogenicity 185, 186 reduction in plaque cariogenicity 189±90 carotenes 684±5, 686±7, 700 carotenoids 685±7 case-control studies 631, 682±3 caspases 505, 622 cataracts 67 catechins 123 Celebrex 543±4 cell biomarkers 548, 558±60 cell culture studies 651, 653±4 cell proliferation, inhibition by phytooestrogens 656±7 central biomarkers 285 ceramides 504±6 cereals 62 chaperones 494 cheese 190, 192, 195 chemoprevention of cancer 543±4 phenolics and 669±80 chemotaxis 367 chemotherapy 550±1 chewing gum 190, 192, 193±4, 204±5, 206, 207, 215 chitin 503 chitosan 503 chlorogenic acid 676 cholecalciferol 140, 687 cholecystokinine (CCK) 280, 308 cholesterol 32±3 choline 27, 34 ChREBP transcription factor 268±70 chromatography 68 chromosome mutation tests 588±9 chronic disorders 741±4 chronic inflammatory diseases 351, 491 citrus flavonoids 675 citrus fruit cultivars 64 claims 162 types of 8±10 see also health claims climate 57±8 clinical studies see intervention (clinical) studies Clostridia 315, 425, 727 Clostridium difficile associated diarrhoea (CDAD) 398, 404±5 Codex Alimentarius Commission 155±6 coenzyme Q10 (ubuquinone) 489 cognitive decline 67 cohort studies 631, 682±3 colon, microbiota in 397, 530±1

colonic crypt homeostasis 539±40 colonisation resistance (barrier effect) 427±8 colonoscopy 546 colorectal cancer 524±80, 742, 744 diagnosis and treatment 546±52 diet and other measures for preventing 451±4, 542 fibre and 630±9 future trends 561±2 gut microflora 528±33 nutrition and 311 phyto-oestrogens and 654±5 pre-, pro- and synbiotic influences on carcinogenesis 552±8 predicting tumour formation 558±61 screening 544±6 susceptibility in old age 408 symptoms 546 types, occurrence and causes 533±41 colostrum-derived preparations 315 combinations of nutrients 382 comet assay 559, 586±7, 588, 589, 596, 597±8, 601 `comets' 587, 588, 589 commensal gut microbes 304, 709 nutrition and 309±11 computed tomography (CT) colonography 547 condensed tannins 670, 672 conjugated linoleic acid (CLA) 357, 358, 360, 366±7, 379, 504 consortia of strains 461 constipation 401±2 copper chelation 508 coronary heart disease (CHD) 17±18, 63 coumestans 115 Crohn's disease 708±9, 710 trials of probiotics 719±21 see also inflammatory bowel disease CROWNALIFE project 398, 561 cruciferous vegetables 615±16 prevention of cancer 617±20, 623 see also glucosinolates culture independent methods 326±34 curcumin 509, 675 cyclooxygenases 543 cyclooxygenase-2 (COX-2) 491, 543 cytochrome P450 enzymes 620±1 cytogenetic experiments 597 cytokines 350, 351, 352, 353 lymphocyte-derived 368±9 monocyte-derived 368

Index daidzein 122, 509, 651 dairy products 190, 191, 192, 194±5 fermented 406, 525 databases food composition databases 59 of probiotic strains 734 of small subunit rRNA sequences 327±8 deficiencies of vitamins carotenoids 686 folate, vitamin B12 and vitamin B6 696±7 niacin 699 riboflavin 693±4 vitamin A 684±5 vitamin C 690 vitamin D 143±4, 688 vitamin E 692±3 deficiency diseases, reducing 155±6 degenerative disease 485±523 angiogenesis, body function and 495±500 anti-angiogenic food compounds 501±11 diet and prevention of see diet environmental/exogenous risk factors 493±5 future trends 511±12 genetic/endogenous risk factors 486±93 mechanisms of 485±6 delayed-type hypersensitivity 369 denaturing gradient gel electrophoresis (DGGE) 340±1, 532, 738 dendritic cells 302, 553, 712 Denmark 157 dental caries 185±6, 188, 200±19 aetiology 185±6 and carbohydrates 185, 186, 188±9, 200±1 cariogenicity 185, 186, 189±90 early prevention 206±7 prevention of intrafamiliar transmission 205±6 prevention in orthodontic patients 208 sucrose and 208±16 xylitol and prevention of 202±8 dental erosion 186 dental plaque 185±6, 203±4 reduction in cariogenicity 189±90 dentine 185 detection limit 333 diabetes 19, 142 gestational diabetes mellitus 25

757

diallyl disulphide 506 diarrhoea 398, 404±5, 740±1 dicalcium phosphate 175 diet 493±4 and age-related diseases 66±8 changing dietary habits 175±6 and gastrointestinal diseases 726±9 and gut microflora in old age 403±4 and oral health 184±6 strategies 188±92 prevention of colorectal cancer 451±4, 542 prevention of degenerative disease 17±56 effects of supplement intake 30±3 foetal nutritional requirements 21±7 influence in early life 17±21 neonatal nutritional requirements 21±2, 27±30 nutrition during lactation and pregnancy 33±9 safety concerns of functional foods 39±41 strategies for analysing interaction between genetic background and diet 237±44 vitamin deficiencies see deficiencies of vitamins `Diet and Obesity' project 244 dietary fibre see fibre dietary intake surveys, for vitamin D 147±50 dietary restriction 493, 511 dihomo- -linolenic acid (DGLA) 355, 359 and eicosanoid production 363 dioxin 122 disease-specific probiotics 721 dithiocarbamates 618 DNA adducts 559, 589, 596±7, 598±9 bacterial 712±13, 715 repair processes 585 DNA arrays (DNA chips) 235±6, 337±40, 341±2, 604 DNA-damage 537±8, 581 oxidative 689±90 primary DNA-damage tests 586, 587 DNA protection 582 mechanisms of 583±6 specificity of 601±2 time-kinetics of 602±3 docosahexaenoic acid (DHA) 355, 358, 360, 367, 370

758

Index

dosage optimum dose 735 simplifying 70 dose-effect relationships 603 dot blot hybridisation 330 double contrast barium enema 547 drugs 40±1 influence on gut microbiota in old age 404±5 multiple drug resistance (MDR) 539 obesity treatments 245 Dukes' staging 550 duodenum, microbiota in 530 E-selectin 353±4 eating sequence 189 education 153, 154 eggs 34 eicosanoids 361±4 mechanisms of action 364±72 eicosapentaenoic acid (EPA) 355, 358, 360, 363±4, 367, 370 8OHdG 599, 689±90 ellagic acid 676 enamel 185 endogenous risk factors 486±93 endometrial cancer 640 endoscopy 545, 546 endpoints 737 limitations of predictive value 600±1 used in antigenotoxicity experiments 583, 586±9 energy balance 230, 231 energy density of foods 283±4 maternal nutrition 23±4 regulatory role of lipogenesis in energy metabolism 263 energy drinks 181 energy intake 278 caloric restriction 493, 511 reduction and management of obesity 245±6 enteric nervous system (ENS) 295, 299±300 enterobacteria 399, 401 Enterococcus spp. 734 enterocolitis 741 enterocytes 300±1 enterodiol (END) 657±8 enterolactone (ENL) 310, 657±8 environment environmental/exogenous risk factors 493±5

obesity and 223±4, 230±4 restrictive 230±1 enzymes glucosinolates induction of phase II enzymes 621 modulation of carcinogen activating phase I enzymes 620±1 inhibition and angiogenesis 501 inhibition by phyto-oestrogens 657±8 regulation of glycolytic/lipogenic enzymes 264±6 synbiotics and cancer prevention altering metabolic activity of gut microbiota 556±7 modulating activity of host enzymes 557±8 eosinophils 351 epidemiological (observational) studies 13±14, 682±3 early origin of adult disease 17±20 glucosinolates 617±18 nutrient-gene interaction 237±9 phyto-oestrogens 649±51, 652±3 protective role of fibre 634±7 vitamin D and fracture risk 145±7 epithelium 296±8, 712 equol 126 ergocalciferol 140 ergosterol 507 erythritol 213±14 Escherichia coli (E. coli) 430, 710 antibacterial activity of bifidobacteria 456±8 antibacterial activity of lactobacilli 435±46 probiotics and IBD 715±17, 719 etheno-modified DNA-bases 600 ethnomics/ethnobotanomics 71 European Prospective Investigation into Cancer and Nutrition (EPIC) 59 European Union (EU) 1±16, 157 approval and substantiation of health claims 13±14 food legislation 1±4 medicines and legislation 14±15 member states 2, 3 national regulatory initiatives 10±13 PROEUHEALTH cluster 744 regulation of food health claims 7±10, 40, 739 regulation of novel foods and novel ingredients 4±7 White Paper on Food Safety 2, 3 evaluation 162

Index excess weight 542 see also obesity excessive angiogenesis 496±8, 499 exogenous risk factors 493±5 expression profiling 235±6, 241±2 extrinsic primary afferent neurons (EPANs) 299 facultative bacteria 397 faecal bulk 633±4 faecal occult blood testing (FOBT) 544±5 faecal water activity 560±1 familial adenomatous polyposis (FAP) 535, 546 family history 545 famine 18 fat intake 232±3, 238 satiating efficiency 282±3 substitutes 38±9 fat synthesis 260±77 and nutrition 260±4 process 260±2 see also lipogenesis fatty acids 238, 349±93 eicosanoids 361±4 mechanisms of action 364±72 functional foods enriched with 378±82 and inflammatory diseases 375±8 maternal nutrition 25±6 modulation of fatty acid composition of cells of immune system 358±61 neonatal nutrition 28 nomenclature, sources and intakes 354±8 and osteoporosis 99±100 other mechanisms of action 372±5 polyunsaturated see polyunsaturated fatty acids (PUFAs) regulation of lipogenic gene expression 270±1 short chain (SCFAs) 307, 309, 311, 507, 532±3, 632±3, 727 supplementation 32±3 fatty fish 380 Fearon-Vogelstein cascade 533±4 fermentable carbohydrates 185, 186 reduction 188±9 fermentation of fibre in gut 632±3 fermented dairy products 406, 525 fibre 420 defining 628±30 and gut health 306±7, 309, 310, 311, 313

759

in old age 404, 407 methods of measurement 630 and prevention of cancer 628±44 breast cancer 639±40 clinical studies 638±9 epidemiological evidence 634±7 fibre intake and cancers of GIT 630±4 hormonally related cancers 637±8, 640 and satiety 283 fibroblast growth factor (FGF) 498 Finland 157 fish oil 367±70, 376±8, 380 flavin adenine dinucleotide (FAD) 693, 695 flavin mononucleotide (FMN) 693, 695 flavonoids 115±16, 307, 670, 672 flavonols 115±16, 123 flaxseed 652 flexible sigmoidoscopy 545 flow cytometry (FCM) 335±7, 342 fluidity of plasma membrane 372±3 fluorescence in situ hybridisation (FISH) 330±4, 532, 588, 737 combined with FCM 335, 336±7, 342 limitations 333±4 fluoride 190, 194 fluorogenic substrates 343±4 fluorouracil 550±1 foetal origin of adult diseases 17±21 foetus, nutritional requirements of 21±7 folate 544, 695±8 folic acid 26±7 supplementation 31 follicle-associated epithelium (FAE) 301±2 follow-up 552 Food and Agriculture Organisation (FAO) 744±5 Food Composition Databases (FCDB) 59 food fortification see fortification food intake 278±81 dietary intake surveys 147±50 and ideal food for fortification 159±61 long-term regulation 281 non-physiological factors influencing 279 physiological factors influencing 279±81 food poisoning 726 food safety see safety fortification, food 139 calcium 174±7, 180±1

760

Index

food vehicle for 157±8 general principles 156 ideal food for 159±61 issues in vitamin D fortification 156±63 reducing deficiency diseases 155±6 Fourier transform spectrometer 69 fractures, osteoporitic 84±5, 144, 145 vitamin D and risk of 145±7 France 12±13 fructans 272, 273 fructooligosaccharides (FOS) 407, 426±7 fructose 213, 262 fruits 62, 94±5, 701 cultivars 64 functional fibre 629 functional foods 192 approaches to 33±4 probiotics and 711 functional magnetic resonance imaging (fMRI) 279, 285 galactitol 202

-linolenic acid (GLA) 355, 359, 365, 376, 379 ganoderic acid F 510 garlic 65±6, 635 garlic oil 506 gastric atrophy 402±3 gastric cancer 654±5 gastrointestinal diseases, diet and 726±9 gastrointestinal tract 295, 296 distribution of microbiota 303, 396±7, 422, 529±31 gastroplasty (gastric banding) 245 GC-clamp primer 340±1 gel electrophoresis D/TGGE 340±1, 352, 738 SCGE assay 559, 586±7, 588, 589, 596, 597±8, 601 gene expression biomarkers 559±60 nutrient-sensitive genes 235±6 profiling on a genome-wide basis 241±2 regulation of lipogenic gene expression by fatty acids 270±1 gene mutation assays 588±9 gene polymorphism 239±42, 316 Generally Regarded As Safe (GRAS) status 733±4 genetic engineering 39, 70, 316±17, 463, 715 genetics colorectal cancer 535

genetic/endogenous risk factors 486±93 influences on obesity 224±34 candidate genes and mechanism 226±30, 231 interaction between genes and lifestyle 230±4 lipogenesis molecular mechanisms involved in controlling glycolytic/lipogenic genes 266±70 regulation of lipogenic gene expression by fatty acids 270±1 prevention of osteoporosis 100±5 candidate genes 100±1 interaction of genotype and diet 101±5 strategies for analysing interaction between genetic background and diet 237±44 see also nutrient-gene interactions genistein 60±1, 509, 658 and breast cancer 651±2 and osteoporosis 121, 122±3 genome wide scanning 227±8, 241±2 genomics 71, 604 genotoxic damage 559 gestational diabetes mellitus 25 ghrelin 281 gingival recession 187 ginseng 510 glucagon-like peptides (GLPs) 280, 308 D-glucitol (sorbitol) 202, 213 glucokinase 264 glucokinase regulatory protein (GKRP) 264 glucose 24, 213, 266 regulation of glycolytic/lipogenic genes 268±70 glucosinolates 615±27 chemistry and hydrolysis 616±17 future trends 623 mechanisms of action 620±3 occurrence 615±16 role in prevention of cancer 617±20 glutamate 305±6 glutamine 29±30, 305 glutathione 307, 693 glutathione-S-transferase (GST) 557, 582, 600, 621 glycolytic/lipogenic enzymes, regulation of 264±6 glycolytic/lipogenic genes 266 molecular mechanisms involved in controlling 266±70

Index glycolytic pathway 261±2 glycosphingolipids 505 grapes 63 green tea polyphenols 674±5 growth factors 307 gut health 295±324 functional foods and promoting 312±13, 314 future trends 313±17 gut microbiota and 302±5 gut renewal and absorption 313±15 immune system see immune system improving in the elderly 394±415 nutrients and gut function 305±8 nutrition and 309±12 probiotics, prebiotics and 416±82 effectiveness 427±61 improving effectiveness 461±2 types of probiotics and prebiotics and their influence on gut health 420±7 structure of the gut and immune system 296±305 gut microbiota 325±48, 528±33, 726±7 age-specific intestinal microbiota 315±16 and allergy 315 altering the composition of 554 altering metabolic activity of 556±7 characterising 334±41 composition 303, 395, 727, 728 old age 397±9 development 529 distribution in the gastrointestinal tract 303, 396±7, 422, 529±31 functions of 532±3 in the elderly 394±415 colon cancer 408 factors affecting 402±5 immunosenescence 405±7 key research areas 408±9 modification 399±402 and health 302±5 molecular based methods for identifying 326±34 need for isolation and phenotypic characterisation 341±4 role in healthy intestine 709 role in IBD 709±10 successional development 302±3, 395±9, 529 gut microbiota in old age 397±9 interactions with host physiology 396±7

761

unculturables 531±2 gut wall 296±8 haemotrophic nutrition 22±3 hamartomatous polyposis syndromes 538±9 hard caramels 204±5, 206 health claims 162, 739 approval and substantiation 13±14 EU food law and 7±10, 40, 739 national regulatory initiatives 10±13 `health foods' 181 heart disease 17±18, 63 Helicobacter pylori 396, 402±3, 429 antibacterial activity of lactobacilli 430±5, 436±7 heparin 502±3 hepatoma cell lines mutagenicity test 592, 594±5 herbs 65 hereditary nonpolyposis colorectal cancer (HNPCC) 535±6, 546 hexitols 202 high-sodium high-protein intake 103 hip fractures 84±5, 97, 145 histriotrophic nutrition 22 homocysteine 27, 695±6 hormonally related cancers 637±8, 640 hormone replacement therapy (HRT) 117, 542±3, 544 hormones, gut 298±9 nutrition and 308 host-mediated assay 595±6 host-microbe crosstalk 302±5 Howell-Jolly bodies 696 human breast milk 27±30, 395 human studies 13±14, 737 biomonitoring 597±600 phyto-oestrogen action on bone 125±6 see also epidemiological (observational) studies; intervention (clinical) studies humulone 510 hybridisation 328±9 dot blot 330 FISH 330±4, 532, 588, 737 hydrolysis of glucosinolates 616±17 hydrolyzable tannins 670 hyperglycemia 494 hyperoxia 492 hypochlorhydia 402±3 hypoxia 491±2 ileum (small intestine) 396±7, 530

762

Index

image analysis 334±5 immune system 295±6, 349±93, 401, 429 adhesion molecules 353±4 ageing and 352±3 bifidobacteria and immunomodulatory mechanisms 458±9 in disease 351±2 eicosanoids 361±4 mechanisms of action 364±72 fatty acid-enriched functional foods 378±82 fatty acids and inflammatory disease 375±8 fatty acids and modulation of fatty acid composition of cells 358±61 gut microbiota and health 302±5 in health 349±51 immunogenicity of colon cancer 540±1 nutrients and 308±9 other mechanisms of action 372±5 response to probiotics 552±3 specific features 300±2 vitamin D role in modulating defence mechanisms 142 immunosenescence 405±7, 409 in situ detection of gut microbiota 342±4 in vitro studies 14, 463, 735±6 antimicrobial function of probiotics and prebiotics 430±56 antimutagens 589±95, 600±1 in vivo phage display 512 in vivo test for antimutagenicity 595±7 indole-3-carbinol (I3C) 618, 621 indoles 617, 618±20, 622±3 infants and children gut microbiota 302±3, 395 development 529 neonatal nutritional requirements 21±2, 27±30 role of functional foods 33±9 rotaviral diarrhoea 740±1 inflammatory bowel disease (IBD) 312±13, 708±25 analysing effectiveness of probiotics 713±21 future trends 721 mechanisms of probiotic effect 712±13 role for biotic bacteria 711 role of enteric flora 709±10 screening patients with 545 inflammatory cytokines 351, 352, 353 inflammatory diseases chronic 351, 491 fatty acids and 375±8

fatty acid-enriched functional foods 378±82 innate immunity 300 insoluble fibre 629±30 insufficient angiogenesis 496, 498, 499 insulin 266 and colon cancer 638 regulation of glycolytic/lipogenic genes 266±8, 269±70 insulin growth factor (IGF) 493 insulin growth factor-I (IGF-I) 652 intake see food intake intercellular adhesion molecule 1 (ICAM-1) 353±4 interferon (IFN) 350±1 interleukin (IL) 350±1 interleukin-10 (IL-10) knock-out murine model 713±15 International Scientific Association for Probiotics and Prebiotics (ISAPP) 745 international studies, large-scale 59, 242±4, 398, 561, 744 intervention (clinical) studies 13±14, 631, 683 antibacterial activity of probiotics 434± 5, 459±61, 463 antimutagenicity 597±600 dental caries 203±4, 208±15 nutrient-gene interaction 239±44 phyto-oestrogen action on bone 125±6 probiotics and IBD 715±21 protective role of dietary fibre 638±9 intestinal bypass surgery 245 intestinal permeability 713 intestinofugal neurons 299±300 intracellular signalling molecules 373±4 intrafamiliar transmission of dental caries 205±6 intrinsic primary afferent neurons (IPANs) 299 inulin 272, 426±7 iron supplementation 31 irritable bowel syndrome (IBS) 742±4 isoflavones 115±16, 125±6, 645, 646, 675 and breast cancer 649±50, 651±2 see also phyto-oestrogens isoflavonoids 60±1 isothiocyanates (ITCs) 61, 616, 617, 618± 20 mechanisms of action 620±3 Japan 739 Joint Health Claims Initiative (JHCI) 11

Index kaempferol 123 kinases 658 krestin (polysaccharide-K or PSK) 504 labelled detector probes 338±9 labelling issues 739 lactic acid bacteria (LAB) 317 and colon cancer 552 gut microbiota 302±3 old age 398±9 probiotics 417, 419, 420±6, 526 vaccine delivery 562 see also bifidobacteria; lactobacilli lactitol 214±15 lactobacilli 406, 562, 727 probiotics 417, 419, 422, 423±5, 526, 729±30 antibacterial activity 430±50 risk of bacteremia 733 Lactobacillus acidophilus 417, 423±4 Lactobacillus casei 424±5 Lactobacillus casei Shirota 417, 445±6, 450 Lactobacillus delbrueckii subsp. bulgaricus 417, 711 Lactobacillus gasseri 423, 424 Lactobacillus johnsonii 423, 424 Lactobacillus plantarum 425 Lactobacillus reuteri 425 Lactobacillus rhamnosus GG 719, 721 Lactobacillus salivarius 424, 719 lactosidase 458 lactulose 214±15 large-scale international studies 59, 242±4, 398, 561, 744 legumes 60±1 lentinan 503±4 leptin 229, 247, 281 leptin receptor gene 229 leukotrienes 361±3 Li-Fraumeni Syndrome (LFS) 538 lifestyle healthy ageing in the Mediterranean 57±60 modification 246 obesity and interaction between genes and 230±4 light 154, 494, 688 lignan precursors 310 lignans 115, 650, 652, 653 lignin 634 linker of activated T-cells (LAT) 373±4 linoleic acid 355, 357, 365 lipid rafts 373

763

lipids anti-angiogenic 504±7 see also fatty acids lipogenic pathway 260±1 lipogenesis 260±77 future trends 272±3 in humans 262 improving using functional foods 270±2 molecular mechanisms involved in controlling glycolytic/lipogenic genes 266±70 nutrition and 263±4 regulation of glycolytic/lipogenic enzymes 264±6 long-term 265±6 short-term 264±5 regulation of lipogenic gene expression by fatty acids 270±1 regulatory role in energy metabolism 263 targeting foods to improve 271±2 lipoproteins 691 liposoluble compounds 504±7 liquid foods 284 Listeria monocytogenes 450 liver 260±2 long chain polyunsaturated fatty acids (LCPUFAs) 308±9 and eicosanoid production 363±4 and inflammatory diseases 376±8, 379±82 see also polyunsaturated fatty acids (PUFAs) low-fat diet 246 lozenges 204±5 lung cancer 655 luteolin 509 lycopene 62 lymphocyte-derived cytokines 368±9 lymphocytes 368±9 lymphoid cells 301±2 M-cells 301 macronutrients composition of diet and obesity 232±3 content and satiety 282±3 magnesium 90 malonyl-CoA 262, 263 maltitol 214±15 mammalian cell lines mutagenicity tests 589±94 D-mannitol 202, 213 mass spectrometry (MS) 69

764

Index

mast cells 351 maternal nutrition 23±7 matrix metalloproteinases (MMPs) 123 meal initiation 280±1 see also satiety meal termination 279±80 see also satiation medicine EU legislation 14±15 food as 57 Mediterranean diet 57±80 determinants of healthy ageing 57±8 food as medicine 57 functional properties of Mediterranean herbs, spices and wild greens 65±6 and healthy lifestyle 58±60 Mediterranean foods and their functional properties 60±4 metabolomics and Mediterranean plant extracts 69±70 melanocortin-4 receptor (MC4±R) 229 melanoma 655 membrane fluidity 372±3 mesalazine 715±17 metabolites, carcinogenic 599 metabolomics 69±70, 285 metal chelators 508 metastatic colorectal cancer, chemotherapy for 551 methiomine 695±6 methylenetetrahydrofolate reductase (MTHFR) 101, 695 MTHFR genotype-vitamin B interactions 103 metronomic dosing 512 micro-array technology 235±6, 337±40, 341±2, 604 microgelelectrophoresis assay 559, 586±7, 588, 589, 596, 597±8, 601 micronucleus (MN) assay 588, 595, 597 microsatellite instability (MSI) 536±7 milk and milk products 191, 194±5 milkfat 504±5 minerals 33, 306, 309, 508 prevention of osteoporosis 87±95 mismatch repair genes 536, 537 mitochondria 486±9 mitochondrial dysfunction 486±9, 494±5 mitochondrial permeability transition pores (mtPtP) 488 modified citrus pectin (MCP) 504 monitoring and control 162 monocyte-derived cytokines 368

monofunctional enzyme inducers 585 monogenic forms of obesity 228±9 mRNAs, in situ detection of 342±3 mucins 297, 540 MUC 2 mucin 540 mucosal cell proliferation 558±9 mucus 296±8 adhesion to see adhesion gut microbiota and intestinal 315±16 multiple drug resistance (MDR) 539 mutagens 581 inactivating 554 see also antimutagenicity mutations, obesity 229±30 Mycobacterium paratuberculosis 710 myeloid cells 301±2 myrosinase 616, 617 n-3 PUFAs 313, 315, 354±5, 504 and eicosanoid production 363±4 functional foods enriched with 379±82 and inflammatory diseases 376±8 mechanisms of action 367±72 see also long chain polyunsaturated fatty acids (LCPUFAs) n-6 PUFAs 354±5, 365±6 natural killer cell activity 367 necrotising enterocolitis (NEC) 741 neonatal nutritional requirements 21±2, 27±30 neovastat 502 nerves, gastrointestinal 308 nested cohort studies 682±3 Netherlands, The 12 neural networks 339 neural tube defects (NTD) 697 New Zealand 157 niacin 698±9 nicotinamide 698±9 nicotinic acid 698±9 nisin 431±4 nitrate reductases 556±7 nitric oxide (NO) 306 NO radicals 493 nitrogen 203±4 non-protein 29±30 nitroreductases 556±7 nitrosamine nitrosoproline (NPRO) 599 nitrosamines 602, 689 NO radicals 493 non-alcoholic steato-hepatitis (NASH) 273 non-digestible oligosaccharides (NDOs) 272, 273, 426±7

Index non-neoplastic angiogenesis-dependent diseases 498 non-protein nitrogen 29±30 nonsteroidal anti-inflammatory drugs (NSAIDs) 543±4 non-viable probiotics 463 Norway 157 novel foods, regulation of 4±7 novel ingredients, regulation of 4±7 Nuclear Factor kappaB (NFkB) pathway 712 nuclear magnetic resonance (NMR) spectroscopy 69, 285 NUGENOB (Nutrient Gene Interaction in Human Obesity ± Implications for Dietary Guidelines) study 242±4 nutrient function claims 8±9 nutrient-gene interactions 223±59 analysing interaction between genetic background and diet 237±44 and development of obesity 236±44 future trends 248 genetic influences on obesity 224±34 managing obesity 244±8 integrating knowledge of genetics of obesity and nutrient-gene interaction 247±8 nutrient-sensitive genes 234±6 nutrients combinations of 382 and gut function 305±8 and gut immune system 308±9 see also macronutrients nutrigenomics 316 nutrition and colorectal cancer 311 fat synthesis and obesity 260±77 foetal and neonatal requirements 21±30 and gut health 309±12 and lipogenesis 263±4 nutrition claims 8±9, 162 nutrition education 153, 154 nutrition programmes, state-wide 42 nuts 62±3 obesity 18±19 epidemic of 223, 230±4 genetic influences 224±34 managing 244±8 current strategies 244±6 integrating knowledge on genetics of obesity and nutrient-gene interaction 247±8 nutrient-gene interactions in the control

765

of 223±59 nutrition, fat synthesis and 260±77 satiety and 278 satiety and control of 278±91 selecting optimal obesity phenotype for genetic research 225±6 observational studies see epidemiological (observational) studies oestrogen 544, 655±6 oestrogen receptors 101, 117, 120±1 genotype-phyto-oestrogen interactions 104±5 oleic acid 359, 364±5, 375±6, 378±9, 504 oligofructose 272 oligonucleotide probes, rRNA-targeted 328±9 oligosaccharides 28, 400, 426±7, 462, 730±2 non-digestible 272, 273, 426±7 olive cultivars 63±4 Omegacoeur 70 optimal nutrition 682 carotenoids 686±7 folate, vitamin B12 and vitamin B6 697±8 niacin 699 riboflavin 694 vitamin A 685 vitamin C 690±1 vitamin D 688 oral carcinoma 185 oral cavity 529±30 oral contraceptives 542±3 oral health 184±99 dental caries see dental caries dietary strategies for 188±92 effects of ageing 187±8 functional foods for promoting 192±5 future trends 195±6 key dietary factors 184±6 oral tolerance 301, 302 orexins 298 organic acids 428, 451 orlistat 245 ornithine decarboxylase (ODC) 557±8 orthodontic patients 208 osteoclast apoptosis 122±3 osteomalacia (vitamin D deficiency) 143, 144 osteopenia 83±4, 116 osteoporosis 38, 67, 83±114, 139, 174 bone growth and factors affecting bone mass 85±7 definition 83±4, 144

766

Index

epidemiology 84±5 genetic variation and diet 100±5 candidate genes 100±1 interaction of genotype and diet 101±5 lipids and 99±100 minerals and 87±95 phyto-oestrogens and prevention 115±38 prevention 116±17 proteins and 99 treatment 117±20 vitamins and 95±9 vitamin D fortification 144±7 osteoprotegerin (OPG) 123±4 ovarian cancer 640 ovariectomised (OVX) cynomolgus macaques 125 ovariectomised (OVX) rodents model 124 over-fortification 161±2 oxidative DNA damage 689±90 oxidative phosphorylation (OXPHOS) 486, 489 oxidative processes 700 see also antioxidant effects oxidised DNA bases 586 32 P-postlabelling 586 p53 537±8 pantothenic acid 699±700 parathyroid hormone (PTH) 178 PARP-1 698±9 pathogens: probiotics' antibacterial activity against 429±61 pathological angiogenesis 496±500 pattern recognition 285 PDGF 496 peak bone mass (PBM) 85, 89, 144±5 pectin 504 pellagra 698 peptides 502±3 glucagon-like 280, 308 periodontal (gum) disease 184 permeability, intestinal 713 peroxisome proliferator activated receptors (PPARs) 270±1, 374±5 peroxynitrite 491, 493 peroxynitrous acid 491 Peutz-Jeghers syndrome 538±9 pH 552 phagocytosis 367 pharmabiotics 711 phase I enzymes 620±1 phase II enzymes 621

phenethyl ITC (PEITC) 618±20 phenolics 307, 310, 669±80 anti-angiogenic 509±10 functional properties 670±3 future trends 676 role in prevention of cancer 674±6 see also phyto-oestrogens phenylalanine 237, 670, 671 phenylketonuria (Fùlling's disease) 237, 247 phenylpropanoids 670, 671 phosphates 177, 191 phosphorus 91±2 physical activity 542 physical state 284 physiological angiogenesis 495±6, 497, 498±9 physiological factors changes in gut microflora in the elderly 402±3 genetic effects on obesity 230, 231 influencing food intake 279±81 phytic acid 634 phyto-oestrogens 35, 307, 645±68 in food 646±7 processing and storage induced changes 647±8 future trends 658 mechanisms of action 655±8 oestrogen receptor genotype interactions with 104±5 prevention of cancers 649±55 prevention of osteoporosis 115±38 action on bone cells 122±4 animal studies 124±5 human studies 125±6 mechanisms of action on bone metabolism 120±1 phytosterols 506±7 placenta 22±3 plant-based DNA-protective compounds 584±5 plant extracts 68±70 detection of functional compounds 68±9 extraction 68 metabolomics 69±70 plant phenolics 307, 310 plant sterols 506±7 plaque, dental see dental plaque polymorphisms, gene 239±42, 316 polyphenols 63, 654, 670±3 polyphosphates 191 polysaccharide-K (PSK or krestin) 504

Index polyunsaturated fatty acids (PUFAs) 355±8, 374 eicosanoid production 363±4 and lipogenesis 270±1, 272, 273 long chain (LCPUFAs) 308±9, 363±4, 376±8, 379±82 mechanisms of action 365±6, 367±72 n-3 PUFAs see n-3 PUFAs n-6 PUFAs 354±5, 365±6 population screening 544±5 positron emission tomography (PET) 279, 285 postnatal nutritional requirements 21±2, 27±30 potassium 94±5 pouchitis 717±19 PPAR response elements (PPAR-REs) 270 prebiotics 37±8, 312, 316, 416±82, 524±5, 527, 715, 726±52 defining 420, 730±2 economic and medicinal relevance 728±9 evidence for effects 740±4 acute disorders 740±1 chronic disorders 741±4 future trends 463 gut health in the elderly 407, 409 improving effectiveness in optimising gut health 461±2 types of prebiotics and their influences on 426±7 influences on colon carcinogenesis 552±8 mechanisms of action 427±9 methods for determining mode of action and effectiveness 735±40 modification of gut microflora 399±402 safety issues 733±5 target populations 732±3 targeting for specific probiotics 561±2 types of products and ease of use 732 precautionary principle 3±4 precholecalciferol 140 predictive value, limitations of 600±1 preeclampsia 23 pregnancy and lactation diet supplementation 30±3 nutritional requirements 21±30 role of functional foods 33±9 preload ! test meal studies 282 primary cell mutagenicity tests 591, 594 primary DNA-damage tests 586, 587

767

probiotics 35±7, 416±82, 524±7, 726±52 antimicrobial function antibacterial activity against pathogens 429±61 mechanisms of action 427±9 databases of probiotic strains 734 defining 417±19, 525, 729±30 disease-specific 721 economic and medicinal relevance 728±9 evidence for effects 740±4 acute disorders 740±1 chronic disorders 741±4 future trends 463 gut health 310±11, 312±13, 314, 317 in the elderly 406±7, 409 improving effectiveness in optimising gut health 461±2 types of probiotics and their influences on 420±6 in IBD 708±25 influences on colon carcinogenesis 552±8 methods for determining mode of action and effectiveness 735±40 modification of gut microflora 399±402 safety issues 733±5 survival of probiotics strain 738±9 target populations 732±3 targeting prebiotics for specific probiotics 561±2 types of products and ease of use 732 processing changes in phyto-oestrogens 647±8 simplifying 70 PROEUHEALTH cluster 744 programming 17±21 prohibited claims 9 propionate 532±3, 633 prospective studies 238 prostaglandins 361±3, 491 prostate cancer 640, 652±4 protamine 503 protein kinase C (PKC) 691±2 proteins 20±1 anti-angiogenic 502±3 C-reactive protein (CRP) 370 foetal requirements 26 GKRP 264 and gut function 305±6 neonatal requirements 29 prevention of osteoporosis 99 stress and protein denaturation 494

768

Index

proteomics 285, 511±12 protocatechuic acid 676 prunes 34±5 psyllium 637 purity 739±40 PYY 281 quantitative trait loci (QTL) 228 quercetin 123, 502 quinone reductase 621 radiotherapy 550±1 raloxifene 119±20 RANKL 123±4 reactive oxygen species (ROS) 672, 673 and degenerative disease 485, 486±7, 489, 492±3 real time quantitative PCR 738 recommended daily allowances (RDAs) 42, 630 calcium 174, 175 vitamins 681 vitamin D 150, 151±2 regulation 156±7, 739 EU see European Union reoxygenation 492 resistant starch (RS) 307, 427 respiratory burst 367 restrictive environment 230±1 resveratrol 122, 502, 510, 675±6 retinoic acid 510 retinol 683±5 see also vitamin A retrospective studies 631 reutericyclin 445 reuterin 445 rheumatoid arthritis 68, 351, 375±7 riboflavin 693±4 ribosomal RNA (rRNA) 326±9 generation of a database of small subunit rRNA sequences 327±8 rRNA-targeted oligonucleotide probes 328±9 rice bran 35 rickets 96, 139, 143, 688 risk analysis 3±4 risk-benefit analysis 735 rosemary 65 rotaviral diarrhoea, in children 740±1 S14 gene 265±6 Saccharomyces boulardii 717, 719 safety 2±4 concerns of functional foods 39±41

issues in the use of probiotics, prebiotics and synbiotics 733±5 level of intake of vitamin D 150±3 safety review 5±6 sage 65 saliva 186, 203 stimulation of salivary flow 191±2 salivary gland dysfunction 187±8 Salmonella antibacterial activity of bifidobacteria 456 antibacterial activity of lactobacilli 441, 442±3, 446±50 Salmonella enterica serovar Typhimurium 430 saponins 510 satiation 278, 279±80, 284 satiety 278±91 developing biomarkers of 284±5 factors influencing satiation and 278±81 impact of different food components on 282±4 and obesity 278 using biomarkers to assess weightcontrol foods 286 screening 544±6 secondary dentine 187 selective estrogen-receptor modulators (SERMs) 119±20 selective tissue oestrogen activity regulators 120 selenium 508 sequence, eating 189 shark cartilage 502 short chain fatty acids (SCFAs) 307, 309, 311, 507, 532±3, 632±3, 727 short-term studies 203±4 sibutramine 245 side effects 734 signalling defects in signalling transduction 492±3 intracellular signalling molecules 373±4 silymarin 509 single cell gel electrophoresis (SCGE) assay (comet assay) 559, 586±7, 588, 589, 596, 597±8, 601 16S ribosomal RNA (16S rRNA) gene sequences 326±34, 531±2, 737±8 culture independent identification of gut bacteria 330±4 database 327±8

Index oligonucleotide probes 328±9 T/DGGE 340±1, 532, 738 small intestine (ileum) 396±7, 530 smoking 542 sodium 92±4 solid foods 284 soluble adhesion molecule concentrations 353±4, 369±70 soluble fibre 629±30 somatic mutation and recombination tests (SMART) 588, 597 sorbitol (D-glucitol) 202, 213 soy isoflavones 125±6, 645 see also phyto-oestrogens Spain 12 specificity disease-specific probiotics 721 of DNA protection 601±2 sphingolipids 504±6 sphingosine 504±5 sporadic cancer 537 spot test 588 squalamine 507 SREBP-1c transcription factor 266±8, 269±70 staging 549±50 state-wide nutrition programmes 42 stearidonic acid 381 steatosis 273 stomach 396, 529±30 stool bulk 633±4 stool weight 637 storage 647±8 strain consortia of strains 461 databases of probiotic strains 734 survival of probiotics strain 738±9 stress 494±5 substantiation of health claims 13±14 suckling-weaning transition 264 sucrose 208±16 sugar-free chewing gum 192, 193±4 sulindac 543 sunlight 154, 494, 688 supplementation 682 calcium 176, 177, 180 developing supplements for healthy ageing 70±1 pregnancy and lactation 30±3 vitamin D 153±4 suppressing agents 673 surgery 245, 549 survival of probiotics strain 738±9 Sweden 10±11, 157

769

sweeteners 189, 200±19 dental caries and carbohydrates 200±1 sucrose and dental caries 208±16 xylitol and prevention of dental caries 202±8 synbiotics 38, 462, 524±5, 527±8, 561±2, 726±52 defining 420, 732 economic and medicinal relevance 728±9 evidence for effects 740±4 acute disorders 740±1 chronic disorders 741±4 influences on colon carcinogenesis 552±8 methods for determining mode of action and effectiveness 735±40 modification of gut microflora 399±402 safety issues 733±5 target populations 732±3 types of products and ease of use 732 SYNCAN project 560, 561 synephrine 64 synergy 71 T-cells 302 immunogenicity of colon cancer 541 linker of activated T-cells (LAT) 373±4 tamoxifen 119 tannins 670, 672 targeted arrays 604 taurine 29±30 supplementation 31±2 TCC see calcium citrate tea polyphenols 674±5 teeth dental caries see dental caries reduction of tooth susceptibility 190±1 tooth wear 186, 187 see also oral health telomerase 491 telomere shortening, oxidative induction of 490±1 temperature gradient gel electrophoresis (TGGE) 340±1, 532, 738 terpenes 510 Th1, Th2 paradigm 350±1 thiamin 699±700 thrifty gene hypothesis 231 thyme 65 tibolone 120 time-kinetics 602±3

770

Index

TIMP-1 (tissue inhibitor of metalloproteinase 1) 548 tissue biomarkers 548, 558±60 tissue cultures 736 TNM classification 550 tocopherols 62, 691 tooth wear 186, 187 total fibre 629, 630 total mesorectal excision (TME) 549 toxicants, environmental 494 TPA (tissue polypeptide antigen) 548 TPS (tissue polypeptide-specific antigen) 548 trace elements 95 traceability 3 transcriptomics 285 transgenic animals, gene mutations in 588, 596 transit time, gastrointestinal 401±2 traveller's diarrhoea 740 triacylglycerol 354 tricalcium phosphate 175 Trichinella spiralis 450 troponin I 502 tumour associated antigens (TAA) 541 tumour markers 547±8 twin overfeeding study 239 tyrosine 670, 671 ubuquinone (coenzyme Q10) 489 UDP-glucuronosyl transferase (UGT or UDPGT) 600, 621 UDS test 586 ulcerative colitis (UC) 708, 710, 741±2, 743 trials of probiotics 715±17 see also inflammatory bowel disease (IBD) ultraviolet (UV) light 154, 494 unculturable gut microbiota 531±2 under-fortification 161±2 United Kingdom (UK) 11, 157 United States (US) 157, 739 urea 194 urinary calcium excretion (calciuria) 92±3, 94, 99 vaccine delivery 562 valproic acid 504 vancomycin 734 vascular cell adhesion molecule 1 (VCAM-1) 353±4 vascular endothelial growth factor (VEGF) 495, 498

vasculogenesis 495 vegetables 61±2, 94±5, 701 cruciferous 615±16, 617±20, 623 vertebral fractures 84±5 vitamin A 161, 306, 681 prevention of cancer 683±5 prevention of osteoporosis 97±8 vitamin B interactions with MTHFR genotype 103 riboflavin (vitamin B2) 693±4 vitamin B3 (niacin) 698±9 vitamins B6 and B12 695±8 vitamin C prevention of cancer 689±91 prevention of osteoporosis 98±9 vitamin D 89, 139±73, 681 deficiency 143±4, 688 dietary intake 147±53 recommendations 150, 151±2 safe level of intake 150±3 function 141±2 interaction with VDR genotype 102±3 issues in fortification 156±63 bioavailability 158 characteristics of fortificant and food vehicle 157±8 characteristics of a successful fortification programme 162±3 claims 162 effectiveness of fortification of vitamin D status 158±9 ideal food for fortification 159±61 monitoring, control and evaluation 162 over- and under-fortification 161±2 metabolism 141 new roles 142 prevention of cancer 142, 687±8 prevention of osteoporosis 95±6 fortification 144±7 sources 140 status 142±3 strategies to improve supply 153±4 vitamin D binding protein (DBP) 141 vitamin D insufficiency 143±4 vitamin D receptor (VDR) gene 100±1, 141±2 interactions with calcium 102 interactions with calciuric factors 103 interaction with vitamin D 102±3 vitamin E 691±3 vitamin K interaction with apolipoprotein E genotype 103±4

Index prevention of cancer 699±700 prevention of osteoporosis 96±7 vitamins 33, 582 anti-angiogenic 508±9 cancer prevention 681±707 future trends 700±1 role 683±700 deficiencies see deficiencies of vitamins gut health 306, 309 prevention of osteoporosis 95±9 see also under individual vitamins VSL#3 450, 717±19 weaning 264 weight excess 542 of foods 283±4 weight-control foods 286 wheat bran 633±4 whole rice 35 wild greens 66 wine 63 wireless capsule endoscopy 546 World Health Organisation (WHO) 744±5

771

xylitol 190, 201, 202±8, 209, 213, 216 biochemical and bioinorganic mechanisms 204 chemical characterisation 202±3 in chewing gum 190, 192, 193±4, 204±5, 206 combination with other adjuvants 208, 209 early prevention of dental caries 206±7 food products 204±5, 206 long-term effects 207 prevention of dental caries in orthodontic patients 208 prevention of intrafamilial transmission of dental caries 205±6 short-term and laboratory caries studies 203±4 Yakult 417 yeasts 397 YY peptide 308 zinc 306